<p>Figure S1. Related to Figure 1. Characterization of FAP-drozitumab BsAbs and antitumor efficacy in the presence of FAP-expressing fibroblasts in in vitro co-culture models; Table S1. BsAb binding to DR5 and FAP antigens; Table S2. Anti-tumor efficacy of FAP-drozitumab bispecific molecules is FAPdependent.</p>
Abstract Myeloid cells represent the most abundant immune cell type within the tumor microenvironment of certain tumor entities, including tumor associated macrophages (TAMs). Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for TAMs is macrophage colony stimulating factor 1 (CSF1). We generated a monoclonal antibody (RG7155) that binds to the secondary dimerization interface of CSF1 receptor (CSF1R) as a specific and potent allosteric inhibitor. In vitro, RG7155 treatment results in cell death of CSF1-differentiated macrophages. In animal models, CSF1R inhibition reduced the F4/80+ TAMs infiltrate by 90% and was accompanied by an increase of the CD8+/CD4+ T cell ratio. The ability of RG7155 to reduce TAMs is currently evaluated in a first-in-man phase I clinical study in patients suffering either from pigmented villonodular synovitis (PVNS), a neoplastic disorder characterized by CSF1 overexpression, or other tumor entities. The associated biomarker program involves mandatory paired pre- and on-treatment biopsies of tumor and surrogate skin tissue as well as pharmacodynamic marker assessment in circulating blood. In patients treated with RG7155 an increase of CSF1 associated with a sustained decrease of CD14+CD16+ alternatively activated monocytes in peripheral blood was detected. In PVNS patients administration of RG7155 led to striking reductions of CSF1R+ and CD163+ macrophages in tumor tissue resulting in objective clinical responses according to RECIST (Response Evaluation Criteria in Solid Tumors) in 5 out of 6 patients. All six evaluable PVNS patients showed partial metabolic response in FDG-PET imaging and significant symptomatic improvement as early as 4 weeks after treatment initiation. Furthermore, TAM reduction was also observed in paired tumor samples of patients with various advanced solid malignancies, suggesting broad applicability of this therapeutic approach. This abstract is also presented as Poster A50. Citation Format: Michael Cannarile, Sabine Hoves, Ann-Marie Broeske, Joerg Benz, Katharina Wartha, Valeria Runza, Flora Rey-Giraud, Leon P. Pradel, Friedrich Feuerhake, Irina Klaman, Tobin Jones, Ute Jucknischke, Stefan Scheiblich, Ingo H. Gorr, Antje Walz, Keelara Abiraj, Philippe Cassier, Antonio Sica, Carlos Gomez-Roca, Christophe Le Tourneau, Jean-Pierre Delord, Antoine Italiano, Hyam Levitsky, Jean-Yves Blay, Dominik Ruettinger, Carola H. Ries. Targeting tumor-asoociated macrophages with a novel anti-CSF1R antibody in cancer patients. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR04. doi:10.1158/1538-7445.CHTME14-PR04
The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer.
Abstract Background: Activation of the extrinsic apoptosis pathway in tumor cells through agonistic death receptor 5 (DR5) antibodies has been evaluated in the clinic with limited success so far. In this context, several reports show that DR5 activation is strongly dependent on receptor hyperclustering on the cell surface. Therefore a therapeutic principle that induces DR5 hyperclustering specifically at the tumor site may provide superior efficacy, potency and safety compared to conventional DR5 agonistic antibodies. Fibroblast activation protein (FAP) is a marker for activated fibroblasts and abundantly expressed in cancer associated fibroblasts of various epithelial tumor indications and as a tumor antigen on tumors of mesenchymal origin. Due to its relative absence from normal tissues, FAP can be used as a tumor targeting antigen. Here, we are using the broad expression of FAP in tumor stroma for crosslinking of DR5 by a bispecific antibody. Aim: In order to achieve superior tumor targeting and tumor located DR5 hyperclustering we have generated a bispecific antibody, RG7386, comprised of an agonistic DR5 binder and a FAP targeting moiety. Results: RG7386 shows potent and selective binding to FAP and DR5 and can simultaneously bind to both targets. In in vitro co-culture assays, using human DLD1 colon tumor cells and FAP expressing fibroblasts, RG7386 induces potent, FAP dependent DR5 hyperclustering and apoptosis induction in DR5 positive tumor cells (IC50: 0.05 nM). In preclinical in vivo models with co-injection of DLD-1 tumor cells and fibroblasts as well as patient-derived colorectal cancer models, RG7386 shows FAP dependent efficacy and apoptosis induction superior to conventional DR5 antibodies. Furthermore the superior induction of apoptosis could be confirmed by in vivo and ex vivo analysis of cleaved Caspase-3 with imaging, Luminex and histopathology. Conclusion: RG7386 is a promising novel therapeutic entity for the treatment of solid tumors with FAP positive tumor stroma inducing DR5 activation by FAP dependent DR5 hypercrosslinking which results in potent anti-tumor activity. Citation Format: Katharina Wartha, Barbara Weiser, Thomas Friess, Meher Majety, Valeria Runza, Frank Herting, Thomas Weber, Werner Scheuer, Suzana Vega Harring, Hadassah Sade, Huifeng Niu. A novel bispecific FAP-DR5 antibody inducing potent and tumor-specific death receptor 5 (DR5) activation by fibroblast activation protein (FAP) dependent crosslinking. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A59. doi:10.1158/1538-7445.CHTME14-A59
