Background: Tyrosine kinase inhibitors (TKIs) have been developed against EGFR. However, in NSCLC (Non-small Cell Lung Cancer) and some other cancers, the occurrence of activating mutations in EGFR leads to acquired resistance. Anomalous cMET signaling additionally contributes to developing resistance to targeted therapies, including ones directed at EGFR. A number of bispecific antibodies targeting EGFR and cMET are in clinical trials and JNJ-61186372 (Amivantamab) from Janssen labs, has been given fast track approval by the FDA. In this study we created bispecific and trispecific antibody formats which target both cMET and EGFR. Trispecific (TsAb), unlike the bispecific (BsAb), binds to two different epitopes on cMET and one on EGFR resulting in enhanced internalization of the receptors. This study demonstrates that our antibodies exhibit enhanced capabilities than the benchmark at clinical stage in cell and non-cell based in vitro assays. Methods: The two cMET binding arms of TsAb were designed to block the cMET-HGF (ligand) interaction and enhance the internalization of the receptors. Cell based assays were performed using a NSCLC cell line NCI-H1975 (ATCC CRL-5908) and HGF-expressing NCI-H1975. Receptor binding and receptor/ligand blocking (cMET/HGF and EGFR/EGF) assays were performed using both ELISA and flow cytometry. Phosphorylation assays were performed using ELISA and Western blotting. Receptor internalization studies were executed using the pHrodo labelling kit and were further assessed through fluorescence microscopy and quantified through flow cytometry. ADCC assays (Antibody dependent cellular cytotoxicity) were performed through the Crystal violet method. Results: BsAb was more effective than benchmark in terms of its binding capabilities to cMET and EGFR receptors and also in blocking the interaction of cMET and EGFR with their ligands. Moreover, functional assays revealed that the new BsAb is in most cases better than benchmark in successfully inhibiting the phosphorylation of not only the cMET and EGFR receptors but also the downstream signaling pathways which are involved in cancer cell proliferation, survival and obstructing the apoptotic pathways. Furthermore, the new TsAb was found to be more effective than BsAb and benchmark in cMET/EGFR receptor internalization which may lead to additional extenuation of the downstream pathways for these receptors. Cancer cell killing assays for ABT BsAb show ADCC comparable to the benchmark. Conclusion: Comparing to BsAbs, TsAb induces enhanced internalization of the cMET/EGFR receptors leading to further extenuation of the downstream oncogenic signaling pathways. These data suggest the development of ABT BsAb and specifically TsAb as a new drug target for patients with lung cancer and NSCLC in particular.

Citation Format: Mohd Zaman, Cai Huang, Sachith Gallolu Kankanamalage, Amit K. Chaudhary, Jianbo Dong, Yue Liu. Development of cMET/cMET/EGFR Trispecific antibody as therapeutic modality for Non-small Cell Lung Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB069.