Clinical experience with monoclonal antibodies to epidermal growth factor receptor

Recent knowledge about the intermediate steps and final consequences of ligand-dependent epidermal growth factor receptor (EGFR) activation has clearly supported the notion that EGFR plays a fundamental role in regulating the proliferation and survival of malignant neoplasms. Among the rationally designed target-based therapeutics that are being assessed, those targeting EGFR appear to be some of the most clinically relevant. The strategy of using monoclonal antibodies (mAbs) to block ligand binding to the extracellular domain of the EGFR has led to the development of therapeutics that robustly arrest malignant cell proliferation and, in some cases, induce profound tumor regression. The chimeric mAb against EGFR, cetuximab, has already been approved by regulatory agencies worldwide to treat patients with advanced colorectal cancer. Other mAbs against EGFR, particularly panitumumab (ABX-EGF), h-R3, and EMD72000, are in advanced stages of clinical development.     

Epidermal growth factor receptor as a target for therapy with antireceptor monoclonal antibodies.

The epidermal growth factor (EGF) receptor is a potential target for antitumor therapy. Recent studies from many laboratories have found that this receptor is expressed in high levels on a variety of human tumor cells. Furthermore, the EGF receptor has been implicated in autocrine stimulation of cell growth in a number of experimental studies. We have produced anti-EGF receptor monoclonal antibodies (MAbs), which block the binding of EGF and transforming growth factor alpha (TGF-alpha), and can prevent ligand-stimulated activation of EGF receptor tyrosine kinase. These MAbs have been useful in studies of EGF receptor function. Experiments utilizing the MAbs to block ligand binding have demonstrated that autocrine stimulation of EGF receptor phosphorylation can occur via an extracellular pathway, involving TGF-alpha-mediated activation of EGF receptor on the surface of the cell. The capacity of anti-EGF receptor MAbs to inhibit cell proliferation has provided evidence of an autocrine stimulatory pathway in cultures of malignant human skin, breast, colon, and lung cells. Growth of a variety of human tumor xenografts can be inhibited in situations where autocrine dependency is demonstrable in cell culture. Imaging studies with anti-EGF receptor MAb labeled with indium 111 (111In) demonstrated selective uptake in xenografts expressing high receptor levels. Based on these observations, a phase I trial was carried out with 111In-labeled anti-EGF receptor MAb 225 IgG1 in patients with advanced squamous cell lung carcinoma, a tumor that invariably expresses large numbers of EGF receptors. In the case of squamous lung carcinoma, there is evidence that overexpression of EGF receptors correlates with worse clinical stage and worse prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The epidermal growth factor receptor as a target for therapy with antireceptor monoclonal antibodies

The epidermal growth factor (EGF) receptor is a potential target for antitumor therapy, because it is expressed at high levels on many human tumor cells and appears to be involved in autocrine stimulation of cell growth in a number of experimental studies. Anti-EGF receptor monoclonal antibodies (MAbs) which block ligand binding can prevent the growth in culture of cells that are stimulated by EGF or TGF-alpha. Growth of human tumor xenografts bearing high levels of EGF receptors is also inhibited. A Phase I trial in patients with squamous cell carcinoma of the lung has demonstrated the capacity of a single dose of 120 mg anti-EGF receptor MAb to localize in such tumors and to achieve saturating concentrations in the blood for more than 3 days, without causing toxicity.     

Effects of anti-epidermal growth factor (EGF) receptor antibodies and an anti-EGF receptor recombinant-ricin A chain immunoconjugate on growth of human cells

The effects of antiepidermal growth factor (EGF) receptor monoclonal antibodies (MAbs) 528 and 225 and a 528-ricin A conjugate on the growth of normal and malignant human cells were tested in vitro. Malignant human cell lines with EGF receptor numbers ranging from 0 to 4 X 10(5) receptors/cell, human fetal fibroblasts, and normal marrow granulocyte/macrophage progenitors (CFU-gm) showed no effect when grown with 10(-12) M to 10(-7) M MAb 225 or 528. MAbs 225 and 528 and EGF also had no effect on the ability of marrow stromal cells to maintain CFU-gm viability in long-term marrow cultures. Reversible growth inhibition of A431 epidermoid and MDA-468 breast carcinoma cells with 2 and 3 X 10(6) EGF receptors/cell, respectively, was observed with both antibodies and with 10(-8) M EGF. In contrast, an immunoconjugate prepared with MAb 528 and recombinant ricin A chain (528-rRA) showed dose-dependent killing over a concentration range of 10(-12) M to 10(-8) M against cells with greater than or equal to 1.2 X 10(5) EGF receptors/cell [concentration that causes 50% inhibition of growth (IC50) values, approximately 10(-12) M to 10(-10) M]. Human fetal fibroblasts (5.6 X 10(4) EGF receptors/cell), melanoma cells without detectable EGF receptors, and human CFU-gm showed IC50 values of greater than 10(-8) M. Killing of KB epidermoid carcinoma cells and 547 ovarian carcinoma cells with 4 and 1.2 X 10(5) EGF receptors/cell by 10(-10) or 10(-11) M 528-rRA was time dependent, but cytotoxicity to 547 cells was not complete even with 48 hours of immunotoxin treatment. Cytotoxicity of 528-rRA was not enhanced by chloroquine or verapamil. In vitro, anti-EGF receptor MAbs cause reversible antiproliferative effects only against malignant cell lines with amplified EGF receptor expression. In contrast, 528-rRA shows potent, specific toxicity to cells with greater than 50,000 EGF receptors/cell. However, kinetics of cell killing with 528-rRA are protracted, suggesting that prolonged exposure may be required for in vivo antitumor effects.     

The epidermal growth factor (EGF)

This review summarizes briefly some of the historical aspects of the discovery, isolation, and mode of action of epidermal growth factor (EGF). Also considered are some of the key experiments which relate the biochemical mechanisms of EGF action to the mechanism involved in oncogenic transformation by the retroviruses.     

Side effects of anti-cancer molecular-targeted therapies (not monoclonal antibodies)

PURPOSE OF REVIEW: Major advances have been achieved in the field of biologically based therapies for cancer in the last few years, and some of the recently approved 'molecular-targeted therapies' are now being used in daily clinical practice. We aim to review some aspects of the toxicity and safety of small-molecule anti-cancer molecular-targeted therapies, with some insights into the physiopathology and predictive factors of toxicity, its correlation with response, and how to prevent and overcome it. RECENT FINDINGS: As a whole, small-molecule molecular-targeted therapies are well tolerated. Their toxic profile is favorable, but during the drug development process some severe (sometimes lethal) toxicities have been observed, such as interstitial lung disease in patients treated with drugs targeting the epidermal growth factor receptor. Pharmacogenomic studies can help us to identify those patients with well characterized polymorphisms, and to define the best-tolerated and most effective treatments. SUMMARY: Molecular-targeted therapies have a good toxicity profile in general; however, some patients are exquisitely sensitive to developing particular and severe toxicities related to these drugs.     

Hypomagnesaemia and targeted anti-epidermal growth factor receptor (EGFR) agents

Currently, targeted anti-epidermal growth factor receptor (EGFR) agents have an important role in the treatment of various cancers. These drugs, particularly anti-EGFR monoclonal antibodies, may induce electrolyte disorders, such as hypomagnesaemia and hypocalcaemia. Early symptoms of magnesium deficiency can easily go unrecognized. However, hypomagnesaemia can in rare cases lead to serious clinical manifestations, including cardiac arrhythmias or convulsions. The elective tubular expression of renal EGF/EGFR explains the mechanism of this class-related drug side effect. Inhibition of the EGFR induces a mutated-like transient receptor potential cation channel, subfamily M, member 6 (TRPM6) syndrome, characterized by urinary magnesium and calcium wasting. The risk of hypomagnesaemia is associated with treatment duration. It is a reversible toxicity; the recovery of magnesium serum levels is usually seen 4–6 weeks of stopping the anti-EGFR antibody. Using literature from peer-reviewed journals, this review reports the clinical trials findings and discusses the mechanisms and the treatment of hypomagnesaemia induced by anti-EGFR targeted agents.     

Generation and characterization of monoclonal antibodies to human keratinocyte growth factor receptor

Keratinocyte growth factor receptor (KGFR) and fibroblast growth factor receptor (FGFR) 2c share identical amino acid sequences, except for a 46-amino acid domain in the extracellular region. Monoclonal antibodies (MAbs) specific to KGFR have not been reported nor are commercially available. In this study, we generated murine MAbs specific to KGFR in non-obese diabetic (NOD) mice using a modified Repeated Immunizations at Multiple Sites (RIMMS) technology. Stable cell lines expressing the full-length human KGFR or FGFR2c were produced to facilitate the identification of KGFR-specific MAbs. Following the initial screening of hybridoma clones with a fluorescence-based, confocal cell detection method and ELISA, KGFR-specific MAbs were selected and confirmed by flow cytometry and Western blot analyses. Antagonistic MAbs were identified using a cell-based functional assay. These KGFR MAbs will be important reagents for studying the biological function and tissue distribution of this receptor in normal and pathological conditions.     

Fulvestrant regulates epidermal growth factor (EGF) family ligands to activate EGF receptor (EGFR) signaling in breast cancer cells

Estrogen receptor-α (ER) targeted therapies are routinely used to treat breast cancer. However, patient responses are limited by resistance to endocrine therapy. Breast cancer cells resistant to the pure steroidal ER antagonist fulvestrant (fulv) demonstrate increased activation of epidermal growth factor receptor (EGFR) family members and downstream ERK signaling. In this study, we investigated the effects of fulv on EGFR signaling and ligand regulation in several breast cancer cell lines. EGFR/HER2/HER3 phosphorylation and ERK1,2 activation were seen after 24–48 h after fulvestrant treatment in ER-positive breast cancer cell lines. 4-Hydroxy-tamoxifen and estradiol did not cause EGFR activation. Fulvestrant did not affect EGFR expression. Cycloheximide abolished the ability of fulv to activate EGFR suggesting the autocrine production of EGFR ligands might be responsible for fulvestrant induced EGFR signaling. qRT-PCR results showed fulv differentially regulated EGFR ligands; HB-EGF mRNA was increased, while amphiregulin and epiregulin mRNAs were decreased. Fulvestrant induced EGFR activation and upregulation of EGFR ligands were ER dependent since fulv treatment in C4-12, an ER-negative cell line derivative of MCF-7 cells, did not result in EGFR activation or change in ligand mRNA levels. ER downregulation by siRNA induced similar EGFR activation and regulation of EGFR ligands as fulvestrant. Neutralizing HB-EGF antibody blocked fulv-induced EGFR activation. Combination of fulv and EGFR family tyrosine kinase inhibitors (erlotinib and lapatinib) significantly decreased EGFR signaling and cell survival. In conclusion, fulvestrant-activated EGFR family members accompanied by ER dependent upregulation of HB-EGF within 48 h. EGF receptor or ligand inhibition might enhance or prolong the therapeutic effects of targeting ER by fulvestrant in breast cancer.     

Epidermal growth factor receptor monoclonal antibodies inhibit the growth of lung cancer cell lines.

The ability of monoclonal antibody (MAb 108), an immunoglobulin G (IgG)2a against the epidermal growth factor receptor (EGF-R), to interact with lung cancer cell lines was investigated. 125I-EGF bound with high affinity to non-small-cell lung cancer (NSCLC) cells, and MAb 108 inhibited specific binding of nine NSCLC cell lines in a dose-dependent manner (IC50 = 0.3-3 micrograms per ml). 125I-MAb 108 bound with high affinity (kd = 2 nM) to a single class of sites (Bmax = 70,000 per cell) using NSCLC neuroendocrine cell line NCI-H460. Specific 125I-MAb 108 binding was inhibited with high affinity by MAb 108 but not by a control antibody IgG using large-cell carcinoma cell line NCI-H1299. 125I-MAb 108 binding was not internalized at 37 degrees C using NSCLC neuroendocrine cell line NCI-H460 and adenocarcinoma cell line NCI-H23. Also, 1 microgram per ml of MAb 108 but not of a control IgG inhibited the clonal growth of NCI-H23 and squamous cell carcinoma cell line NCI-H157 in vitro. Also, MAb 108 inhibited xenograft formation of cell lines NCI-H460, NCI-H157, and NCI-H727 in nude mice in vivo. After a palpable tumor had formed using NCI-H460 cells, injection of 100 micrograms of MAb 108 (intraperitoneally three times weekly) inhibited xenograft volume in nude mice by approximately 50%. These data suggest that MAb 108 may interact with EGF receptors on lung cancer cell lines and inhibit NSCLC proliferation.     

Production and characterization of monoclonal antibodies against insulin-like growth factor type 1 receptor

The type 1 insulin-like growth factor receptor (IGF-1R) has been extensively reported to play an important role in cancer. Activation of the IGF-1R by IGF-I and IGF-II binding to the extracellular domains of the receptor induces mitogenic and anti-apoptotic effects, which are important events in tumor growth and survival. Several cancer cell types overexpress IGF-1R, suggesting a possible use of monoclonal antibodies (MAbs) against IGF-1R as diagnostic reagents. Here, we report the production and characterization of two independent MAbs, namely 7C2 and 9E11, generated by immunizing mice with the soluble extracellular part of this receptor (amino acids 1-906). Both MAbs bind to membrane bound IGF-1R and do not cross-react with insulin receptor isoforms, IR-A and IR-B expressed on IGF-1R() cells. MAbs 7C2 and 9E11 stained the IGF- 1R on frozen or paraffin-embedded tissue sections or frozen cells. The MAbs 7C2 and 9E11 immunoprecipitated the IGF-1R from P6 cell lysates (cells overexpressing human IGF-1R) and could detect non-reduced intact IGF-1R on immunoblots. However, the MAbs were not able to detect reduced and denatured receptor alpha and beta chains. Sequencing of the heavy- and light-chain variable regions revealed that the 7C2 and 9E11 CDR amino acid sequences are different but result in antibodies with similar properties. MAbs 7C2 and 9E11 are therefore potentially useful diagnostic tools and could be of therapeutic use for humans in the future.     

Approaches to optimize the use of monoclonal antibodies to epidermal growth factor receptor

Preclinical and clinical studies consistently demonstrate the antitumor activity of the various monoclonal antibodies (mAbs) that block ligand binding to the extracellular domain of the epidermal growth factor receptor (EGFR). However, the objective antitumor activities of these agents are disproportionately lower than would be expected based on the high rates of expression, overexpression, and aberrations of EGFR in human malignancies, particularly carcinomas. Several technologic and clinical approaches are being explored to optimize the potency of these antibodies, such as humanization of the murine parent molecules, and conjugation and widening of the specificity of the mAb. Also, combined therapy with the different types of EGFR-interacting or other targeted agents may lead to a heightened potential. This review highlights the results of current approaches for improvement of the therapeutic indices of these agents.     

First-line use of anti-epidermal growth factor receptor monoclonal antibodies in metastatic colorectal cancer

Several molecular pathways have been shown to play key roles in the development and progression of colorectal cancer (CRC). This enhanced understanding of tumor biology has provided the rationale for the design and development of novel agents that are directed against important targets, including growth factors, receptors, and tumor-specific/tumor-selective antigens. The epidermal growth factor receptor (EGFR) signaling pathway has received much attention over the past 5-10 years because it is overexpressed in more than 85% of tumors from patients with metastatic CRC. Cetuximab and panitumumab are monoclonal antibodies presently approved for use by the FDA in the refractory disease setting, and they have provided significant advances in the treatment of advanced CRC. However, much focus has shifted toward using these biologic therapies in combination with cytotoxic chemotherapy in up-front settings, such as first-line therapy and in the neoadjuvant therapy of liver-limited disease. The clinical studies conducted to date suggest that cetuximab can be safely and effectively combined with oxaliplatin- and irinotecan-based chemotherapy in the first-line treatment of metastatic CRC. Moreover, the results of the CRYSTAL phase III study provide support for the use of the combination of FOLFIRI (5-fluorouracil/leucovorin/irinotecan) and cetuximab in the neoadjuvant setting and allow for R0 surgical resection with curative intent. Much work continues to investigate the critical molecular biomarkers that can be used to predict clinical response to chemotherapy and/or targeted therapies as well as to identify which patients might be at increased risk for developing drug-specific side effects.     

Novel C6-substituted 1,3,4-oxadiazinones as potential anti-cancer agents

The insulin-like growth factor 1 receptor (IGF-1R) is a membrane receptor tyrosine kinase over-expressed in a number of tumors. However, combating resistance is one of the main challenges in the currently available IGF-1R inhibitor-based cancer therapies. Increased Src activation has been reported to confer resistance to anti-IGF-1R therapeutics in various tumor cells. An urgent unmet need for IGF-1R inhibitors is to suppress Src rephosphorylation induced by current anti-IGF-1R regimens. In efforts to develop effective anticancer agents targeting the IGF-1R signaling pathway, we explored 2-aryl-1,3,4-oxadiazin-5-ones as a novel scaffold that is structurally unrelated to current tyrosine kinase inhibitors (TKIs). The compound, LL-2003, exhibited promising antitumor effects in vitro and in vivo; it effectively suppressed IGF-1R and Src and induced apoptosis in various non-small cell lung cancer cells. Further optimizations for enhanced potency in cellular assays need to be followed, but our strategy to identify novel IGF-1R/Src inhibitors may open a new avenue to develop more efficient anticancer agents.     

Fully human monoclonal antibodies to hepatocyte growth factor with therapeutic potential against hepatocyte growth factor/c-Met-dependent human tumors

c-Met is a well-characterized receptor tyrosine kinase for hepatocyte growth factor (HGF). Compelling evidence from studies in human tumors and both cellular and animal tumor models indicates that signaling through the HGF/c-Met pathway mediates a plethora of normal cellular activities, including proliferation, survival, migration, and invasion, that are at the root of cancer cell dysregulation, tumorigenesis, and tumor metastasis. Inhibiting HGF-mediated signaling may provide a novel therapeutic approach for treating patients with a broad spectrum of human tumors. Toward this goal, we generated and characterized five different fully human monoclonal antibodies that bound to and neutralized human HGF. Antibodies with subnanomolar affinities for HGF blocked binding of human HGF to c-Met and inhibited HGF-mediated c-Met phosphorylation, cell proliferation, survival, and invasion. Using a series of human-mouse chimeric HGF proteins, we showed that the neutralizing antibodies bind to a unique epitope in the beta-chain of human HGF. Importantly, these antibodies inhibited HGF-dependent autocrine-driven tumor growth and caused significant regression of established U-87 MG tumor xenografts. Treatment with anti-HGF antibody rapidly inhibited tumor cell proliferation and significantly increased the proportion of apoptotic U-87 MG tumor cells in vivo. These results suggest that an antibody to an epitope in the beta-chain of HGF has potential as a novel therapeutic agent for treating patients with HGF-dependent tumors.