Purpose: Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epidermal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members.Experimental Design/Results: We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing compensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly efficacious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting.Conclusions: Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER represents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic.

clinicaltrialsregister.eu Identifier: 2013-003829-29.

Tumor cell motility is one of the rate-limiting steps of invasion, which defines progression toward a more malignant phenotype. Elevated expression of epidermal growth factor receptor (EGFR) in many cancers is associated with progression of superficial to invasive forms of the disease. The naturally occuring type III mutant epidermal growth factor receptor (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the epidermal growth factor receptor. EGFRvIII is expressed frequently by a number of human solid tumours including those of the lung, breast, prostate, brain and ovary. Our study was designed to investigate the effect of EGFRvIII expression on cell motility and compare it to that of ligand-activated EGFR using transfected fibroblasts. We show here using time-lapse video recording that expression of EGFRvIII greatly enhances the motility of fibroblasts independently of ligand stimulation. In addition, expression of EGFRvIII caused a marked increase in the number of cellular protrusions (lamellipodia) and a reduction in the number of stress fibers and focal adhesions. The EGFR tyrosine kinase inhibitor, AG1478, and the MEK inhibitor, U0126, blocked these cellular effects of EGFRvIII. Two cell lines expressing different levels of EGFR were used for comparison. The low-expressing cell line responded to EGF treatment by increasing motility in a manner very similar to the motility induced by EGFRvIII. In contrast, the high-expressing cell line responded to EGF by detachment from the extracellular matrix and decreased motility. Cellular detachment was correlated to a high phosphorylation of PLC-␥, whereas increased motility was correlated to a high level of ERK phosphorylation. Overall these results indicate that tumor-associated EGFR mutations might be critical for tumor cell motility, invasion and thus progression of disease.

Activation of the receptor tyrosine kinase MET is associated with poor clinical outcome in certain cancers. To target MET more effectively, we developed an antagonistic antibody mixture, Sym015, consisting of two humanized mAbs directed against nonoverlapping epitopes of MET. We screened a large panel of well-annotated human cancer cell lines and identified a subset with highly elevated MET expression. In particular, cell lines of lung cancer and gastric cancer origin demonstrated high MET expression and activation, and Sym015 triggered degradation of MET and significantly inhibited growth of these cell lines. Next, we tested Sym015 in patient- and cell line-derived xenograft models with high MET expression and/or exon 14 skipping alterations, and in models harboring amplification as a mechanism of resistance to EGFR-targeting agents. Sym015 effectively inhibited tumor growth in all these models and was superior to an analogue of emibetuzumab, a monoclonal IgG4 antibody against MET currently in clinical development. Sym015 also induced antibody-dependent cellular cytotoxicity (ADCC) , suggesting that secondary effector functions contribute to the efficacy of Sym015.Retrospectively, all responsive, high MET-expressing models were scored as highly-amplified by hybridization, pointing to amplification as a predictive biomarker for efficacy. Preclinical toxicology studies in monkeys showed that Sym015 was well tolerated, with a pharmacokinetic profile supporting administration of Sym015 every second or third week in humans. The preclinical efficacy and safety data provide a clear rationale for the ongoing clinical studies of Sym015 in patients with -amplified tumors..

Tumor growth in the context of EGFR inhibitor resistance may remain EGFRdependent and is mediated by mechanisms including compensatory ligand upregulation and de novo gene alterations. Sym004 is a two-antibody mixture targeting nonoverlapping EGFR epitopes. In preclinical models, Sym004 causes signifi cant EGFR internalization and degradation, which translates into superior growth inhibition in the presence of ligands. In this phase I trial, we observed grade 3 skin toxicity and hypomagnesemia as mechanism-based dose-limiting events during dose escalation. In dose-expansion cohorts of 9 and 12 mg/kg of Sym004 weekly, patients with metastatic colorectal cancer and acquired EGFR inhibitor resistance were enrolled; 17 of 39 patients (44%) had tumor shrinkage, with 5 patients (13%) achieving partial response. Pharmacodynamic studies confi rmed marked Sym004-induced EGFR downmodulation. MET gene amplifi cation emerged in 1 patient during Sym004 treatment, and a partial response was seen in a patient with EGFR S492R mutation that is predictive of cetuximab resistance. SIGNIFICANCE:Potent EGFR downmodulation with Sym004 in patients with metastatic colorectal cancer and acquired resistance to cetuximab/panitumumab translates into signifi cant antitumor activity and validates the preclinical hypothesis that a proportion of tumors remains dependent on EGFR signaling. Further clinical development and expanded correlative analyses of response patterns with secondary RAS/EGFR mutations are warranted. Cancer Discov; 5(6);[598][599][600][601][602][603][604][605][606][607][608][609]