Colorectal cancer (CRC) is the third most common cancer world-wide with 1.2 million patients diagnosed yearly. In late stage CRC, the most commonly used targeted therapies are monoclonal antibodies cetuximab and panitumumab, which inactivate EGFR1. Recent studies have identified alterations in KRAS2–4 and other genes5–13 as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in CRC and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in CRC on response to anti-EGFR antibody therapy, we performed complete exome sequence and copy number analyses of 129 patient-derived tumorgrafts and targeted genomic analyses of 55 patient tumors, all of which were KRAS wild-type. We analyzed the response of tumors to anti-EGFR antibody blockade in tumorgraft models or in clinical settings. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumors with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumorgraft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluate response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and provide new avenues for intervention in the management of CRC.

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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.

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..

HER2 plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved, trastuzumab and pertuzumab. It has been suggested that optimal inhibition of HER2 is achieved when utilizing two or more antibodies targeting nonoverlapping epitopes. Superior clinical activity of the trastuzumab plus pertuzumab combination in metastatic breast cancer supports this hypothesis. Because trastuzumab and pertuzumab were not codeveloped, there may be potential for further optimizing HER2 targeting. The study herein evaluated functional activity of anti-HER2 antibody combinations identifying optimal epitope combinations that provide efficacious HER2 inhibition. High-affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2-dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anticancer activity. Mol Cancer Ther; 14(3); 669-80. Ó2015 AACR.

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Predicting the outcome of immunotherapy is essential for efficient treatment. The recent clinical success of immunotherapy is increasingly changing the paradigm of cancer treatment. Accordingly, the development of immune-based agents is accelerating and the number of agents in the global immuno-oncology pipeline has grown 60-70% over the past year. However, despite remarkable clinical efficacy in some patients, only few achieve a lasting clinical response. Treatment failure can be attributed to poorly immunogenic tumors that do not attract tumor infiltrating lymphocytes (TILs). Therefore, we developed positron emission tomography (PET) radiotracers for non-invasive detection of CD4+ and CD8a+ TILs in syngeneic mouse tumor models for preclinical studies.Methods: Seven syngeneic mouse tumor models (B16F10, P815, CT26, MC38, Renca, 4T1, Sa1N) were quantified for CD4+ and CD8a+ TILs using flow cytometry and immunohistochemistry (IHC), as well as for tumor growth response to Sym021, a humanized PD-1 antibody cross-reactive with mouse PD-1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator and radiolabeled with Zirconium-89 (89Zr-DFO-CD4/89Zr-DFO-CD8a). Tracers were optimized for in vivo PET/CT imaging in CT26 tumor-bearing mice and specificity was evaluated by depletion studies and isotype control imaging. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET/CT imaging was conducted in the panel of syngeneic mouse models prior to immunotherapy with Sym021.Results: Syngeneic tumor models were characterized as “hot” or “cold” according to number of TILs determined by flow cytometry and IHC. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a were successfully generated with a radiochemical purity >99% and immunoreactivity >85%. The optimal imaging time-point was 24 hours post-injection of ~1 MBq tracer with 30 µg non-labeled co-dose. Reduced tumor and spleen uptake of 89Zr-DFO-CD8a was observed in CD8a+ depleted mice and the uptake was comparable with that of isotype control (89Zr-DFO-IgG2b) confirming specificity. PET imaging in syngeneic tumor models revealed a varying maximum tumor-to-heart ratio of 89Zr-DFO-CD4 and 89Zr-DFO-CD8a across tumor types and in-between subjects that correlated with individual response to Sym021 at day 10 relative to start of therapy (p=0.0002 and p=0.0354, respectively). The maximum 89Zr-DFO-CD4 tumor-to-heart ratio could be used to stratify mice according to Sym021 therapy response and overall survival was improved in mice with a 89Zr-DFO-CD4 ratio >9 (p=0.0018).Conclusion: We developed 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET radiotracers for specific detection and whole-body assessment of CD4+ and CD8a+ status. These radiotracers can be used to phenotype preclinical syngeneic mouse tumor models and to predict response to an immune checkpoint inhibitor. We foresee development of such non-invasive in vivo biomarkers for prediction and evaluation of clinical efficacy of immunotherapeutic agents, such as Sym021.

These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.