TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations

Abstract

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.

The making of bispecific antibodies

“…2, box 8 ). 196 The scFab format was also combined with the LUZ-Y Fc heterodmerization strategy ( Fig. 2, box 7 ).…”

Santamarine Inhibits NF-κB Activation and Induces Mitochondrial Apoptosis in A549 Lung Adenocarcinoma Cells via Oxidative Stress

“…At present, the most effective chemotherapeutic drugs for NSCLC are gefitinib, erlotinib, and afatinib, which are highly targeted EGFR-directed tyrosine kinase inhibitors (TKIs) [ 4 ]. However, genetic instability, aberrant activation of signaling pathways, and development of secondary drug resistance have become the major limitations of such drugs [ 5 ]. Exploring novel therapeutic agents with promising anticancer activity and their molecular mechanisms is, therefore, necessary for improving the outcome of NSCLC treatment.…”

Molecular mechanisms of perineural spread of cutaneous carcinoma: a potential role for receptor tyrosine kinases and other biomarkers

“…Recently, the superiority of a tetraspecific antibody to individual monospecific antibodies was demonstrated in a heterogenous tumour population. (200) Beyond inducing antibody dependent cell cytotoxicity by recruiting FcγR positive immune cells, some research groups have looked at targeting the immune system against cancer by designing dual antibody constructs that cross-link tumour cells and T cells via their CD3 receptors. (199) In terms of the ErbB family specifically, where the HER2 receptor is completely dependent and the HER3 is a partially impaired RTK, preventing heterodimerisation is a key strategy.…”

“…Furthermore, comparable to dual EGFR/HER2 targeting, several studies have now shown that dual inhibition of HER3 and EGFR is efficacious in multiple tumour models. (200) Moreover, inhibition of HER3 phosphorylation has been correlated with anti-proliferative activity of tyrosine kinase inhibitors in colorectal, pancreatic and NSCLC cell lines, suggesting that optimal EGFR signalling inhibition requires simultaneous inhibition of HER3 signalling. (210) We were unfortunately unsuccessful in our initial attempts to optimise a HER3 immunostaining protocol for FFPE tissue to allow assessment of HER3 expression in perineural spread cSCCHN.…”