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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.

BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT.

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.

Hepatocyte growth factor (HGF) exerts a cytostatic effect on HepG2 and B16-F1 cell lines. To evaluate the possible involvement of the apoptotic process in this effect, we performed studies at cellular and molecular levels. HGF induced apoptosis only in HepG2 hepatoma cells at day 3 in about 20% of the cells undergoing growth inhibition, while hallmarks of apoptosis did not occur in B16-F1 melanoma cells. During the first 24 h after HGF treatment, enhanced expression of the pro-apoptotic genes bax and c-Myc was observed at level of mRNA and protein. Concomitant induction of antizyme (AZ) might lower ornithine decarboxylase (ODC) protein level though a huge increase in ODC mRNA level took place. This was suggested as a signal for apoptosis decisional phase. The levels of the proteins examined except that of AZ fell down thereafter when HepG2 cells underwent apoptosis. In B16-F1 cells, only ODC and AZ protein levels were elevated probably in relation to the initial elevated growth rate and the absence of apoptosis involvement in the following cytostatic effect of HGF in melanoma cells. Consistent with this hypothesis, bax mRNA and protein levels were unchanged or even lower relative to control values.

Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development.

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