Helle Jacobsen

Sym004: A Novel Synergistic Anti–Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy

Epidermal growth factor receptor (EGFR) is a validated therapeutic target in cancer and EGFR antagonists with greater effectiveness than existing clinical agents remain of interest. Here, we report a novel approach based on Sym004, a mixture of two anti-EGFR monoclonal antibodies directed against distinct nonoverlapping epitopes in EGFR extracellular domain III. Like anti-EGFR monoclonal antibodies in current clinical use, Sym004 inhibits cancer cell growth and survival by blocking ligand-binding receptor activation and phosphorylation and downstream receptor signaling. However, unlike the other antibodies, Sym004 induces rapid and efficient removal of the receptor from the cancer cell surface by triggering EGFR internalization and degradation. Compared with reference anti-EGFR monoclonal antibodies, Sym004 exhibited more pronounced growth inhibition in vitro and superior efficacy in vivo. Together, these findings illustrate a strategy to target EGFR more effectively than existing clinical antibodies.

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.

Pan-HER, an Antibody Mixture Simultaneously Targeting EGFR, HER2, and HER3, Effectively Overcomes Tumor Heterogeneity and Plasticity

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. Clin Cancer Res; 21(18); 4110–22. ©2015 AACR. See related commentary by Yarden and Sela, p. 4030

Preclinical Pharmacokinetics and Safety of Sym004: A Synergistic Antibody Mixture Directed against Epidermal Growth Factor Receptor

Purpose: Sym004 is a novel therapeutic antibody mixture product comprising two unmarketed monoclonal antibodies (mAb) targeting the epidermal growth factor receptor (EGFR). In previous preclinical proof-of-concept studies, Sym004 was shown to elicit superior cancer cell growth inhibition activities compared with marketed anti-EGFR mAbs. This article describes the design and results of the preclinical safety program conducted to support early clinical development of Sym004. Experimental Design: Tissue cryosections from various species were stained with Sym004 to evaluate tissue cross reactivity. The pharmacokinetics of Sym004 were evaluated in a mouse xenograft model and in Cynomolgus monkeys. Monkeys received once weekly intravenous infusions of Sym004 in the range 2 to 24 mg/kg for 6 to 8 weeks. Cetuximab (a marketed anti-EGFR mAb) and the individual antibodies comprising Sym004 were included in the repeat-dose toxicity studies at single-dose level. Results: Sym004 had a staining pattern similar to cetuximab in tissue panels from both human and non-human primates. Once weekly dosing of Sym004 to Cynomolgus monkeys did not cause accumulation, whereas administration of the individual antibodies resulted in prolonged half-life and accumulation. In direct comparisons with cetuximab, Sym004 did not induce any distinct or novel adverse findings in the animals. However, an early onset of pronounced, reversible, and anticipated anti-EGFR–mediated pharmacologic effects, such as skin rash, dehydration, and liquid feces, was observed. Only minor adverse effects were recorded in animals treated with the individual antibodies comprising Sym004. Conclusion: Sym004 was well tolerated and did not induce any unexpected toxicities. The preclinical safety data enabled initiation of the ongoing clinical development. Clin Cancer Res; 17(18); 5962–72. ©2011 AACR.

Targeting Three Distinct HER2 Domains with a Recombinant Antibody Mixture Overcomes Trastuzumab Resistance.

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. ©2015 AACR.

In vivo imaging of therapy response to a novel Pan-HER antibody mixture using FDG and FLT positron emission tomography

Purpose Overexpression of the human epidermal growth factor receptor (HER) family and their ligands plays an important role in many cancers. Targeting multiple members of the HER family simultaneously may increase the therapeutic efficacy. Here, we report the ability to image the therapeutic response obtained by targeting HER family members individually or simultaneously using the novel monoclonal antibody (mAb) mixture Pan-HER. Experimental design and results Mice with subcutaneous BxPC-3 pancreatic adenocarcinomas were divided into five groups receiving vehicle or mAb mixtures directed against either EGFR (HER1), HER2, HER3 or all three receptors combined by Pan-HER. Small animal positron emission tomography/computed tomography (PET/CT) with 2′-deoxy-2′-[18F]fluoro-D-glucose (FDG) and 3′-deoxy-3′-[18F]fluorothymidine (FLT) was performed at baseline and at day 1 or 2 after initiation of therapy. Changes in tumor uptake of tracers were quantified and compared to reduction in tumor size. Imaging results were further validated by immunohistochemistry and qPCR. Mean FDG and FLT uptake in the Pan-HER treated group decreased by 19±4.3% and 24±3.1%, respectively. The early change in FDG and FLT uptake correlated with tumor growth at day 23 relative to day 0. Ex vivo molecular analyses of markers associated with the mechanisms of FDG and FLT uptake confirmed the in vivo imaging results. Conclusions Taken together, the study supports the use of FDG and FLT as imaging biomarkers of early response to Pan-HER therapy. FDG and FLT PET/CT imaging should be considered as imaging biomarkers in clinical evaluation of the Pan-HER mAb mixture.

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.

Abstract 4562: Superior targeting of the human epidermal growth factor receptor 2 (HER-2) with recombinant monoclonal antibody mixtures

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Human epidermal growth factor receptor 2 (HER2) is involved in development and maintenance of malignant phenotypes of several human cancers and therefore represent an attractive therapeutic target. Trastuzumab is currently the only anti-HER2 antibody approved for treatment of human cancers and although succesful more efficacious treatments are warranted. Mixtures of recombinant monoclonal antibodies are promising candidates as the next generation of antibody therapeutics due to their multipotent activities. The aim of the present study was to identify mixtures of anti-HER2 antibodies with superior activity to existing monoclonal antibodies. Anti-HER2 antibodies were raised in mice by immunizations with HER-2 in different antigen presenting formats. A large antibody repertoire consisting of approximately 150 unique anti-HER-2 antibodies was cloned from the mice using the mSymplex™ technology. Based on a thorough sequence and binding analysis, 40 antibodies were selected for functional evaluation. The 40 antibodies were tested as individual antibodies and in mixtures of two and three for the ability to inhibit the growth of four human cancer cell lines using a standard viability assay. In total, more than 1300 mixtures were evaluated for ability to inhibit growth of the four cell lines. The 20 mixtures with the highest levels of growth inhibition were further characterized with regard to potency (IC50) and ability to engage in ADCC and CDC. The results demonstrated that HER2 mixtures were superior to trastuzumab, pertuzumab and the mixture of the two at inhibiting the growth of seven of the 10 cell lines investigated. Interestingly, the mixtures also inhibited the growth of the trastuzumab resistant breast cancer cell line HCC202, reflecting the differentiated mechanisms of anti-HER2 antibody mixtures. In vivo, anti-HER2 mixtures had superior activity compared to the monoclonal anti-HER2 antibody trastuzumab in two models of human gastric cancer. Based on these results, a candidate mixture was selected and referred to as Sym005. In conclusion, these data demonstrate that the novel antibody mixture Sym005 has a unique set of HER2 inhibitory mechanisms, which translates into superior anti-cancer activity in HER2-positive tumor xenografts. Furthermore, the results provide a clear rationale for evaluation of Sym005 in clinical trials on patients with HER2 positive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4562. doi:10.1158/1538-7445.AM2011-4562

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.

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.