A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity.

Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling.

Molecular characterization of novel trispecific ErbB-cMet-IGF1R antibodies and their antigen-binding properties.

Therapeutic antibodies are well established drugs in diverse medical indications. Their success invigorates research on multi-specific antibodies in order to enhance drug efficacy by co-targeting of receptors and addressing key questions of emerging resistance mechanisms. Despite challenges in production, multi-specific antibodies are potentially more potent biologics for cancer therapy. However, so far only bispecific antibody formats have entered clinical phase testing. For future design of antibodies allowing even more targeting specificities, an understanding of the antigen-binding properties of such molecules is crucial. To this end, we have generated different IgG-like TriMAbs (trispecific, trivalent and tetravalent antibodies) directed against prominent cell surface antigens often deregulated in tumor biology. A combination of surface plasmon resonance and isothermal titration calorimetry techniques enables quantitative assessment of the antigen-binding properties of TriMAbs. We demonstrate that the kinetic profiles for the individual antigens are similar to the parental antibodies and all antigens can be bound simultaneously even in the presence of FcγRIIIa. Furthermore, cooperative binding of TriMAbs to their antigens was demonstrated. All antibodies are fully functional and inhibit receptor phosphorylation and cellular growth. TriMAbs are therefore ideal candidates for future applications in various therapeutic areas.

Ret oncogene signal transduction via a IRS-2/PI 3-kinase/PKB and a SHC/Grb-2 dependent pathway: possible implication for transforming activity in NIH3T3 cells.

Multiple endocrine neoplasia 2A (MEN 2A) is an inherited disease caused by mutations of the Ret proto-oncogene. Although many different Ret mutations have been described, little is known about the signaling pathways triggered by the Ret oncogene. In this study, we have determined the signaling properties of a Ret-9bp duplication encoding amino acids 634-636, which was recently identified in a patient with all clinical features of the MEN 2A syndrome. The Ret-9bp duplication leads to constitutive activation of the Ret tyrosine kinase. Furthermore, Ret-9bp increased mitogenic and transforming activity demonstrated by thymidine incorporation as well as colony formation in soft agar. Studying intracellular signaling pathways, which may be involved in malignant transformation of Ret-9bp expressing NIH3T3 cells, we could demonstrate Ret-9bp dependent phosphorylation of insulin receptor substrate-2 (IRS-2) with consecutive activation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT). Moreover, Ret-9bp induces phosphorylation of SHC resulting in growth factor receptor binding protein-2 (Grb-2) binding and activation of the mitogen activating protein (MAP) kinase pathway. In addition to these postreceptor cytoplasmic signaling events, we have studied nuclear signal by Ret-9bp and found activation of c-jun and jun-D, two members of the jun/AP-1 family of transcription factors. In summary, an oncogenic 9bp duplication of Ret causes Ret dimer formation and ligand independent activation of the tyrosine kinase. Besides the signaling steps leading to MAPK activation, we could demonstrate that Ret-9bp induced constitutive activation of a signaling pathway involving IRS-2, PI 3-kinase and PKB/AKT which could transduce the oncogenic Ret signal to increased gene transcription via activation of the jun/AP-1 transcription factor family.

Activation of phosphatidylinositol 3-kinase is necessary for differentiation of FDC-P1 cells following stimulation of type III receptor tyrosine kinases.

Signaling molecules that are responsible for proliferation and differentiation of hematopoietic cells following ectopic expression of receptor tyrosine kinases (RTKs) were investigated in the interleukin 3 (IL-3)-dependent hematopoietic cell line, FDC-P1. Cells were transfected with human platelet-derived growth factor receptor (PDGF-R), macrophage colony stimulating factor-1 receptor (CSF-1R), epidermal growth factor receptor (EGF-R), and chimeras consisting of the extracellular domain of EGF-R and the transmembrane and cytoplasmic domains of either HER2 (HER1-2) or c-kit (EK-R). All FDC-P1 transfectants proliferated in response to the corresponding growth factor in the absence of IL-3. However, only cells expressing PDGF-R, CSF-1R, and EK-R (type III RTKs) differentiated along the monocyte-macrophage lineage after treatment with their activating ligands. Analysis of proteins from these RTK-expressing cells revealed that a Mr 85,000 protein showed in vitro phosphorylation, and V8 protease peptide mapping showed that this protein was p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase). Accordingly, activation of PDGF-R-, CSF-1R-, and EK-R-expressing cells led to an increase in PI3-kinase activity. Expression of EK-R mutant Y721F, which lacked the known p85 binding site, blocked differentiation and activation of PI3-kinase, without affecting proliferation. Last, addition of wortmannin to cells expressing PDGF-R, CSF-1R, and EK-R blocked ligand-induced differentiation in a concentration-dependent manner, and this effect correlated with wortmannin's ability to inhibit PI3-kinase. Thus, ectopic expression of both type I and III RTKs could stimulate FDC-P1 proliferation in the absence of IL-3; however, only activation of type III RTKs led to differentiation via selective coupling to p85 and PI3-kinase activation.

Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3.

Amplification and/or overexpression of HER2/neu and HER3 genes have been implicated in the development of cancer in humans. The fact that these receptor tyrosine kinases (RTKs) are frequently coexpressed in tumor-derived cell lines and that heterodimers form high affinity binding sites for heregulin (HRG) suggests a novel mechanism for signal definition, diversification or amplification. In cells expressing HER2 and HER3, tyrosine phosphorylation of HER3 is markedly increased upon exposure to recombinant HRG. ATP binding site mutants of HER2 and HER3 demonstrate transphosphorylation of HER3 by HER2, but not vice versa. HRG-induced transphosphorylation of HER3 results in a substrate phosphorylation pattern distinct from HER2 cells and enhances association of the receptor with SHC and phosphoinositol 3-kinase in transfected 293 and mammary carcinoma-derived MCF-7 cells. The physiological relevance of HER2/HER3 heterodimerization is demonstrated by HRG-dependent transformation of NIH 3T3 cells coexpressing the two receptors. These findings demonstrate the acquisition of expanded signaling capacities for HER2 by HRG-induced heterodimerization with HER3 and provide a molecular basis for the involvement of receptor heteroactivation in the development of human malignancies.

Inhibition of glioma cell growth by a truncated platelet-derived growth factor-beta receptor.

Abnormal expression of platelet-derived growth factor (PDGF) receptors has been observed in malignant glioma and other tumors such as osteosarcomas and malignant melanomas. However, their role in the development and maintenance of the tumors is not understood. Signaling through the PDGF receptors is activated by ligand-induced dimerization. Thus, introduction of mutant receptors that are kinase deficient but still dimerization competent is one strategy to study the importance of PDGF receptors in glioma cell growth. A truncated PDGF-beta receptor was introduced into C6 rat glioma cells and the PDGF-mediated signaling and subsequent cell growth studied. In clones expressing the mutant receptor, PDGF-BB-induced tyrosine phosphorylation of the endogenous receptor was significantly reduced. In addition, these cells grew to lower density in culture and formed smaller colonies in soft agar than the C6 parental cells. Furthermore, the ability of cells expressing the truncated receptor to grow as xenografts in nude mice was significantly impaired. These results support the important role for the PDGF-beta receptor in C6 glioma cell growth. They also demonstrate the usefulness of dominant-negative mutants of the PDGF receptor for the evaluation of the role of the receptor in tumorigenesis.