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.
Highlights
Receptor tyrosine kinases (RTKs) are prominent targets in tumor cell biology
We already explored in the past the properties of trispecific, trivalent Fc-containing antibodies-targeting receptor tyrosine kinases (RTKs) and could demonstrate that such constructs can be produced, are non-agonistic and inhibit tumor growth in a similar fashion as the combination of parental antibodies (Castoldi et al, 2012). We extend this approach to tetraspecific, tetravalent scFab and single-chain Fv (scFv)-based Fc-containing knob-into-hole antibodies, which can simultaneously bind to four different RTKs, namely HER1, HER3, IGF1R and cMet for the aforementioned reasons of receptor crosstalk and resistance
We report the development and in vitro characterization of novel tetravalent tetraspecific antibodies for simultaneous targeting of HER1, HER3, IGF1R and cMet on tumor cells with the potential to overcome resistance mechanisms mediated by compensatory signaling between different RTK signaling pathways
Summary
Receptor tyrosine kinases (RTKs) are prominent targets in tumor cell biology. Fifty-eight human RTKs falling in 20 subfamilies are known and for many of them it has been shown that aberrant activation promotes growth, apoptosis resistance and metastasis of tumor cells (Lemmon and Schlessinger, 2010). We have recently published a study on a bispecific HER1and cMet-targeting antibody, which inhibits receptor crosstalk (Castoldi et al, 2013). IGF1R can counteract inhibition of HER1 and render monospecific inhibition futile (Hendrickson and Haluska, 2009) These findings have already led to the generation of bispecific antibodies (BsAbs) targeting HER1 and IGF1R in various formats such as a tetravalent single-chain Fv (scFv)-based IgG1 antibody (Croasdale et al, 2012) and a bivalent one arm single-chain. We extend this approach to tetraspecific, tetravalent scFab and scFv-based Fc-containing knob-into-hole antibodies, which can simultaneously bind to four different RTKs, namely HER1, HER3, IGF1R and cMet for the aforementioned reasons of receptor crosstalk and resistance. We demonstrate that the presented tetraspecific antibodies can be produced in eukaryotic cells, are fully functional, non-agonistic and in all in vitro cellular models superior to BsAbs targeting either HER3 and cMet or HER1 and IGF1R
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
