Michael M. Grandal

Pan‐HER – an antibody mixture targeting EGFR, HER2, and HER3 abrogates preformed and ligand‐induced EGFR homo‐ and heterodimers

The human epidermal growth factor receptor (HER)‐family is involved in development of many epithelial cancers. Therefore, HER‐family members constitute important targets for anti‐cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER‐targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan‐HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan‐HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan‐HER compared with single HER‐targeting with single and dual mAbs on HER‐family cross‐talk and dimerization focusing on EGFR. The effect of Pan‐HER on cell proliferation and HER‐family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non‐overlapping antibodies. Furthermore, changes in EGFR‐dimerization patterns after treatment with Pan‐HER were investigated by in situ proximity ligation assay and co‐immunoprecipitation, demonstrating that Pan‐HER and the EGFR‐targeting mAb mixture efficiently down‐regulate basal EGFR homo‐ and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan‐HER and the EGFR‐targeting mAb mixture also blocked EGF‐binding and thereby ligand‐induced changes in EGFR‐dimerization levels. These results suggest that Pan‐HER reduces the cellular capability to switch HER‐dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan‐HER in resistant settings.

Efficacy of Sym004 in Patients With Metastatic Colorectal Cancer With Acquired Resistance to Anti-EGFR Therapy and Molecularly Selected by Circulating Tumor DNA Analyses: A Phase 2 Randomized Clinical Trial

Importance Acquired resistance to anti-EGFR therapy (epidermal growth factor receptor) is frequently due to RAS and EGFR extracellular domain (ECD) mutations in metastatic colorectal cancer (mCRC). Some anti-EGFR–refractory patients retain tumor EGFR dependency potentially targetable by agents such as Sym004, which is a mixture of 2 nonoverlapping monoclonal antibodies targeting EGFR. Objective To determine if continuous blockade of EGFR by Sym004 has survival benefit. Design, Setting, and Participants Multicenter, phase 2, randomized, clinical trial comparing 2 regimens of Sym004 with investigator’s choice from March 6, 2014, through October 15, 2015. Circulating tumor DNA (ctDNA) was analyzed for biomarker and tracking clonal dynamics during treatment. Participants had wild-type KRAS exon 2 mCRC refractory to standard chemotherapy and acquired resistance to anti-EGFR monoclonal antibodies. Interventions Participants were randomly assigned in a 1:1:1 ratio to Sym004, 12 mg/kg/wk (arm A), Sym004, 9 mg/kg loading dose followed by 6 mg/kg/wk (arm B), or investigator’s choice of treatment (arm C). Main Outcomes and Measures Overall survival (OS). Secondary end points included preplanned exploratory biomarker analysis in ctDNA. Results A total of 254 patients were randomized (intent-to-treat [ITT] population) (median age, 63 [range, 34-91] years; 63% male; n = 160). Median OS in the ITT population was 7.9 months (95% CI, 6.5-9.9 months), 10.3 months (95% CI, 9.0-12.9 months), and 9.6 months (95% CI, 8.3-12.2 months) for arms A, B, and C, respectively (hazard ratio [HR], 1.31; 95% CI, 0.92-1.87 for A vs C; and HR, 0.97; 95% CI, 0.68-1.40 for B vs C). The ctDNA revealed high intrapatient genomic heterogeneity following anti-EGFR therapy. Sym004 effectively targeted EGFR ECD-mutated cancer cells, and a decrease in EGFR ECD ctDNA occurred in Sym004-treated patients. However, this did not translate into clinical benefit in patients with EGFR ECD mutations, likely owing to co-occurring resistance mechanisms. A subgroup of patients was defined by ctDNA (RAS/BRAF/EGFR ECD-mutation negative) associated with improved OS in Sym004-treated patients in arm B compared with arm C (median OS, 12.8 and 7.3 months, respectively). Conclusions and Relevance Sym004 did not improve OS in an unselected population of patients with mCRC and acquired anti-EGFR resistance. A prospective clinical validation of Sym004 efficacy in a ctDNA molecularly defined subgroup of patients with refractory mCRC is warranted. Trial Registration clinicaltrialsregister.eu Identifier: 2013-003829-29

A combination of two antibodies recognizing non‐overlapping epitopes of HER2 induces kinase activity‐dependent internalization of HER2

The human epidermal growth factor receptor 2 (HER2/ErbB2) is overexpressed in a number of human cancers. HER2 is the preferred heterodimerization partner for other epidermal growth factor receptor (EGFR) family members and is considered to be resistant to endocytic down‐regulation, properties which both contribute to the high oncogenic potential of HER2. Antibodies targeting members of the EGFR family are powerful tools in cancer treatment and can function by blocking ligand binding, preventing receptor dimerization, inhibiting receptor activation and/or inducing receptor internalization and degradation. With respect to antibody‐induced endocytosis of HER2, various results are reported, and the effect seems to depend on the HER2 expression level and whether antibodies are given as individual antibodies or as mixtures of two or more. In this study, the effect of a mixture of two monoclonal antibodies against non‐overlapping epitopes of HER2 was investigated with respect to localization and stability of HER2. Individual antibodies had limited effect, but the combination of antibodies induced internalization and degradation of HER2 by multiple endocytic pathways. In addition, HER2 was phosphorylated and ubiquitinated upon incubation with the antibody combination, and the HER2 kinase activity was found to be instrumental in antibody‐induced HER2 down‐regulation.

Sym015: A Highly Efficacious Antibody Mixture against MET-Amplified Tumors

Purpose: 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. Experimental Design/Results: 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 MET exon 14 skipping alterations, and in models harboring MET 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) in vitro, suggesting that secondary effector functions contribute to the efficacy of Sym015. Retrospectively, all responsive, high MET-expressing models were scored as highly MET-amplified by in situ hybridization, pointing to MET 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. Conclusions: The preclinical efficacy and safety data provide a clear rationale for the ongoing clinical studies of Sym015 in patients with MET-amplified tumors. Clin Cancer Res; 23(19); 5923–35. ©2017 AACR.

Simultaneous Targeting of Two Distinct Epitopes on MET Effectively Inhibits MET- and HGF-Driven Tumor Growth by Multiple Mechanisms

Increased MET activity is linked with poor prognosis and outcome in several human cancers currently lacking targeted therapies. Here, we report on the characterization of Sym015, an antibody mixture composed of two humanized IgG1 antibodies against nonoverlapping epitopes of MET. Sym015 was selected by high-throughput screening searching for antibody mixtures with superior growth-inhibitory activity against MET-dependent cell lines. Synergistic inhibitory activity of the antibodies comprising Sym015 was observed in several cancer cell lines harboring amplified MET locus and was confirmed in vivo. Sym015 was found to exert its activity via multiple mechanisms. It disrupted interaction of MET with the HGF ligand and prompted activity-independent internalization and degradation of the receptor. In addition, Sym015 induced high levels of CDC and ADCC in vitro. The importance of these effector functions was confirmed in vivo using an Fc-effector function–attenuated version of Sym015. The enhanced effect of the two antibodies in Sym015 on both MET degradation and CDC and ADCC is predicted to render Sym015 superior to single antibodies targeting MET. Our results demonstrate strong potential for use of Sym015 as a therapeutic antibody mixture for treatment of MET-driven tumors. Sym015 is currently being tested in a phase I dose escalation clinical trial (NCT02648724). Mol Cancer Ther; 16(12); 2780–91. ©2017 AACR.

Sym004-induced EGFR elimination is associated with profound anti-tumor activity in EGFRvIII patient-derived glioblastoma models

BackgroundSym004 is a mixture of two monoclonal antibodies (mAbs), futuximab and modotuximab, targeting non-overlapping epitopes on the epidermal growth factor receptor (EGFR). Previous studies have shown that Sym004 is more efficient at inducing internalization and degradation of EGFR than individual components, which translates into superior cancer cell inhibition. We investigated whether Sym004 induces removal of EGFRvIII and if this removal translates into tumor growth inhibition in hard-to-treat glioblastomas (GBMs) harboring the mutated, constitutively active EGFR variant III (EGFRvIII).MethodsTo address this question, we tested the effect of Sym004 versus cetuximab in eight patient-derived GBM xenograft models expressing either wild-type EGFR (EGFRwt) and/or mutant EGFRvIII. All models were tested as both subcutaneous and orthotopic intracranial xenograft models.ResultsIn vitro studies demonstrated that Sym004 internalized and removed EGFRvIII more efficiently than mAbs, futuximab, modotuximab, and cetuximab. Removal of EGFRvIII by Sym004 translated into significant in vivo anti-tumor activity in all six EGFRvIII xenograft models. Furthermore, the anti-tumor activity of Sym004 in vivo was superior to that of its individual components, futuximab and modotuximab, suggesting a clear synergistic effect of the mAbs in the mixture.ConclusionThese results demonstrate the broad activity of Sym004 in patient-derived EGFRvIII-expressing GBM xenograft models and provide a clear rationale for clinical evaluation of Sym004 in EGFRvIII-positive adult GBM patients.

Abstract 1219: Sym015, a novel antibody mixture targeting non-overlapping epitopes of MET, effectively inhibits growth of MET dependent tumors and overcomes resistance to a single monoclonal antibody

The tyrosine kinase receptor MET is involved in progression of a variety of human cancers and constitutes a promising therapeutic target. Particularly, subsets of tumors originating from lung or gastric tissues appear to be truly MET dependent. MET dependency is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, kinase activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents in lung and colorectal cancers. Sym015, a novel antibody mixture comprising two monoclonal antibodies targeting non-overlapping epitopes on the SEMA domain of MET, was shown to effectively inhibit cell growth in vitro through effective MET degradation. In the present study, we screened a large panel of highly annotated human cancer cell lines for sensitivity to Sym015 in order to identify potential markers of response. Sym015 effectively inhibited growth of cell lines with MET-amplification, MET-exon 14 deletion, and autocrine HGF production, including MET-amplified cell lines with acquired resistance to EGFR targeting agents. To validate the in vitro findings, a range of cell line- and patient-derived xenograft models with MET amplification or Exon 14 deletion were tested for sensitivity to Sym015 and an analogue of the clinical stage anti-MET monoclonal antibody emibetuzumab (LY2875358). Sym015 effectively inhibited growth of tumors with autocrine HGF production, MET-amplification, and/or Exon 14 deletion, and had superior activity compared to the emibetuzumab analogue in many of the models. Importantly, tumors with a partial response to the emibetuzumab analogue were strongly inhibited by subsequent treatment with Sym015 in two MET-amplified models, one of which also harbors a MET-exon 14 deletion. In summary, our findings demonstrate a potent antitumor effect of Sym015 in MET-dependent models. The data thus strongly support initiation of clinical trials for patients with MET-amplification and Exon 14 deletions. Citation Format: Thomas T. Poulsen, Michael M. Grandal, Helle J. Jacobsen, Dorte S. Hansen, Trine Lindsted, Mikkel W. Pedersen, Ivan D. Horak, Michael Kragh, Johan Lantto. Sym015, a novel antibody mixture targeting non-overlapping epitopes of MET, effectively inhibits growth of MET dependent tumors and overcomes resistance to a single monoclonal antibody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1219.

Abstract 1218: A novel synergistic antibody pair targeting non-overlapping epitopes of MET effectively inhibits MET-driven cancer models

The receptor tyrosine kinase MET (Hepatocyte Growth Factor Receptor, HGFR) has been associated with development and progression of a range of human tumors due to its regulation of cell proliferation, migration, invasion, and angiogenesis. A subset of human tumors, particularly of lung or gastric origin, appear to have a primary dependency on MET, which is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents. Sym015 is a mixture of two humanized monoclonal antibodies against non-overlapping epitopes on MET. The specific pair of antibodies was identified in a high-throughput cell based screen searching for antibody mixtures with superior growth inhibitory activity against four MET-dependent cell lines. Both antibodies bind the SEMA domain of MET with high affinity. Synergistic activity was confirmed by dose-response curves in several MET-dependent cell lines and cell line and patient-derived xenograft models. Mechanistically, Sym015 blocks HGF binding to MET, induces MET internalization and degradation effectively diminishing MET oncogenic signaling. Sym015 also induces higher levels of antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro compared to individual mAbs. In conclusion, Sym015, a novel monoclonal antibody mixture against MET, shows strong inhibitory activity against MET driven cell lines and xenografts due to a combined effect of MET degradation and secondary effector functions. These data support initiation of clinical trials. Citation Format: Michael M. Grandal, Thomas T. Poulsen, Klaus Koefoed, Karsten W. Eriksen, Anna Dahlman, Paolo Conrotto, Thomas Bouquin, Helle Jacobsen, Ivan D. Horak, Michael Kragh, Johan Lantto, Mikkel W. Pedersen. A novel synergistic antibody pair targeting non-overlapping epitopes of MET effectively inhibits MET-driven cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1218.