Main antibody provided inside a RTU-dispenser at a concentration on 1?g/ml was incubated for 1?hour at 37C. 2C3?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 manifestation and induces strong ADCC in vitro. A comparison of Ginkgolide A pancreatic and colorectal malignancy 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 effectiveness in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly total tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific Ginkgolide A anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a medical development candidate for the treatment of pancreatic malignancy. KEYWORDS: ADCC, Bispecific antibody, EGFR, IGF-1R, pancreatic malignancy Intro The epidermal growth element receptor (EGFR) and the insulin-like growth element-1 receptor (IGF-1R) are frequently over-expressed receptor tyrosine kinases that display enhanced activation in a variety of human tumors. EGFR and IGF-1R contribute to tumor development and progression by enhancing cell proliferation, inhibiting apoptosis and inducing angiogenesis.1,2 Both receptor tyrosine kinases mediate tumor growth via the PI3K-AKT and RAS-RAF-MAPK pathway, and cross-talk between EGFR and IGF-1R signaling was observed on receptor and downstream signaling levels. Interestingly, several preclinical and medical studies shown that IGF-1R signaling may induce resistance to EGFR inhibitors3,4 and EGFR-dependent signaling can confer resistance to IGF-1R inhibitors.5-8 Hence, targeting EGFR and IGF-1R simultaneously is a promising strategy to achieve enhanced tumor growth inhibition. The first-generation EGFR kinase inhibitors erlotinib or gefitinib are regularly used in the medical center for treatment of tumor malignancies.9 More recently, novel EGFR kinase inhibitors have been approved for cancer therapy, namely osimertinib (AZD9291) for patients Ginkgolide A with EGFR T790M mutation-positive metastatic non-small cell lung cancer (NSCLC),10 and afatinib, a covalent EGFR-HER2 multikinase inhibitor for treatment of (EGFR mutation positive) NSCLC.11,12 Other (irreversible) EGFR kinase inhibitors are in late-stage clinical development, e.g., dacomitinib (PF-00299804), BI 1482694 (HM61713), rociletinib (CO-1686) (examined in ref.13,14). In addition, monoclonal antibodies such as cetuximab and panitumumab, which block the binding of intrinsic receptor ligands (e.g., EGF) to EGFR and inhibit uncontrolled growth of Sirt4 tumor cells are applied in medical practice.15,16 Recently, the novel IgG1 isotype EGFR antibody necitumumab17-19 was authorized for the treatment of metastatic NSCLC. In order to enhance the immune effector function, glycoengineered EGFR antibodies such as imgatuzumab (GA201)20-23 and CetuGEX, a glycoengineered version of cetuximab, have been developed and have been or are in medical tests. Imgatuzumab is not in active medical development based on the bad outcome of a Phase 2 trial where it was compared to cetuximab in combination with FOLFIRI in metastatic colorectal malignancy.24 As an alternative approach, a synergistic combination of EGFR antibodies, SYM004, is currently in clinical development.25,26 Several monoclonal antibodies focusing on IGF-1R, such as R1507 (teprotumumab),27,28 figitumumab (CP-751871),29-32 ganitumab (AMG479),33-35 dalotuzumab (MK-0646)36-38 or cixutumumab (IMC-A12)39-41 came into late-stage clinical development in combination with different chemotherapeutic medicines, (EGFR) kinase inhibitors or antibodies, but are no longer in development due to limited/lack of efficacy in these clinical tests (reviewed in ref. 42-44). Particularly, medical development Ginkgolide A Ginkgolide A of R1507 is not being pursued based on its limited medical effectiveness in sarcoma45 and a Phase 2 study in NSCLC in combination with erlotinib.46 Based on the clinical effects with monospecific receptor tyrosine kinase (RTK) antibodies such as trastuzumab or cetuximab, the generation of bispecific RTK antibodies has attracted high interest. Several bispecific antibodies recently came into, or are about to enter, medical tests, including: 1) MM-141, a bispecific antibody against IGF-1R and HER3, is currently in medical development;47 2) a bispecific, dual action Fab (DAF)-based IgG antibody recognizing EGFR and HER3 simultaneously has been studied in medical tests;48-50 and 3) bispecific c-Met-EGFR antibodies.51,52 In addition, we as well as others have described novel tri- and tetraspecific antibodies targeting oncogenic RTKs.53,54 Taken together, clinical screening of IGF-1R and EGFR-targeted therapeutic antibody combinations focused on the treatment of lung or colorectal cancer resulted in discouraging results.36,40 Heterogeneity with regard to mutational status and signaling pathways, e.g., in colorectal malignancy, requires a obvious hypothesis for patient selection to detect the medical effectiveness of combined EGFR and IGF-1R inhibition. Despite the bad outcome of these medical studies investigating IGF-1R/EGFR combination therapy, innovative drug design such as combining potent signaling inhibition and enhanced engagement of immune effector functions through glycoengineering of the Fc region inside a bispecific IGF-1R/EGFR antibody may help to conquer primary.