mAbs to receptor tyrosine kinases such as EGF receptor͞ErbB-1 and HER2͞ErbB-2 inhibit the tumorigenic growth of certain cancer cells, but although recombinant versions of such Abs are already used in oncology wards, the mechanism underlying immunotherapy remains unknown. We report that anti-EGF receptor Abs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining mAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, mAb combinations are more effective than single Abs in inhibiting HER2 signaling in vitro and tumorigenesis in animals. We present a model attributing efficacy of immunotherapy to the size of Abreceptor lattices formed at the cell surface, which dictates the rate of endocytic clearance and extent of signaling blockade.ErbB ͉ growth factor ͉ oncogene ͉ signal transduction ͉ antibody T he four receptor tyrosine kinases of the ErbB family and their multiple ligand molecules form a layered signaling network, which is implicated in human cancer (reviewed in ref.1): overexpression of ErbB-1͞EGF receptor (EGFR) has been implicated as a feature of poor prognosis in various human malignancies. Moreover, deletion mutants of EGFR exist in brain tumors and point mutations have recently been reported in lung cancer (2). By contrast, ErbB-2͞HER2 is rarely mutated in solid tumors. Instead, the erbB-2 gene is frequently amplified in breast, ovarian, and lung cancer (3). Because of their oncogenic potential and accessibility, ErbB proteins have emerged as attractive targets for pharmaceutical interventions. One major strategy involves the use of mAbs. Early studies uncovered the tumor-inhibitory potential of mAbs directed at ErbB-1 and ErbB-2 (4, 5), and later studies indicated that anti-ErbB mAbs are effective when combined with various chemotherapeutic agents (6, 7). Indeed, the clinical benefit of combining mAbs with certain chemotherapeutic agents was notable, which led to the approval of mAbs to ErbB-2 (Herceptin) and EGFR (C225͞ Cetuximab) for the treatment of breast and colorectal cancer, respectively.Two types of mechanisms have been implicated in ErbBdirected immunotherapy. The first involves mAb-mediated recruitment to tumors of natural killer cells through the Fc-␥ activation receptors of these immune effector cells (8). The second type of mechanisms relates to intrinsic mAb activities, which include blockade of ligand binding or receptor heterodimerization (9), inhibition of downstream signaling to Akt (10), and acceleration of receptor internalization (11,12). The latter mechanism is particularly attractive because ligand-induced endocytosis and degradation of active receptor tyrosine kinases (RTKs) is considered a major physiological process underlying attenuation of growth-promoting signals (13).Several studies reported cooperative effects of mAb combinations (9, 12, 14-16), whereas others found that bivalent, Fc-lacking versions of anti-ErbB mAbs inhibit tumorigenic growth in animals (17, 18). These observations ...
Monoclonal antibodies (mAbs) to ErbB-2/HER2 or to its sibling, the epidermal growth factor receptor (EGFR), prolong survival of cancer patients, especially when combined with cytotoxic therapies. However, low effectiveness of therapeutic mAbs and the evolution of patient resistance call for improvements. Here we test in animals pairs of anti-ErbB-2 mAbs and report that pairs comprising an antibody reactive with the dimerization site of ErbB-2 and an antibody recognizing another distinct epitope better inhibit ErbB-2-overexpressing tumors than other pairs or the respective individual mAbs. Because the superiority of antibody combinations extends to tumor cell cultures, we assume that nonimmunological mechanisms contribute to mAb synergy. One potential mechanism, namely the ability of mAb combinations to instigate ErbB-2 endocytosis, is demonstrated. Translation of these lessons to clinical applications may enhance patient response and delay acquisition of resistance.cancer ͉ growth factor ͉ immunotherapy ͉ signal transduction ͉ tyrosine kinase E rbB-2/HER2 is a member of the epidermal growth factor receptor (EGFR) family. When transactivated, ErbB-2/ HER2 stimulates several downstream signaling cascades, including the mitogen-activated protein kinase cascade (1). This ligand-less receptor is moderately expressed in normal adult tissues, where it regulates cell growth and differentiation. By contrast, amplification of the corresponding gene and consequent overexpression of the HER2/ErbB-2 protein have been reported in 20-30% of tumors of the breast (2-4) and ovary (4). In general, erbB-2 gene amplification associates with enhanced metastatic potential and poor prognosis. Because ErbB-2 is expressed at relatively low levels in normal tissues, it makes an attractive target for immunotherapy. This was originally demonstrated in animals by Greene et al. (5), who targeted Neu, the rodent form of ErbB-2, and later developed this into a widely used clinical strategy (6). The molecular mechanisms underlying the growth-inhibitory effects of anti-ErbB-2 monoclonal antibodies (mAbs) may involve indirect tumor cell cytotoxicity through immunological mechanisms, including antibodydependent cellular cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), increased cancer cell apoptosis, as well as direct interference with signaling cascades (6).Clinical studies established that Trastuzumab (Herceptin), a humanized mAb directed against ErbB-2, is active against ErbB-2-overexpressing metastatic breast cancer, leading to its approval for clinical use (7). The objective response rates to Trastuzumab monotherapy is relatively low (Ϸ15%) and short lived (median duration, 9 months) (8). On the other hand, mAbs seem to display a synergistic effect when combined with chemotherapy, probably because of interruption of ErbB-2-driven survival pathway (9). Yet another strategy, relevant to pancreatic cancer, combines antibodies to EGFR and to ErbB-2 (10). The present study explores an alternative strategy to enhance the therapeutic acti...
Aptamers, oligonucleotides able to avidly bind cellular targets, are emerging as promising therapeutic agents, analogous to monoclonal antibodies. We selected from a DNA library an aptamer specifically recognizing human epidermal growth factor receptor 2 (ErbB-2/HER2), a receptor tyrosine kinase, which is overexpressed in a variety of human cancers, including breast and gastric tumors. Treatment of human gastric cancer cells with a trimeric version (42 nucleotides) of the selected aptamer (14 nucleotides) resulted in reduced cell growth in vitro, but a monomeric version was ineffective. Likewise, when treated with the trimeric aptamer, animals bearing tumor xenografts of human gastric origin reflected reduced rates of tumor growth. The antitumor effect of the aptamer was nearly twofold stronger than that of a monoclonal anti-ErbB-2/HER2 antibody. Consistent with aptamer-induced intracellular degradation of ErbB-2/HER2, incubation of gastric cancer cells with the trimeric aptamer promoted translocation of ErbB-2/HER2 from the cell surface to cytoplasmic puncta. This translocation was associated with a lysosomal hydrolase-dependent clearance of the ErbB-2/HER2 protein from cell extracts. We conclude that targeting ErbB-2/HER2 with DNA aptamers might retard the tumorigenic growth of gastric cancer by means of accelerating lysosomal degradation of the oncoprotein. This work exemplifies the potential pharmacological utility of aptamers directed at cell surface proteins, and it highlights an endocytosis-mediated mechanism of tumor inhibition.T he epidermal growth factor related protein (ErbB) family of receptor tyrosine kinases plays an important role in epitheliogenesis and, accordingly, serves as a major therapeutic target in several cancers. The family comprises four transmembrane receptors and 11 ligands that induce homodimerization or heterodimerization upon binding to the respective receptor (1). ErbB-1 (also called the epidermal growth factor receptor; EGFR) and ErbB-4 share some ligands, whereas no similar ligand is so far known for ErbB-2. Overexpression and mutations of ErbB family members lead to a multitude of malignancies. To date, synthetic tyrosine kinase inhibitors (e.g., Erlotinib and Gefitinib), as well as monoclonal antibodies (mAbs; e.g., Cetuximab and Trastuzumab), have been developed to inhibit pathological signaling or recruit the immune system to cancer cells (2). Aptamers might represent an alternative therapeutic modality. These molecules are small, singlestranded DNA or RNA molecules (3). RNA aptamers were described for the first time in 1990 by two laboratories (4, 5). Since then, aptamers against a multitude of different organic and inorganic, small and macromolecular, targets were developed. In addition, high-affinity aptamer binding ranging from picomolar to low nanomolar concentrations have been documented (6). Aptamers are selected in an evolutionary process called systematic evolution of ligands by exponential enrichment (SELEX). A DNA or RNA library containing single-stranded random ...
Vascular endothelial growth factor (VEGF) is one of the key growth factors regulating tumor angiogenesis and thus it is one of the primary targets for antiangiogenic therapy. The long-term effects of VEGF include induction of proliferation and migration of endothelial cells, tube formation and maintenance of the immature capillaries. The early effects of VEGF include vasodilation and increased permeability. We hypothesize that the early responses to VEGF can serve to develop a quantitative measure of the activity of VEGF, and therefore may be applicable for monitoring the efficacy of systemic suppression of VEGF signaling during antiangiogenic therapy. For that end we tested the ability of MRI and fluorescence microscopy to detect the early response to intradermal VEGF165 in nude mice. VEGF-induced local vasodilation and increased permeability was detected by intravenous administration of macromolecular biotin-BSA-GdDTPA 23 30 min after intradermal administration of VEGF. Contrast leak showed saturation kinetics. Delayed contrast administration (90 min after intradermal administration of VEGF) resulted in low contrast leak and demonstrated that the saturation kinetics is not due to contrast equilibration between plasma and the interstitial space, but rather is due to suppression of vascular permeability. Permeability was restored by a second bolus of VEGF, showing that the saturation kinetics is primarily due to inactivation of the growth factor. Confocal microscopy of fluorescent BSA-FITC confirmed the permeability changes monitored by MRI. Moreover, confocal microscopy showed efficient lymphatic uptake of the extravasated contrast material specifically in regions of VEGF induced hyper-permeability.
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