1
From: Cancer Drug Discovery and Development:
Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ
1
SUMMARY
The epidermal growth factor (EGF) receptor (EGFR) is a tyrosine kinase receptor of the ErbB family that is abnormally activated in many epithelial tumors.
In human tumors, receptor overexpression correlates with a more aggressive clinical course. These observations suggest that the EGFR is a promising target for cancer therapy, and monoclonal antibodies directed at the ligand-binding extracellular domain and low molecular weight (MW) inhibitors of the receptor’s tyrosine kinase inhibitors (TKI) are currently in advanced stages of clinical development. These agents prevent ligand-induced receptor activation and down- stream signaling, which results in cell-cycle arrest, promotion of apoptosis, and inhibition of angiogenesis. In preclinical models, these agents markedly enhance the antitumor effects of chemotherapy and radiation therapy. In patients, anti-
Targeting of the EGFR As a
Modulator of Cancer Chemotherapy
Jose Baselga, MD
CONTENTS SUMMARY
THE EGF RECEPTOR AS A TARGET FOR CANCER THERAPY
ANTI-EGF RECEPTOR STRATEGIES AND MECHANISMS OF ACTION
CLINICAL DEVELOPMENT OF ANTI-EGF RECEPTOR
MONOCLONAL ANTIBODIES
LOW-MW EGF RECEPTOR TYROSINE KINASE INHIBITORS
CHALLENGES IN THE DEVELOPMENT OF ANTI-EGF RECEPTOR
COMPOUNDS
DIRECTIONS IN THE COMBINED TREATMENT WITH ANTI-EGF AGENTS AND CHEMOTHERAPY IN THE TREATMENT OF CANCER
EGFR agents can be given safely at doses that fully inhibit receptor signaling, and single-agent activity has been observed against a variety of tumor types, including colon carcinoma, non-small-cell lung cancer (NSCLC), head and neck cancer, ovarian carcinoma, and renal-cell carcinoma. However, their antitumor activity is significant but modest, and to improve their efficacy, ongoing research efforts are being directed at the selection of patients with EGFR-dependent tumors, identification of the differences among the various classes of agents, and new clinical development strategies. One such strategy, derived from the preclinical models, is to combine these agents with chemotherapy. Clinical trials of anti- EGFR agents in combination with chemotherapy have been conducted or are underway in a variety of tumor types and in different clinical settings. In NSCLC, a series of well-supported multinational phase III clinical trials have shown that the combined therapy with chemotherapy and anti-EGFR TKI is not superior to chemotherapy alone. On the other hand, in advanced colorectal cancer, the com- bined treatment with anti-EGFR monoclonal antibodies and conventional che- motherapy was found to be statistically superior in terms of disease-free survival when compared with chemotherapy alone. In addition, in smaller trials, the addi- tion of anti-EGFR monoclonal antibodies to chemotherapy does result in a higher antitumor response rate than with chemotherapy alone. Taken together, anti- EGFR agents are active antitumor agents, and the optimal way to combine these agents with conventional chemotherapy is still to determined and likely to be agent and tumor-type dependent. Intensive clinical research on how best to inte- grate these agents into treatment is warranted.
THE EGF RECEPTOR AS A TARGET FOR CANCER THERAPY
The epidermal growth factor (EGF) receptor (EGFR) was the first identified of a family of receptors known as the type I receptor tyrosine kinases, or ErbB receptors. This receptor family is comprised of four related receptors: the EGFR itself (ErbB1/EGFR/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) (1–3). These receptors trigger downstream signaling pathways that are not linear but consist of a rich, multilayered network, which allows for horizontal interactions and permits multiple combinatorial responses which may explain the specificity of cellular outcomes to receptor activation. Deregulation of these tightly regulated ErbB receptor signaling pathways leads frequently to malignant transformation. In order to simplify our understanding of EGFR signaling, it may be useful to dissect the process into sequential levels starting at the cell surface, subsequently moving into intracellular-signaling pathways that lead to gene transcription, and ending in a variety of cellular responses (1).
The cell surface is where the initial ligand–receptor and receptor–receptor interactions occur. ErbB receptors are composed of an extracellular ligand-bind- ing domain, a transmembrane segment, and an intracellular protein kinase domain
with a regulatory carboxyl-terminal segment. ErbB receptors become activated by several mechanisms. Under physiological conditions, a variety of EGFR fam- ily ligands drive the formation of homo- or heterodimeric complexes among the four ErbB receptors, which provides for signal amplification and diversification (Fig. 1) (1). In tumor cells, these receptors can be activated by additional mecha- nisms. First, receptor overexpression in the tumor may lead to ligand-independent receptor dimerization. In some tumors, such as glioblastoma, mutant forms of the EGFR that arise from gene rearrangements result in ligand-independent consti- tutive receptor activation and impaired receptor downregulation (4). Heterolo- gous ligand-dependent mechanisms are also at play, as demonstrated by the finding that stimulation of G protein-coupled receptors results in EGFR activa- tion via metalloproteinase-mediated cleavage of precursor membrane-bound EGF ligands (5). Recently, a ligand-independent mechanism of EGFR activation via the urokinase plasminogen receptor has been identified (6). These findings sug- gest that tumor cells may have additional EGFR activation mechanisms beyond receptor overexpression, mutations, and autocrine ligand production.
At the signal-processing level, activation of the intrinsic receptor protein tyrosine kinase and tyrosine autophosphorylation occurs. These events result in the recruitment and phosphorylation of several intracellular substrates, as well as binding of docking and adaptor molecules to specific phosphotyrosine sites on receptor molecules (Fig. 2) (7). A major downstream signaling route of the ErbB family is via the Ras-Raf-MAP-kinase pathway (8). Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs, ERK1, and ERK2 (9). ERK1/2 regulate transcription of molecules that are linked to cell proliferation, survival, and transformation in laboratory studies (9). An- other important target in EGFR signaling is phosphatidylinositol 3-kinase (PI3K) and the downstream protein-serine/threonine kinase Akt (10–12). Akt trans- duces signals that trigger a cascade of responses from cell growth and prolifera- tion to survival and motility (12). Another route for signaling is via the stress-activated protein kinase pathway, involving protein kinase C and Jak/Stat.
The activation of these pathways translates in the nucleus into distinct transcrip- tional programs that mediate a variety of cellular responses, including cell divi- sion, survival (or death), motility, invasion, adhesion, and cellular repair (1).
The EGFR was proposed almost 20 yr ago as a target for cancer therapy for a variety of reasons. First, as already mentioned, the EGFR is frequently overexpressed in human tumors. Examples include cancers of the breast, lung, glioblastoma, head and neck cancer, bladder carcinoma, colorectal cancer, ova- rian carcinoma, and prostate cancer (13). The level of increased expression can reach an order of magnitude or greater. Gene amplification is not a commonly reported finding in tumors, with the exception of the glioblastomas. Further- more, in some glioblastomas a mutant variant of the receptor, denominated EGFR vIII, has a deletion in the extracellular domain leading to constitutive activation of its tyrosine kinase (14–16).
Second, increased EGFR expression correlates with a poorer clinical outcome in a number of malignancies, including bladder, breast, lung, and head and neck cancers (13,17). Third, increased receptor content is often associated with increased production of ligands, such as transforming growth factor (TGF)-_ by the same tumor cells (13,17,18). This establishes conditions conducive to recep- tor activation by an autocrine stimulatory pathway.
And finally, in early studies by one of us (J. M.), a series of monoclonal antibodies (MAbs) directed at the EGFR were shown to inhibit the growth of cancer cells bearing high levels of EGFRs, both in culture and in nude mouse xenografts (19–22).
ANTI-EGF RECEPTOR STRATEGIES AND MECHANISMS OF ACTION
There are several potential strategies for targeting the EGFR, including MAbs that interfere with receptor signaling and MAbs serving as carriers of radionu- clides, toxins, or prodrugs (23); low-molecular-weight (MW) tyrosine kinase inhibitors that interfere with receptor signaling; antisense oligonucleotides or ribozymes, which block receptor translation (24,25); or prevention of receptor Fig. 1. Epidermal growth factor receptor signaling. The process of receptor signaling may be intracell into sequential levels starting at the cell surface, where ligand–receptor and receptor–receptor interactions occur, into key intracellular-signaling pathways that lead to gene transcription and cell-cycle progression. The end result is a variety of cellular responses that promote the malignant phenotype.
trafficking to the cell surface with intracellular single-chain Fv fragments of antibodies (26). Of these approaches, MAbs and the low-MW tyrosine kinase inhibitors are the ones in the most advanced stages of clinical development and will be reviewed in detail.
The antibodies in clinical trials bind to the easily accessible extracellular domain of the receptor and compete with the ligand binding to the receptor. For example, the murine MAb 225 and its chimeric human:murine derivative cetuximab (cetuximab, Erbitux™) bind to the EGFR with high affinity (Kd = 0.39 nM for cetuximab), compete with ligand binding, and block activation of receptor tyrosine kinase by EGF or TGF-_ (19,20,27). In addition, MAb 225/
cetuximab induces antibody-mediated receptor dimerization resulting in recep- tor downregulation, and this effect may be important for its growth-inhibitory capacity (28). The low-MW inhibitors, on the other hand, compete with ATP for binding to the tyrosine kinase portion of the receptor, and thereby abrogate the Fig. 2. Inhibition of signaling pathways by anti-epidermal growth factor (EGF) receptor therapies. In unperturbed conditions, major signaling routes of the EGF receptor are the ones constituted by the Ras-Raf-MAP-kinase and the phosphatidylinositol 3-kinase (PI3K), and the downstream protein-serine/threonine kinase Akt pathway. Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAP- kinases. Akt transduces signals that fall into two main classes: regulation of apoptosis and regulation of cell growth. (B,C): Signal transduction via these pathways is efficiently blocked by anti-EGF receptor therapies as shown here in cultures of A431 cells treated with the EGF receptor tyrosine kinase inhbitor ZD1839. (Adapted from ref. 34.)
receptor’s catalytic activity. Some of these small molecules can induce forma- tion of inactive EGFR homodimers and EGFR/HER2 (ErbB1/ErbB2) hetero- dimers (29,30), which impair EGFR-mediated transactivation of the potent ErbB2 tyrosine kinase. In addition, because of the >80% homology in the kinase domain between the EGFR (ErbB1) and HER2 (ErbB2) (3), some ATP-competi- tive low-MW inhibitory molecules can block the catalytic activity of both recep- tors (reviewed in 31). These small molecules are also able to block the catalytic activity of EGFR mutants lacking the extracellular domain (32) and should be able to prevent ligand-independent activation of EGFR kinase activity as well.
At the level of downstream receptor-dependent signaling pathways, EGFR antibodies and low-MW ATP-competitive inhibitors of the EGFR kinase have similar effects. Both strategies result in an efficient blockade of the main EGFR signal transduction pathways, including the MAPK and PI3K/Akt pathways (33–38) and the Jak/Stat pathway (39) (Fig. 3).
As a result of their effects on the receptor and downstream signaling, anti- EGFR MAbs and the low-MW tyrosine kinase inhibitors interfere with a number of key cellular functions regulated by the receptor that satisfactorily explain their antitumor effects. These are summarized below, with antibody studies described first in most cases because they were reported earlier:
1. Cell-cycle arrest. Initial experiments with MAb 225 demonstrated that the antibody induces G1-phase arrest due to elevated levels of the CDK2 inhibitor p27KIP1, which results in hypophospholyration of Rb protein (40,41). Similarly, low-MW tyrosine kinase inhibitors of the EGFR induce an accumulation of p27KIP1 and of hypophosphorylated Rb protein that leads to a G1 arrest (42,44).
2. Potentiation of apoptosis. In some cases G1 arrest is followed by apoptosis (45).
In DiFi colon carcinoma cells, this can be attributed to induction of Bax and activation of caspase-8 (45–48). Activation of other proapoptotic molecules has also been reported.
3. Inhibition of angiogenesis. Blockade of EGFR activation by cetuximab and by low-MW tyrosine kinase inhibitors results in a significant decrease in tumor-cell production of angiogenic growth factors such as `FGF, VEGF, and IL-8 (45–
48). The decrease in angiogenic growth factors in turn correlates with a signifi- cant decrease in microvessel density and an increase in apoptotic endothelial cells in human tumor xenografts (45).
4. Inhibition of tumor-cell invasion and metastasis. Cetuximab inhibits lung metastasis in mice with established human tumor xenografts (47). Cetuximab and similar MAbs directed against the EGFR have also been shown to inhibit the expression and activity of several matrix metalloproteinases (MMPs) that play a key role in tumor-cell adhesion, including the gelatinase MMP-9. Several studies have correlated this antibody-mediated decrease in MMP production with both a significant reduction in in vitro tumor-cell invasion and the inhibi- tion of tumor growth and metastasis in nude mice (49–51). The inhibitory effects
on invasion, metastasis, and angiogenesis cells may explain why cetuximab treatment is often more effective in vivo than in vitro.
5. Augmentation of the antitumor effects of chemotherapy and radiation therapy.
Based on an initial observation that an anti-EGFR antibody had the capacity to enhance the antitumor activity of cisplatin in a human tumor xenograft model (22), extensive studies of human tumor-cell xenografts were conducted with MAb 225 and cetuximab. The experiments demonstrated that these MAbs mark- edly augment the antitumor effects of different classes of chemotherapeutic agents including cisplatin, doxorubicin, topotecan, and paclitaxel (52–55). In addition to the enhanced antitumor effects when both class of agents are given together, studies in preclinical models have shown that anti-EGFR monoclonal antibodies can reverse chemotherapy resistance (56). In a refractory tumor model, combined treatment with cetuximab and CPT-11 significantly inhibited the growth of CPT-11 refractory DLD-1 and HT-29 colon tumors, whereas either agent alone did not control tumor growth. These findings are highly consistent with the clinical data that have emerged from the colorectal BOND trial (see Subheading 3.).
Fig. 3. Restoration of sensitivity to irinotecan by the addition of cetuximab in a colorectal carcinoma model. Growth inhibition of CPT-11 refractory colorectal tumor xenografts in nude mice. Mice with established HT-29 tumors were treated with two cycles of CPT- 11 therapy (100 mg/kg) on d 0 and 7. Mice with tumors that did not respond to CPT-11 therapy (>2 × initial tumor volume at d 12; shown as dotted vertical line) were selected, randomized, and then treated with cetuximab at 1 mg/dose/q3d (•), continued CPT-11 at 100 mg/kg/wk (white box), or combination therapy (black box). Bars, ±SE (56).
Studies have also demonstrated that the low-MW EGFR tyrosine kinase inhibitors enhance the antitumor activity of conventional chemotherapeutic agents, both in cell culture and in human tumor xenografts (57,58). Similar findings are observed when these agents are given in combination with radiation therapy (59,60).
There is, however, evidence that the mechanisms of action and the antitumor effects of MAbs and the low-MW tyrosine kinase inhibitors are not completely overlapping. Anti-EGFR MAbs (28), but not the low-MW tyrosine kinase inhibi- tors (35), have the capacity to form receptor-containing complexes that result in receptor internalization, an important mechanism for attenuating receptor signal- ing. In addition, cetuximab can elicit antibody-dependent cellular cytotoxicity (ADCC) (61), an antitumor mechanism that also could be important for the action of the anti-erbB2 MAb, trastuzumab (62). In contrast, the inhibition of more than one ErbB receptor type is unique to the low-MW tyrosine kinase inhibitors. There- fore, it is not surprising that in studies with cultured cancer cells maximally inhibited by low-MW EGFR tyrosine kinase inhibitors, the addition of anti-EGFR MAbs can result in further antitumor activity (63). This finding sets the stage for combining anti-EGFR MAbs and low-MW tyrosine kinase inhibitors in the clinic.
CLINICAL DEVELOPMENT OF ANTI-EGF RECEPTOR MONOCLONAL ANTIBODIES
Among available anti-EGFR MAbs (Table 1), the one furthest ahead in clini- cal development is the chimeric human:murine MAb cetuximab (cetuximab, Erbitux™). Cetuximab is a potent inhibitor of the growth of cultured cancer cells that have an active autocrine EGFR loop, and it is capable of inducing complete regressions of well-established human tumor xenografts overexpressing the EGFR (64).
A series of phase I/II studies of cetuximab given alone or in combination either with chemotherapy or radiation have now been completed. In these early studies, cetuximab was found to be safe, and the most prominent side effects included an acneiform skin rash and anaphylactoid or anaphylactic reactions that occurred in 2% of cases (data from ImClone Systems, Inc.). The allergic reactions occurred after the first infusion and responded well to standard therapy (65). Nonneutralizing human antibodies against chimeric antibodies (HACAs) were detected in 4% of patients and were not related to allergic or anaphylactic reactions, and the HACA responses had no effect upon the phar- macokinetics of repeated weekly infusions of cetuximab (66). The optimal biological dose, as determined by saturation of antibody clearance, was found to be in the range of 200 to 400 mg/m2 per week (67). These doses have been confirmed to block EGFR activation and downstream signaling in biopsy specimens from patients (68).
In phase II studies, cetuximab was shown to be active in patients with CPT- 11-treated colorectal cancer and with cisplatin-resistant head and neck tumors.
Following an initial clinical observation that the addition of cetuximab to CPT- 11 induced responses in CPT-11-refractory patients with advanced colorectal carcinoma (69), a phase II study was performed with patients with advanced colorectal carcinoma and progression on CPT-11 treatment. In this study, 120 patients were continued on the same dose and schedule of CPT-11, and cetuximab was added on a full-dose weekly schedule (Table 2). The combination was found to be safe and the response rate was 22.5%, with a median duration of response of 186 d (70). A retrospective analysis of this study has revealed an interesting correlation between the occurrence of skin rash and a greater response rate (71).
Recently, single agent cetuximab in patients with CPT-11-refractory advanced colorectal carcinoma has shown an 11% response rate in a small phase II study (59). These studies by Saltz raised some important questions: first, were the
Table 1
Anti-EGFR MAbs: Clinical Activity
n Response
Cetuximab Colorectal + CPT-11 218 22.9%
(C225) (CPT-11 refractory)
120 17%
Single agent 111 10.8%
11%
Head and + CDDP 22 23%
neck (CDDP refractory)
75 11%
EMD72000 Colorectal Single agent 19 16%
(phase 1)
EBX-EGF Colorectal Single agent 23 13%
Renal Cell Single agent 31 6%
Table 2
IMC-C225 Phase II Trials in Refractory CRC
C225 C225 + Irinotecan
No. patients evaluable 57 121
Response rate 11% (3–19%) 17% (11–24%)
Stable disease 37% 31%
Median duration 182 d 164 d
patients that responded to cetuximab and CPT-11 in the colorectal carcinoma study truly CPT-11 refractory? The design of the phase II colorectal trial of adding cetuximab to CPT-11 by Saltz and colleagues was well suited to answer this question, since a minimal number of cycles of CPT-11 prior to adding cetuximab to CPT-11 was not required. Second, even if we assume that the patients were chemotherapy refractory, the observed antitumor activity with cetuximab could be the result of several possibilities: cetuximab may reverse chemotherapy resistance—an indication of synergy in vivo, an indication of single agent activity by cetuximab, or both: single activity on one hand and a capacity to reverse resistance to chemotherapy on the other. As mentioned above, a follow-up study demonstrated that cetuximab has single-agent activity in colorectal carcinomas, so the question has been partially answered (72). The second possibility has been recently addressed by a European study in which patients refractory to CPT-11 were randomized to receive cetuximab as a single agent or cetuximab plus CPT-11 at the same dose and schedule that they had progressed on. This study, named BOND (bowel oncology with cetuximab an- tibody), evaluated the antitumor activity of cetuximab treatment alone (111 patients) or in combination with CPT-11 (218 patients) in advanced colorectal cancer patients with EGFR-positive disease that had progressed on CPT-11 (73).
In this truly CPT-11-refractory population, the antitumor activity was greater in the patients that were given the same dose and schedule of CPT-11 in combina- tion with cetuximab (response rate 22.9%) that in patients treated with cetuximab alone (response rate 10.8%) (p = 0.0074). Similarly, a significantly better disease control (partial responses plus disease stabilization) and a prolonged median time to progression were observed in the combination as compared to the cetuximab combination arm ( 55.5% vs 32.4%, p = 0.0001; and 4.1 vs 1.5 mo, p < 0.0001). Survival was not increased in the combination arm (8.6 vs 6.9 mo), although this is likely to be related to the fact that upon progression on the cetuximab alone arm, crossover to combined treatment was allowed. The en- hanced antitumor activity of the combination did not result in an increase in CPT- 11 specific toxicity. This study also confirmed the lack of correlation of levels between the levels of EGFR expression and antitumor activity of cetuximab, and a greater response rate in those patients that developed a skin rash (73).
In patients with advanced head and neck tumors, a phase II study analyzed the addition of cetuximab to the treatment of patients who had received two cycles of cisplatin-based therapy and had either stable or progressing disease. In the progressing-on-chemotherapy subgroup, 5 responses were seen out of 22 treated patients, for a response rate of 23% (74). In a larger study involving 75 evaluable patients with refractory head and neck cancer who had documented progression after having received at least two cycles of platinum-based therapy, an 11%
response rate was observed when cetuximab was added to the platinum regimen
(75). In the latter study, the potential source of bias of treating with cetuximab with patients not truly refractory to chemotherapy was controlled for, because patients were required to have documented progression after having received at least two cycles of platinum-based therapy, prior to cetuximab being added to the platinum regimen (75).
Additional studies with cetuximab in combination with chemotherapy have also been conducted. A small phase III study in head and neck tumors comparing cisplatin and placebo to cisplatin and cetuximab showed more than doubling of the response rate, but only a modest and nonsignificant improvement in time to disease progression in the cetuximab arm (76). A randomized phase II study in EGFR-positive, advanced NSCLC patients (LUCAS, the Lung Cancer Cetuximab Combination Study), first-line therapy with cisplatin-vinorelbine was compared to the same combination plus cetuximab (77). Interestingly, a higher response rate was observed in the combination arm than in the chemotherapy alone arm ( response rate 50% vs 29%) (77). These results are of interest, taking into consid- eration the absence of benefit of combining an anti-EGFR TKI to two drug- containing chemotherapy regimens in the same patient population.
Additional responses to chemotherapeutic agents given in combination with cetuximab were observed in phase II studies of gemcitabine in patients with advanced pancreatic carcinoma (78) and docetaxel in advanced NSCLC (79).
Cetuximab can also be administered safely in patients with head and neck cancer, when given in combination with radiation therapy, with 13 complete responses and 2 partial responses in 16 patients (80). A phase III study of radiation ± cetuximab in patients with advanced head and neck tumors has recently com- pleted accrual.
Other anti-EGFR MAbs that have a similar mechanism of action to cetuximab are currently under clinical investigation. ABX-EGF is a fully human IgG2 anti- EGFR MAb that binds with high affinity (Kd = 50 pM), inhibits ligand-dependent receptor activation, and effectively inhibits the growth of human tumor xenografts (81). In a phase II study of ABX-EGF in advanced renal-cell carcinoma, 31 patients who had failed or were unable to receive IL-2/IFN-_ completed one 8-wk cycle of ABX-EGF and were evaluable for response. Objective responses were seen in one patient each at 1 and 1.5 mg/kg/wk dose levels. Fifty-eight percent of patients showed minor response/stable disease, and 36% progressed (82).
EMD 72000 is a humanized anti-EGFR monoclonal antibody that also pre- vents ligand-induced receptor activation and is currently in phase I studies (83).
This antibody has shown single-agent activity in a variety of tumor types and has a prolonged half-life that may allow for a less frequent administration schedule than the other antibodies, which are given on a weekly basis. In an ongoing trial, preliminary efficacy and pharmacodynamic data suggest that a more convenient every 2–3 wk administration schedule may actually be fea-
sible with EMD 72000 (84). As with cetuximab, studies of EMD 72000 in combination with chemotherapy are underway in NSCLC. A pilot study has reported that in patients with NSCLC, the combination of paclitaxel and EMD 72000 is feasible and active (85).
Antibody h-R3 is another anti-EGFR monoclonal antibody that has entered clinical trials. The safety profile of all of these antibodies has been good and, not surprisingly, acneiform skin rashes are the most frequent side effect.
LOW-MW EGF RECEPTOR TYROSINE KINASE INHIBITORS There are a large number of low-MW inhibitors of EGFR tyrosine kinase that are under clinical development (Table 3). In an attempt to classify these anti- receptor agents, we have grouped them by their degree of receptor specificity (restricted to the EGFR, or also inhibiting other ErbB kinases) and by their reversibility or irreversibility of action.
Class 1. Reversible EGFR-Specific Tyrosine Kinase Inhibitors These compounds are the furthest ahead in clinical development and can be exemplified by ZD1839 and ERLOTINIB. ZD1839 (gefinitib, Iressa®), inhibits the EGFR kinase in vitro with an IC50 of 0.02 μM and requires a dose almost 200- fold higher to inhibit HER2 (3.7 μM) (86). Preclinical studies with ZD1839 have shown antitumor activity in a variety of cultured tumor cell lines and in human tumor xenografts, both as a single agent and in combination with chemotherapy and radiation therapy (48,57,58,60,86,87). An intriguing finding has been that cultured breast cancer cells that express high levels of HER2, even in the pres- ence of a low number of EGFRs, are exquisitely sensitive to ZD1839 at concen- trations that do not suppress HER2 tyrosine kinase activity (35–38).
Phase I studies have demonstrated that daily administration of ZD1839 is safe, with dose-dependent pharmacokinetics, although with a high degree of interpatient variability (88–90). The most common side effects were an acnei- form skin rash, generally mild and reversible on cessation of treatment, and diarrhea. In these early studies, the effects of ZD1839 on EGFR activation and receptor-dependent events in the skin, an EGFR-dependent tissue, were ana- lyzed (68). ZD1839 significantly suppressed EGFR phosphorylation, inhibited MAPK activation, reduced keratinocyte proliferation, and increased p27KIP1 levels and apoptosis. Marked reduction in EGFR phosphorylation was observed at doses well below doses producing unacceptable gastrointestinal toxicity, a finding that strongly supports the use of an optimal biological dose instead of the maximally tolerated dose for these types of agents. Clinical responses were observed in patients with NSCLC (88–90).
Phase II studies with two dose levels of ZD1839 (250 and 500 mg) have know been completed in patients with NSCLC that had progressed after first- or sec- ond-line chemotherapy for advanced disease. The first study was conducted in 210 patients previously treated with one or two chemotherapy regimens, and showed an 18.7% response rate and marked improvement in disease-related symptoms (91) (Table 4). Interestingly, the 250 mg/d dose was as active as the 500 mg/d dose and had a lower frequency of adverse events. In the second study of 216 patients who had failed at least two prior chemotherapy regimens, tumor response rates of 11.8% and 8.8% were observed for the 250 and 500 mg/d groups, respectively (92). The results of these trials have led to the regulatory approval of ZD1839 in Japan.
Pilot trials of ZD1839 with carboplatin/paclitaxel or gemcitabine/cisplatin demonstrated these combinations to be well tolerated, with antitumor activity in patients with NSCLC (93,94). The preclinical experiments mentioned above and the feasibility of combining chemotherapy with ZD1839 as demonstrated in the pilot studies led to the design of two large phase III studies of chemotherapy ± ZD1839 in patients with advanced chemotherapy-naïve NSCLC. These studies, known as INTACT 1 and 2 (for Iressa NSCLC Trial Assessing Combination Treatment) were randomized, double-blind, placebo-controlled trials of chemo- therapy± ZD1839 (95,96). In the first study, the chemotherapy regimen was a combination of cisplatin and gemcitabine at the usual dose and schedule (six cycles of gemcitabine 1250 mg/m2 on d 1 and 8, plus cisplatin 80 mg/m2 on d 1) (96). Patients were randomized to chemotherapy + placebo, chemotherapy + 250 mg/d ZD1839, or chemotherapy + 500 mg/d ZD1839. A total of 1093 patients were entered. There were no differences in overall survival (median 11.1, 9.9, and 9.9 mo for placebo, 250-mg, and 500-mg arms respectively), progression- free survival, and time to worsening of symptoms across the three arms. In the second trial, 1037 patients were entered into a similarly designed trial although with a chemotherapy consisting of carboplatin ( AUC 6) and paclitaxel (225 mg/
m2) every 3 wk for six cycles (96). There were again no differences in overall Table 3
Cetuximab-BOND Study Design
Patients with colorectal cancer progressed on or within 3 mo of irinotecan-based chemotherapy
Radomized
Irinotecan + cetuximab n = 218
Cetuximab n = 111
Irinotecan + cetuximab PD
survival (median 9.9, 9.8, and 8.7 mo for placebo, 250-mg, and 500-mg arms respectively), progression-free survival, and time to worsening of symptoms across the three arms. The results in these two well-controlled trials have clearly demonstrated that in NSCLC the addition of ZD1839 to conventional chemo- therapy does not result in enhanced clinical benefit over chemotherapy alone.
Single-agent phase II studies with ZD1839 have also been conducted or are still ongoing in other tumor types, including head and neck carcinomas, colon, prostate, gastric, and breast cancer. In tumors of the head and neck, a phase II study has shown an 11% response rate (97). A study in patients with previously treated colorectal carcinoma with ZD1839 at a (high) dose of 750 mg has recently been reported. Although some evidence of antitumor and biological activity were observed, there were no documented responses (98). In a study in hormone- refractory prostate cancer, patients were randomized to receive either ZD1839 at 250 mg or 500 mg/d (99). A total of 40 patients were treated: no objective or PSA responses were seen, and *9 patients had a best response of treatment failure, meeting the protocol criteria for stopping the study.
Erlotinib (OSI-774, Tarceva®, formerly known as CP-358, 774) is an orally available quinazoline that is a selective inhibitor of the EGFR. A phase I study with increasing daily doses of erlotinib demonstrated that the MTD was 150 mg/
d. At this recommended dose level, erlotinib resulted in a steady-state serum concentration higher than the concentration required to achieve full receptor inhibition in preclinical models. Similar to ZD1839, erlotinib inhibited EGFR- dependent processes in skin and tumor biopsies (100). Clinical responses were also seen in phase II studies conducted in patients with NSCLC, head and neck tumors, and ovarian carcinoma (101–103) (Table 3). In NSCLC, 57 patients that had histologically documented stage IIIB/IV EGFR-positive disease that had failed prior systemic therapy were treated with erlotinib at a dose of 150 mg/d (101). There were two complete remissions and five partial remissions for an overall response rate of 12%. In addition, the median survival in that study was 9.3 mo and the 1-yr survival was an impressive 40% (101).
Table 4
Cetuximab-BOND Response Rate/TTP IRC–ITT cohort
Combination Monotherapy
(n = 218) [95% CI] (n = 111) [95% CI] p-value
PR 22.9% [17.5–29.1] 10.8% [5.7–18.1] 0.0074
Deisease control* 55.5% [48.6–62.2] 32.4% [23.9–42.0] 0.0001
Median TTP 4.1 mo 1.5 mo <0.0001
*CR + PR + SD
As with gefitinib, a comprehensive phase III program in first-line NSCLC of erlotinib in combination with chemotherapy was initiated. Two multicenter, ran- domized, controlled phase III studies evaluated erlotinib at 150 mg/d in combina- tion with standard chemotherapy in patients with stage IIIB/IV metastatic NSCLC.
In the TRIBUTE study, 1050 patients were randomized to receive erlotinib plus carboplatin and paclitaxel or chemotherapy alone. In the TALENT study, 1200 patients were randomized to receive either erlotinib in combination with gemcitabine and cisplatin or chemotherapy plus a placebo. In a recent announce- ment by the pharmaceutical company sponsoring the trial (www.gene.com), the results of both studies were reported as failing to meet their primary endpoint (survival) in addition to other secondary endpoints. These results are highly similar to the results of the gefitinib studies in combination with chemotherapy in NSCLC.
In addition to the TALENT and TRIBUTE studies, there is an ongoing 700- patient phase III study in refractory NSCLC being conducted by the National Cancer Institute in Canada, in which patients are being randomized to receive erlotinib vs placebo, with survival as its primary endpoint.
In ovarian carcinoma, 34 patients were treated and 2 patients had a confirmed response (overall response rate of 6%), in addition to 2 patients with unconfirmed responses and 14 patients with stable disease for >2 mo (103). In head and neck, a multicenter trial of 124 patients with locally recurrent and/or metastatic disease that had been previously treated were given erlotinib at the recommended dose of 150 mg/d: there were 6 patients with confirmed responses, for an overall response rate of 5% (102). In a phase II ongoing study in colorectal carcinoma, no clinical responses have been reported to date (104).
Currently, phase II studies with erlotinib are underway in other tumor types, including breast cancer, and phase IB studies are exploring the feasibility of combining erlotinib with a variety of conventional chemotherapeutic agents.
Class 2. Irreversible EGFR-Specific Tyrosine Kinase Inhibitors This class is represented by EKB-569, an EGFR tyrosine kinase inhibitor that binds irreversibly to the EGFR and has an IC50 of 38.5 nM in vitro (105). To demonstrate that EKB-569 bound covalently to the EGFR, 14C-labeled EKB-569 was synthesized and incubated with cellular membranes from cell lines express- ing the receptor; the reaction was terminated under reducing conditions, and continued binding of labeled EKB-569 to the EGFR was observed. EKB-569 exerts far less inhibition of the tyrosine kinase activities of other members of the EGFR family, displaying an IC50 30 times higher for HER2 than for the EGFR.
In the A431 human tumor xenograft model, a single dose of EKB-569 resulted in a 50% inhibition of receptor phosphorylation at 24 h despite a serum half-life of 2 h, a finding consistent with its reported irreversibility (105). In an initial phase I study, EKB-569 has been reported to be safe both on an intermittent and a continuous dose schedule (106). The observed side effects were skin rashes and diarrhea, very similar to those observed with other compounds of this type.
Class 3. Reversible PAN-HER (Human EGF Receptor Family) Tyrosine Kinase Inhibitors
In those situations in which coexpression of the EGFR (ErbB1) and the HER2 (ErbB2) occurs (13), an inhibitor that simultaneously targets both receptors may have therapeutic advantages. GW2016, currently under clinical development, inhibits the kinase activity of the two receptors, with an IC50 of 10 nM for the EGFR and of 9 nM for HER2. However, GW2016 does not inhibit HER4 well, with an IC50 >30-fold higher (107,108).
A relevant question is whether a dual inhibitor will be of greater efficacy than a receptor-specific tyrosine kinase inhibitor, taking into consideration the obser- vation that EGFR-specific tyrosine kinase inhibitors can prevent activation of HER2 in vivo. If these agents, on the other hand, target better HER2 than the more selective EGFR inhibitors, they could also have an improved activity profile in tumors such as breast cancer, which are HER2 dependent. However, this im- proved activity could also be at the cost of additional toxicities.
Class 4. Irreversible EGF Receptor Family Tyrosine Kinase Inhibitors
CI-1033 is a 4-anilinoquinazoline that irreversibly inhibits in vitro the three catalytically active members of the EGFR family: EGFR, HER2, and HER4.
Irreversibility is achieved by virtue of the compound’s ability to covalently modify a specific cysteine residue in the ATP binding site of these receptors (cys- 773) (109). CI-1033 is currently under phase I evaluation (110). Reported adverse events include an acneiform rash, diarrhea, thrombocytopenia, and one episode of a reversible hypersensitivity reaction. One clinical response has been reported in a patient with advanced squamous cell carcinoma (110).
At the present time it is not known whether these different classes of com- pounds—and the different compounds within the same class—will have a differ- ent activity and/or toxicity profile. As an example, it will be of interest to see whether the pan-HER inhibitors will have a greater efficacy in HER2-driven tumors such as breast cancer when compared to selective EGFR inhibitors.
Another discussion point is whether irreversibility of action will be advanta- geous. Because these agents are given orally on a daily basis, a point can be made that reversible inhibitors could also result in permanent receptor inhibition. How- ever, the effects of irreversible and reversible inhibitors on receptor degradation and synthesis are unknown.
CHALLENGES IN THE DEVELOPMENT OF ANTI-EGF RECEPTOR COMPOUNDS
The finding that anti-EGFR agents have antitumor activity and a low toxicity has validated the EGFR as a target for cancer therapy. On the other hand, there is clearly
a need to optimize their utilization, since their single-agent activity is modest and two large phase III combination studies with conventional chemotherapy in NSCLC have been negative. As with other targeted agents in their early stages of develop- ment, continued research in some key areas will hopefully result in an enhanced efficacy. Challenges include how to improve patient selection, identification of the differences among the various classes of agents, a reassessment of the predictive value of the currently used preclinical model, and new study designs.
Patient Selection
The activity of these agents has been mostly observed in an unselected patient population. The majority of the studies have either not preselected their patient population or have just required a positive EGFR expression in the tumor. This latter approach could be questioned, since there is no standardized method to determine EGFR expression and, most importantly, the level of EGFR expres- sion required in the tumor in order to obtain clinical benefit from these therapies is not known at the present time. Although it may be tempting to establish a parallelism between anti-EGFR agents and the anti-HER2 MAb trastuzumab (Herceptin®), which has activity only on cells displaying amplification of the HER2 target, the biology of the EGFR is quite different from that of HER2. The EGFR has a series of well-known ligands, and ligand binding to the receptor triggers homo- and heterodimer formation; in contrast, HER2 is a ligand-less receptor, and receptor overexpression may be required to activate downstream signaling. In addition, the data with cetuximab in colorectal carcinoma showed that the response rates were comparable in patients expressing 1+, 2+, or 3+
levels of the EGFR (70). The same holds true in patients with head and neck carcinomas, with similar response rates to cetuximab in patients with tumors expressing different levels of EGFR (75). No data are available in patients treated with low-MW inhibitors of the EGFR, but in cell lines there is no linear corre- lation between EGFR expression and response to tyrosine kinase inhibitors (46), as opposed to a linear relationship between receptor number and growth inhibi- tion in breast-cancer cell lines treated with trastuzumab (111).
We believe that factors other than receptor number determine whether a par- ticular tumor is dependent upon the EGFR pathway for driving its proliferation and function. Therefore, it will be critical in the new series of clinical trials to analyze not only the level of EGFR expression in the tumor, but also the level of expression of its ligands, such as TGF-_ or EGF, which are required for the maintenance of an active EGFR autocrine loop. In addition, the expression levels of the other members of the same receptor family, the level of tyrosine phospho- rylation on EGFRs and on downstream molecules such as MAPK, PI3K-Akt, p27, Stat3, Ki-67, and others should be measured. In cell culture, inhibition of phosphorylation of the EGFR and MAPK are necessary but not sufficient for cell-growth inhibition. On the other hand, cell lines such as MDA-468 that have
a mutant PTEN and a high level of basally phosphorylated Akt, are more resistant to tyrosine kinase inhibitors (35). In preliminary data from a phase II study of ZD1839 in breast cancer, only patients who had low levels of phosphorylated Akt had a decrease in the proliferation marker Ki-67 (J. Baselga, personal commu- nication). In a similar fashion, in a phase I study of EMD 72000 in patients with advanced colorectal carcinoma, inhibition of EGFR phosphorylation was ob- served in all tumors, irrespective of response. In one responding tumor for which biopsies pre- and posttherapy are available, the level of basally phosphorylated Akt was low and disappeared completely with treatment with EMD 72000. On the contrary, progressing tumors had higher basal phosphorylated Akt and it did not decrease with treatment (71). Therefore, it will be important to test in an orderly fashion whether these assays can identify markers that predict sensitivity to therapy with EGFR inhibitors. Furthermore, taking into consideration the vast complexity of the EGFR signaling network, instead of limiting the search to a limited number of markers, it will also be necessary to analyze much larger gene and protein expression levels at baseline and on therapy. Such an approach will imply the performance of repeated tumor biopsies in patients participating in these trials. While this approach has been shown to be feasible (84,98), new technologies including gene expression profiling in archived paraffin-embedded tumors may allow for easier prospective and retrospective correlations with clinical benefit (84).
Differences Among the Various Classes of Agents
Emerging clinical data suggest that these agents, although they target the same receptor, may have different activity profiles. Interestingly, these differences in activity seem to be tumor-dependent. In advanced colorectal cancer, consistent single-agent activity has been observed with the MAbs cetuximab and EMD 72000 (72,83,84), whereas the tyrosine kinase inhibitors ZD1839 and erlotinib have been shown to be inactive to date (98,104). Likewise, antitumor activity has been reported in renal-cell carcinoma with the MAb ABX-EGF (82) but not with cetuximab and ZD1839. As a third example, in head and neck tumors, activity has been reported with the MAb cetuximab (74,75) and with the tyrosine kinase inhibitors erlotinib and ZD1839 (97,102).
These findings imply that different agents (or classes of agents) may have to be tested separately in individual tumor types. This differential activity profile could also be an indication that these agents do not have completely overlapping mechanisms of action, and it provides a rationale for studying in the clinic the combined treatment with different anti-EGFR compounds such as tyrosine kinase inhibitors and MAbs. In support for this approach, preclinical studies in a panel of cell lines have shown that once maximal growth inhibition is achieved with one type of agent (MAbs or tyrosine kinase inhibitors), the addition of the other agent results in a remarkable additional antitumor effect (63).
DIRECTIONS IN THE COMBINED TREATMENT WITH ANTI-EGFR AGENTS AND CHEMOTHERAPY
IN THE TREATMENT OF CANCER
Combinations of chemotherapy and anti-EGFR agents in a variety of preclini- cal models have consistently shown a synergistic interaction both with mono- clonal antibodies and with low-MW tyrosine kinase inhibitors. However, the results in the clinic have not been uniform, with negative outcomes in some large studies. This point is well exemplified by the recently negative outcome of the two large phase III studies of chemotherapy ± ZD1839 in patients with advanced chemotherapy-naïve non-small-cell lung carcinoma (95,96), despite the exist- ence of supporting preclinical data (57,58). On the other hand, a true reversal of CPT-11 resistance has been observed with cetuximab in advanced colorectal cancer in the BOND trial (73), in results that mimic what was observed in pre- clinical models. Similarly, a beneficial effect of the combined treatment with cetuximab and chemotherapy when compared with chemotherapy alone has been observed in head and neck cancer and in NSCLC (76,77).
The results of these trials, taken together, suggest that there will not be a unifying approach on how to combine anti-EGFR agents and conventional che- motherapy agents. In the case of the negative lung cancer studies with gefitinib and erlotinib, several potential explanations have been proposed. There is the possibility that these results are just being seen with one class of agents and in one tumor type, NSCLC, where three drugs given concomitantly have never been shown to be superior to a two-drug regimen. However, in the case of triplet combinations of cytotoxic agents, it is generally necessary to reduce the dose of at least one of the three drugs in order to maintain tolerability, which could hamper the activity of the combination. This was not the case in the gefitinib trials, where full doses of chemotherapy and gefitinib were administered. In addition, the results of the NSCLC trial with cetuximab and chemotherapy suggest otherwise. There are other potential explanations for the lack of activity of the gefitinib plus cytotoxic combination chemotherapy. In these studies, patients were not selected on the basis of presence of EGFR or other yet unknown determinants of response: it is possible that if only a small number of patients are sensitive to gefitinib, the diluting effect may make small differences undetectable.
The magnitude of the preclinical effects of the combination could have not been large enough to warrant a clinical effect. It is possible that in order to see an effect in the clinic, in the preclinical models a true proof of synergy or, as in the preclinical studies with cetuximab and cisplatin (112) or trastuzumab and paclitaxel (113), a complete and sustained eradication of well established xe- nografts may be required. Another potential explanation is that the cancer cell lines that are being used in cell culture and xenograft models may be too distant from, and therefore not predictive of, the behavior of human tumors. The impli-
cation is that new models resembling more closely the clinical reality, such as tumor explants, would be highly desirable. However, with results from these models not yet available, we may have to consider different clinical development approaches based preferentially on single-agent studies in an enriched patient population of EGFR-sensitive tumors. In addition, for the combination studies, a new clinical trial methodology may be required. In light of the phase III studies with gefitinib and erlotinib, the type of pilot efficacy evidence required for a given combination may have to be redefined before proceeding to large phase III ran- domized trials. Likewise, other approaches of combining chemotherapy and anti- receptor agents would need to be explored, such as sequential administration, in a fashion similar to how hormonal therapy and chemotherapy have been inte- grated in the treatment of breast cancer. In the case of gefitinib and erlotinib, given their clinical activity as single agents in second and third line in advanced NSCLC, a direction to be explored is the sequential administration of chemotherapy first, followed by the administration of the anti-EGFR TKIs as a maintenance therapy.
In support of this approach is the result of one subset analysis that was performed in the INTACT2 trial (96). In this study, there was a trend towards improved survival in patients who had received chemotherapy *90 d, suggesting a possible effect of gefitinib monotherapy as maintenance therapy. The question of sequen- tial administration of chemotherapy and gefitinib will be addressed in a phase III study that will compare gefitinib 250 mg/d vs placebo following chemoradiation and consolidation docetaxel in patients with inoperable stage IIIA/B NSCLC.
In conclusion, the combined effect of anti-EGFR agents and chemotherapeu- tic agents will have to be carefully explored based on improved preclinical models, specific agents being studied, tumor type, and stage of disease. In addi- tion, special attention may be required to the dose and scheduling of the tested combinations. At the end, it is likely that each individual combination will have to be tested in the clinic. If this is the case, it would seem more appropriate to conduct exploratory randomized phase II studies or to build in early stopping rules for large phase III trials prior to committing to full-scale studies.
REFERENCES
1. Yarden Y, Sliwkowski M. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2:127–137.
2. Klapper L, Waterman H, Sela M, et al. Tumor-inhibitory antibodies to HER-2/ErbB-2 may act by recruiting c-Cbl and enhancing ubiquitination of HER-2. Cancer Res 2000;60:3384–3388.
3. Olayioye M, Neve R, Lane H, et al. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000;19:3159–3167.
4. Kuan CT, Wikstrand CJ, Bigner DD. EGF mutant receptor vIII as a molecular target in cancer therapy. Endocr Relat Cancer 2001;8:83–96.
5. Prenzel N, Zwick E, Daub H, et al. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 1999;402:884–888.
6. Liu D, Ghiso JA, Estrada Y, et al. EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer Cell 2002;1:445–457.
7. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–225.
8. Alroy I, Yarden Y. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett 1997;410:83–86.
9. Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascades. Adv Cancer Res 1998;74:49–139.
10. Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 1999;68:965–1014.
11. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001;411:355–365.
12. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase-Akt pathway in human cancer.
Nat Rev Cancer 2002;2:489–501.
13. Salomon D, Brandt R, Ciardiello F, et al. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995;19:183–232.
14. Tang CK. Epidermal growth factor receptor vIII enhances tumorigenicity in human breast cancer. 2000;60:3081–3087.
15. Moscatello DK. Frequent expression of a mutant epidermal growth factor receptor in mul- tiple human tumors. Cancer Res 1995;55:5536–5539.
16. Nishikawa R. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA 1994;91:7727–7731.
17. Grandis JR, Melhem MF, Gooding WE, et al. Levels of TGF-_ and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Cancer Natl Inst 1998;90:824–832.
18. Rusch V, Baselga J, Cordon-Cardo C, et al. Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res 1993;53:2379–2385.
19. Kawamoto T, Sato JD, Le A, et al. Growth stimulation of A431 cells by EGF: identification of high affinity receptors for epidermal growth factor by an anti-receptor monoclonal anti- body. Proc Natl Acad Sci USA 1983;80:1337–1341.
20. Sato JD, Kawamoto T, Le AD, et al. Biological effect in vitro of monoclonal antibodies to human EGF receptors. Mol Biol Med 1983;1:511–529.
21. Rodeck U, Herlyn M, Herlyn D, et al. Tumor growth modulation by a monoclonal antibody to the epidermal growth factor receptor: immunologically mediated and effector cell-inde- pendent effects. Cancer Res 1987;47:3692–3696.
22. Aboud-Pirak E, Hurwitz, E., Pirak, M.E., Bellot, F., Schlessinger, J. and Sela, M.. Efficacy of antibodies to epidermal growth factor receptor against KB carcinoma in vitro and in nude mice. J Natl Can Inst 1988;80:1605–1611.
23. Azemar M, Schmidt M, Arlt F, et al. Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapide regression in vivo. Int J Cancer 2000;86:269–275.
24. Ciardiello F, Caputo R, Troiani T, et al. Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis, and cooperate with cytotoxic drugs in human cancer cell lines. Int J Cancer 2001;93:172–178.
25. Yamazaki H, Kijima H, Ohmishi Y, et al. Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expresssion. J Natl Cancer Inst 1998;90:558–559.
26. Jannot CB, Beerli RR, Mason S, et al. Intracellular expression of a single-chain antibody directed to the EGFR leads to growth inhibition of tumor cells. Oncogene 1994;13:275–282.
27. Gill GN, Kawamoto T, Cochet C, et al. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epi- dermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem 1984;259:7755–7760.
28. Fan Z, Lu Y, Wu X, et al. Antibody-induced epidermal growth factor dimerization mediates inhibition of autocrine proliferation of A431 squamous carcinoma cells. J Biol Chem 1994;269:27,595–27,602.
