Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100641
ISSN1083-351X
AutoresJoost Neijssen, Rosa M.F. Cardoso, Kristen Chevalier, Luus Wiegman, Thomas Valerius, Geoffrey M. Anderson, Sheri L. Moores, Janine Schuurman, Paul W.H.I. Parren, William R. Strohl, Mark L. Chiu,
Tópico(s)HER2/EGFR in Cancer Research
ResumoA bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling. A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling. Aberrant activations of both epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) signaling pathways have been implicated in driving tumor cell growth and proliferation in lung cancer (1Birchmeier C. Birchmeier W. Gherardi E. Vande Woude G.F. Met, metastasis, motility and more.Nat. Rev. Mol. Cell Biol. 2003; 4: 915-925Crossref PubMed Scopus (2160) Google Scholar, 2Liu X. Yao W. Newton R.C. Scherle P.A. Targeting the c-MET signaling pathway for cancer therapy.Expert Opin. Investig. Drugs. 2008; 17: 997-1011Crossref PubMed Scopus (114) Google Scholar, 3Bean J. Brennan C. Shih J.Y. Riely G. Viale A. Wang L. Chitale D. Motoi N. Szoke J. Broderick S. Balak M. Chang W.C. Yu C.J. Gazdar A. Pass H. et al.MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 20932-20937Crossref PubMed Scopus (1384) Google Scholar, 4Dulak A.M. Gubish C.T. Stabile L.P. Henry C. Siegfried J.M. HGF-independent potentiation of EGFR action by c-Met.Oncogene. 2011; 30: 3625-3635Crossref PubMed Scopus (84) Google Scholar). In a subgroup of non–small cell lung cancer (NSCLC), activating mutations in the EGFR gene, mainly L858R mutation and exon 19 deletions, result in ligand-independent activation of the EGFR kinase activity (5Arrieta O. Cardona A.F. Martin C. Mas-Lopez L. Corrales-Rodriguez L. Bramuglia G. Castillo-Fernandez O. Meyerson M. Amieva-Rivera E. Campos-Parra A.D. Carranza H. Gomez de la Torre J.C. Powazniak Y. Aldaco-Sarvide F. Vargas C. et al.Updated frequency of EGFR and KRAS mutations in NonSmall-cell lung cancer in Latin America: The Latin-American Consortium for the investigation of lung cancer (CLICaP).J. Thorac. Oncol. 2015; 10: 838-843Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Tyrosine kinase inhibitors (TKIs) targeting EGFR are the standard of care for patients with EGFR-mutated NSCLC (6Sequist L.V. Yang J.C. Yamamoto N. O'Byrne K. Hirsh V. Mok T. Geater S.L. Orlov S. Tsai C.M. Boyer M. Su W.C. Bennouna J. Kato T. Gorbunova V. Lee K.H. et al.Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations.J. Clin. Oncol. 2013; 31: 3327-3334Crossref PubMed Scopus (2306) Google Scholar, 7Soria J.C. Ohe Y. Vansteenkiste J. Reungwetwattana T. Chewaskulyong B. Lee K.H. Dechaphunkul A. Imamura F. Nogami N. Kurata T. Okamoto I. Zhou C. Cho B.C. Cheng Y. Cho E.K. et al.Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer.N. Engl. J. Med. 2018; 378: 113-125Crossref PubMed Scopus (2001) Google Scholar); however, many patients will acquire resistance to TKIs (8Yu H.A. Arcila M.E. Rekhtman N. Sima C.S. Zakowski M.F. Pao W. Kris M.G. Miller V.A. Ladanyi M. Riely G.J. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers.Clin. Cancer Res. 2013; 19: 2240-2247Crossref PubMed Scopus (1607) Google Scholar, 9Oxnard G.R. Hu Y. Mileham K.F. Husain H. Costa D.B. Tracy P. Feeney N. Sholl L.M. Dahlberg S.E. Redig A.J. Kwiatkowski D.J. Rabin M.S. Paweletz C.P. Thress K.S. Janne P.A. Assessment of resistance mechanisms and clinical implications in patients with EGFR T790M-positive lung cancer and acquired resistance to osimertinib.JAMA Oncol. 2018; 4: 1527-1534Crossref PubMed Scopus (314) Google Scholar). In addition, MET pathway activation via increased expression of receptor or ligand is frequently implicated in TKI resistance (10Turke A.B. Zejnullahu K. Wu Y.-L. Song Y. Dias-Santagata D. Lifshits E. Toschi L. Rogers A. Mok T. Sequist L. Lindeman N.I. Murphy C. Akhavanfard S. Yeap B.Y. Xiao Y. et al.Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC.Cancer Cell. 2010; 17: 77-88Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar, 11Engelman J.A. Zejnullahu K. Mitsudomi T. Song Y. Hyland C. Park J.O. Lindeman N. Gale C.M. Zhao X. Christensen J. Kosaka T. Holmes A.J. Rogers A.M. Cappuzzo F. Mok T. et al.MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.Science. 2007; 316: 1039-1043Crossref PubMed Scopus (3731) Google Scholar, 12Yano S. Yamada T. Takeuchi S. Tachibana K. Minami Y. Yatabe Y. Mitsudomi T. Tanaka H. Kimura T. Kudoh S. Nokihara H. Ohe Y. Yokota J. Uramoto H. Yasumoto K. et al.Hepatocyte growth factor expression in EGFR mutant lung cancer with intrinsic and acquired resistance to tyrosine kinase inhibitors in a Japanese cohort.J. Thorac. Oncol. 2011; 6: 2011-2017Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Treatment strategies targeting both receptors using a combination of single-agent EGFR and MET inhibitors do not cover the wide range of resistance mechanisms (13Scagliotti G.V. Moro-Sibilot D. Kollmeier J. Favaretto A.G. Cho E.K. Grosch H. Kimmich M. Girard N. Tsai C.-M. Hsia T.-C. Brighenti M. Schumann C. Wang X.A. Wijayawardana S.R. Gruver A.M. et al.A randomized, controlled, open label phase II study of erlotinib (E) with or without the MET antibody emibetuzumab (Emi) as first-line treatment for EGFRmt non-small cell lung cancer (NSCLC) patients who have disease control after an 8-week lead-in treatment with erlotinib.J. Clin. Oncol. 2017; 35: 9019Google Scholar, 14Scagliotti G.V. Shuster D. Orlov S. von Pawel J. Shepherd F.A. Ross J.S. Wang Q. Schwartz B. Akerley W. Tivantinib in combination with erlotinib versus erlotinib alone for EGFR- mutant NSCLC: An exploratory analysis of the phase 3 MARQUEE study.J. Thorac. Oncol. 2018; 13: 849-854Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), hence the need for novel approaches to overcome resistance and to achieve clinical benefit. Simultaneous engagement of both EGFR and MET, through a bispecific antibody (BsAb), is a potential strategy to overcome resistance and achieve greater efficacy (15Puri N. Salgia R. Synergism of EGFR and c-Met pathways, cross-talk and inhibition, in non-small cell lung cancer.J. Carcinog. 2008; 7: 9Crossref PubMed Scopus (155) Google Scholar). Identification of an antagonist antibody targeting MET can be challenging as the mechanism of action depends on the valency of the antibody for the tumor target antigen. Such antibodies are referred to as anti-MET, which modulate the activity of c-Met, also called tyrosine-protein kinase Met or hepatocyte growth factor receptor, which is a protein encoded by the MET gene. Upon ligand binding, MET dimerizes and initiates signaling pathway activation (16Luraghi P. Schelter F. Kruger A. Boccaccio C. The MET oncogene as a therapeutical target in cancer invasive growth.Front. Pharmacol. 2012; 3: 164Crossref PubMed Scopus (10) Google Scholar). Therefore, antibodies that induce dimerization of MET may have agonistic activity (17Husain B. Ellerman D. Expanding the boundaries of biotherapeutics with bispecific antibodies.BioDrugs. 2018; 32: 441-464Crossref PubMed Scopus (45) Google Scholar), although antagonistic bivalent MET monoclonal antibodies (mAbs) have been reported (18Liu L. Zeng W. Wortinger M.A. Yan S.B. Cornwell P. Peek V.L. Stephens J.R. Tetreault J.W. Xia J. Manro J.R. Credille K.M. Ballard D.W. Brown-Augsburger P. Wacheck V. Chow C.K. et al.LY2875358, a neutralizing and internalizing anti-MET bivalent antibody, inhibits HGF-dependent and HGF-independent MET activation and tumor growth.Clin. Cancer Res. 2014; 20: 6059-6070Crossref PubMed Scopus (73) Google Scholar, 19Gonzalez A. Broussas M. Beau-Larvor C. Haeuw J.F. Boute N. Robert A. Champion T. Beck A. Bailly C. Corvaia N. Goetsch L. A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors.Int. J. Cancer. 2016; 139: 1851-1863Crossref PubMed Scopus (11) Google Scholar). An antibody with a monovalent anti-MET binding arm may prevent MET dimerization-based agonism (20Merchant M. Ma X. Maun H.R. Zheng Z. Peng J. Romero M. Huang A. Yang N.Y. Nishimura M. Greve J. Santell L. Zhang Y.W. Su Y. Kaufman D.W. Billeci K.L. et al.Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E2987-E2996Crossref PubMed Scopus (157) Google Scholar, 21Xiang H. Bender B.C. Reyes 2nd, A.E. Merchant M. Jumbe N.L. Romero M. Davancaze T. Nijem I. Mai E. Young J. Peterson A. Damico-Beyer L.A. Onartuzumab (MetMAb): Using nonclinical pharmacokinetic and concentration-effect data to support clinical development.Clin. Cancer Res. 2013; 19: 5068-5078Crossref PubMed Scopus (24) Google Scholar). However, an antibody with this property, such as onartuzumab, did not have a favorable clinical profile (22Surati M. Patel P. Peterson A. Salgia R. Role of MetMAb (OA-5D5) in c-MET active lung malignancies.Expert Opin. Biol. Ther. 2011; 11: 1655-1662Crossref PubMed Scopus (41) Google Scholar, 23Spigel D.R. Edelman M.J. O'Byrne K. Paz-Ares L. Mocci S. Phan S. Shames D.S. Smith D. Yu W. Paton V.E. Mok T. Results from the phase III randomized trial of onartuzumab plus erlotinib versus erlotinib in previously treated stage IIIB or IV non-small-cell lung cancer: METLung.J. Clin. Oncol. 2017; 35: 412-420Crossref PubMed Scopus (162) Google Scholar, 24Spigel D.R. Ervin T.J. Ramlau R.A. Daniel D.B. Goldschmidt Jr., J.H. Blumenschein Jr., G.R. Krzakowski M.J. Robinet G. Godbert B. Barlesi F. Govindan R. Patel T. Orlov S.V. Wertheim M.S. Yu W. et al.Randomized phase II trial of onartuzumab in combination with erlotinib in patients with advanced non-small-cell lung cancer.J. Clin. Oncol. 2013; 31: 4105-4114Crossref PubMed Scopus (374) Google Scholar), likely due to (1) inability to induce Fc-mediated effector functions; (2) reduced MET downmodulation via internalization by monovalent molecules; and (3) solely targeting MET, which may trigger development of resistance via oncogenic EGFR signaling. Thus, we embarked on discovering a molecule with a different molecular format and distinct epitope to improve efficacy. BsAbs that target EGFR and MET through distinct epitopes and architecture have had varying clinical results (25Lee B.S. Kim H.J. Hwang J.W. Cheong K.H. Kim K.A. Cha H.Y. Lee J.M. Kim C.H. The dual inhibition of met and EGFR by ME22S, a novel met/EGFR bispecific monoclonal antibody, suppresses the proliferation and invasion of laryngeal cancer.Ann. Surg. Oncol. 2016; 23: 2046-2053Crossref PubMed Scopus (13) Google Scholar, 26Patnaik A. Gordon M. Tsai F. Papadopoulos K.P. Rasco D. Beeram M. Fu S. Janku F. Hynes S.M. Gundala S.R. Willard M.D. Zhang W. Lin A.B. Hong D. A phase I study of LY3164530, a bispecific antibody targeting MET and EGFR, in patients with advanced or metastatic cancer.Cancer Chemother. Pharmacol. 2018; 82: 407-418Crossref PubMed Scopus (29) Google Scholar, 27Lee D. Sung E.S. Ahn J.H. An S. Huh J. You W.K. Development of antibody-based c-Met inhibitors for targeted cancer therapy.Immunotargets Ther. 2015; 4: 35-44PubMed Google Scholar, 28Lee J.M. Lee S.H. Hwang J.W. Oh S.J. Kim B. Jung S. Shim S.H. Lin P.W. Lee S.B. Cho M.Y. Koh Y.J. Kim S.Y. Ahn S. Lee J. Kim K.M. et al.Novel strategy for a bispecific antibody: Induction of dual target internalization and degradation.Oncogene. 2016; 35: 4437-4446Crossref PubMed Scopus (16) Google Scholar). To maximize inhibition of EGFR and MET pathways, we aimed at discovering a novel BsAb that combines all the previously described mechanisms of action for EGFR and MET antibodies but without inducing receptor dimerization and activation. The BsAb would have two binding arms: one monovalent arm that engages EGFR and the other monovalent arm that engages MET. To enable the selection of the optimal bispecific molecule, we screened a panel of BsAbs in an empirical approach that led to the selection of amivantamab (JNJ-61186372), an EGFR × MET BsAb that has activity in EGFR TKI-resistant NSCLC models (29Moores S.L. Chiu M.L. Bushey B.S. Chevalier K. Luistro L. Dorn K. Brezski R.J. Haytko P. Kelly T. Wu S.J. Martin P.L. Neijssen J. Parren P.W. Schuurman J. Attar R.M. et al.A novel bispecific antibody targeting EGFR and cMet is effective against EGFR inhibitor-resistant lung tumors.Cancer Res. 2016; 76: 3942-3953Crossref PubMed Scopus (95) Google Scholar). Here, we describe a versatile selection strategy, provide structural insights in the binding of amivantamab, and present novel functional in vivo antitumor data. The controlled Fab-arm exchange (cFAE) platform was used to generate a panel of 40 (5 MET parental mAbs with 8 EGFR parental mAbs) MET × EGFR BsAbs in the DuoBody format (30Labrijn A.F. Meesters J.I. de Goeij B.E. van den Bremer E.T. Neijssen J. van Kampen M.D. Strumane K. Verploegen S. Kundu A. Gramer M.J. van Berkel P.H. van de Winkel J.G. Schuurman J. Parren P.W. Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 5145-5150Crossref PubMed Scopus (187) Google Scholar) (Fig. 1A). The BsAb quality was confirmed as being monodisperse by size-exclusion chromatography and purity by SDS-PAGE. The anti-gp120 antibody b12 was included as a nonbinding arm of control BsAbs that had either an EGFR or MET binding arm to generate monovalent control antibodies. The parental antibodies were chosen to cover a broad epitope space for both MET and EGFR mAbs from different cross-block groups, sequence diversity (unique heavy chain complementarity-determining region 3 [CDR-H3] sequence clusters), binding affinity (human–cynomolgus monkey cross-reactivity for EGFR mAbs; human–rhesus monkey cross-reactivity for MET mAbs), and ligand-blocking ability (Table S1). Since residual parental MET homodimer mAbs can result in an agonistic response, an excess of EGFR homodimer mAb was used in the cFAE reaction to minimize the levels of residual MET homodimer mAbs. The cFAE preparations were analyzed by cation exchange chromatography to confirm the generation of BsAb species and to determine the percentage of residual homodimer in the batches. The homodimer materials were included as reference in the same run. The cation exchange chromatography showed successful generation of a BsAb species was observed after the exchange process when there was a peak located in between the elution peaks of the two-originating parental mAb homodimer peaks. The percentage of residual homodimers are listed in Table S2. We used an elimination strategy to discard BsAb molecules with unwanted properties at predefined decision points: sufficient monovalent binding affinity (EC50 <1 μg/ml, which is within the range of serum concentration of a therapeutic mAb (31Lobo E.D. Hansen R.J. Balthasar J.P. Antibody pharmacokinetics and pharmacodynamics.J. Pharm. Sci. 2004; 93: 2645-2668Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar)), minimal induction of EGFR and MET phosphorylation, and minimal cell proliferation via direct or cross talk activation of EGFR and MET. The first phase of the selection process focused on the binding properties of the antibodies (Fig. 1B). A key requirement of the BsAb was the ability to bind both targets in a monovalent format with affinity EC50 1 μg/ml were considered low or poor binders and discarded (Fig. 2A). Functionally monovalent MET×b12 BsAbs bound well to all cell lines tested, whereas the reactivity of the EGFR panel was more diverse with monovalent EGFR C, EGFR D, EGFR F, and EGFR G BsAbs binding poorly (EC50 >1 μg/ml). Binding for monovalent EGFR BsAbs was ranked EGFR H > EGFR E > EGFR B > EGFR A; for monovalent MET BsAbs, binding was ranked MET B > MET A > MET C > MET D > MET E. None of the MET×EGFR BsAb combinations had enhanced binding as compared with their respective monovalent controls (data not shown). In A549 cells, MET was not phosphorylated under steady-state conditions in the absence of HGF. An agonistic bivalent antibody MET 5D5 IgG1 and a functionally monovalent bispecific version of MET 5D5 (MET 5D5×b12) were used as positive and negative controls for MET phosphorylation, respectively (Fig. 2B; Western blots quantified agonistic activity of antibodies Fig. S1). BsAbs with a score ≥3 and 4 were deselected (Fig. 2C). None of the tested bivalent EGFR parental mAbs induced MET phosphorylation. Although all bivalent MET parental mAbs induced MET phosphorylation, no agonistic activity of the METxb12 BsAb combinations was observed, confirming the hypothesis that MET agonistic antibodies can be converted to antagonistic molecules by using (functionally) monovalent formats (20Merchant M. Ma X. Maun H.R. Zheng Z. Peng J. Romero M. Huang A. Yang N.Y. Nishimura M. Greve J. Santell L. Zhang Y.W. Su Y. Kaufman D.W. Billeci K.L. et al.Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E2987-E2996Crossref PubMed Scopus (157) Google Scholar). Some bispecific MET×EGFR molecules induced MET phosphorylation, suggesting that binding of EGFR and MET on the same cell could result in MET dimerization and activation or phosphorylation of MET in trans. BsAbs containing the MET C arm that were identified as MET agonists, as with variants with MET arms B, D, and E with scores of 3 to 4, were excluded from further analysis (Fig. 2C). No combinations containing EGFR H were agonistic, even when combined with MET C, which induced medium to high phosphorylation with other EGFR binders. Furthermore, molecules containing MET A did not induce MET phosphorylation. We excluded BsAbs containing the EGFR A, C, D, F, and G arms since they failed the required specifications for affinity and/or induced MET phosphorylation in the A549 phosphorylation assay. We examined the impact of receptor phosphorylation via proliferation assays of cell lines with differing levels of EGFR and MET. The remaining EGFR×MET BsAb panel was further tested for (1) inhibition of HGF-driven KP4 cell proliferation expressing an autocrine HGF–MET loop and (2) inhibition of H1975 cell proliferation driven by the EGFR signaling pathway. The percentage of cell proliferation relative to the isotype (negative) control b12 IgG1 in KP4 cells was evaluated at 0.014, 0.37, and 10 μg/ml BsAb concentrations. All BsAbs showed a dose-dependent inhibition of proliferation, except molecules containing the MET D and MET E arms (Fig. 3A). This was expected as the parental MET mAbs were unable to block HGF binding to MET in vitro (data not shown). Molecules containing the MET A, MET B, and MET C arms had the strongest inhibition of proliferation compared with cells treated with the isotype control. The percentage of cell proliferation relative to 10 μg/ml b12 IgG1 negative control and bivalent EGFR-H positive control in H1975 cells was evaluated. The bivalent anti-MET 5D5 IgG1 was included as a control agonistic mAb. Except for the positive control and EGFR H BsAb combinations, none of the BsAbs inhibited proliferation of H1975 (Fig. 3B). A third proliferation assay was conducted using NCI-H441 cell lines with equivalent levels of EGFR and MET (Table S3). The NCI-H441 proliferation assay identified agonistic MET×EGFR BsAb. BsAbs MET A×EGFR B, MET A×EGFR E, MET A×EGFR H, MET D×EGFR B and MET D×EGFR H induced mild proliferation of NCI-H441 cells, albeit less pronounced than the agonist MET 5D5 IgG1 control (Fig. 3C). Based on these proliferation assays, we deselected BsAb combinations containing MET A, MET D, and MET E crossed with EGFR B arms. BsAbs with the MET B and C arms in combination with EGFR H were selected for further characterization. The identified molecules did not induce MET phosphorylation in A549 cells, yet some induced proliferation of NCI-H441 cells (Fig. 3C), suggesting that another signaling pathway could be activated due to induction of EGFR phosphorylation via cross-linking of MET and EGFR (32Agarwal S. Zerillo C. Kolmakova J. Christensen J.G. Harris L.N. Rimm D.L. Digiovanna M.P. Stern D.F. Association of constitutively activated hepatocyte growth factor receptor (Met) with resistance to a dual EGFR/Her2 inhibitor in non-small-cell lung cancer cells.Br. J. Cancer. 2009; 100: 941-949Crossref PubMed Scopus (81) Google Scholar, 33Jo M. Stolz D.B. Esplen J.E. Dorko K. Michalopoulos G.K. Strom S.C. Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.J. Biol. Chem. 2000; 275: 8806-8811Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 34Guo A. Villen J. Kornhauser J. Lee K.A. Stokes M.P. Rikova K. Possemato A. Nardone J. Innocenti G. Wetzel R. Wang Y. MacNeill J. Mitchell J. Gygi S.P. Rush J. et al.Signaling networks assembled by oncogenic EGFR and c-Met.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 692-697Crossref PubMed Scopus (439) Google Scholar). To identify agonistic BsAbs that induced EGFR activation in the absence of EGFR ligand and inhibited ligand-induced EGFR phosphorylation, we lysed cell lines treated with the antibodies and determined EGFR Tyr1068 phosphorylation. In A549 cells, which have low MET and moderate EGFR expression, no EGFR phosphorylation was observed in unstimulated cells. Clear EGFR phosphorylation was observed by treating the cells with EGF and effectively blocked by BsAbs containing EGFR H, partially by BsAb MET C×EGFR E, and not by the MET A×b12 BsAb control (Fig. 3D). In contrast to EGFR H, the EGFR E epitope was located outside the EGF binding site and did not block EGF binding to EGFR (not shown). No EGFR phosphorylation was observed by any of the tested MET×EGFR BsAbs in the absence of EGF, confirming the lack of MET-induced EGFR cross-phosphorylation in A549 cells. Only BsAbs containing the EGFR H arm inhibited H1975 cell proliferation and EGF-induced EGFR phosphorylation in A549 cells. Hence, BsAbs with any other EGFR arm were rejected. BsAbs containing the MET C arm were also discarded as all combinations containing this arm and an EGFR arm with affinity EC50 values <1 μg/ml also induced MET phosphorylation (Fig. 2C). BsAbs containing the MET D and E arms poorly inhibited KP4 cell proliferation, rendering them unsuitable (Fig. 3A). BsAbs containing MET A and D arms were slightly agonistic in the NCI-H441 proliferation assay, although the effect was small compared with the positive control MET 5D5 IgG1 (Fig. 3C). Altogether, the MET B×EGFR H BsAb showed the most optimal properties and with growth in a proprietary cell line for production of parental mAbs with low fucose Fc production, the BsAb became amivantamab. The crystal structure of the MET B Fab arm of amivantamab bound to human MET Sema-PSI region was solved to better understand the potent inhibition of MET signaling. The structure of the Fab–Sema-PSI complex was determined to 3.1-Å resolution with one complex in the P43212 asymmetric unit (Table S4). The structure contained MET residues 40 to 564 with glycans N-linked to residues N45, N106, N149, N202, and N405, Fab heavy chain residues 1 to 222, and Fab light chain residues 1 to 213. The Fab–Sema-PSI combining site was well defined by the electron density, which allowed reliable positioning of the binding residues. The amivantamab Fab bound to the MET Sema domain using all CDRs except CDR-H1 (Fig. 4A). The Sema domain had a seven-bladed β-propeller with four antiparallel β-strands per blade. The Fab bound to the outside wall of the propeller via interactions predominantly with the long loops connecting propeller blades 1 to 2 (loop 1–2; epitope residues D94, F96-D100, and S103-N106) and blades 2 to 3 (loop 2–3; epitope residues F162-P164, I166, and E167). The Fab also had interactions with intra-blade 3 loop C2D3 (epitope residues T222 and D224) and the disulfide-bonded pair (C98-C160) that bridged β-strand D2 with the mid-region of loop 1 to 2. The Fab CDR-H2, -H3, -L2, and -L3 (paratope residues W50H, N55H, Y57H, N59H, L100H -Y105H, D107H, Y49L, A50L, S52L, L55L, S56L, and A91L -F94L) were positioned along the folded mid-section of loop 1 to 2, stabilizing this long loop in a folded down conformation. CDR-L1 (paratope residues S30-W32) interacted exclusively with loops 2 to 3 and C2D3 (Fig. 4, A and B). The large 1000 Å2 interface between the Fab and Sema was dominated by polar interactions (Fig. 4B). Of the 18 epitope residues, 11 had hydrogen bond interactions with the Fab—the paratope counterpart had 10 of the 23 residues having hydrogen bonds with Sema. The tightly packed interface between the Fab and Sema domains involve hydrogen bond interactions between both protein backbone atoms. Epitope residues D94, Q99, K104, P164, and I166 of MET were buried into the Sema–Fab combining site and had extensive interactions with the Fab (Fig. 4, B and C). The side chain of D94 had a hydrogen bond contact with the side chain of R101 (CDR-H3), but these residues could adopt different conformations in solution and form a stronger salt bridge interaction. Q99 and K104 had hydrogen bond contacts with the carbonyl group of G102 (CDR-H3), a glycine strategically located in a crowded region of the combining site (Fig. 4B). P164, F96, P97, F162, and P164 constituted the lining of a shallow cavity on Sema. F96 and F162 formed a cluster of aromatic residues with Fab residues W32 (CDR-L1), Y49 (CDR-L2), and Y105 (CDR-H3). I166 was buried by a portion of this aromatic cluster and the side chain of residues L100, D107 (CDR-H3), and L55 (CDR-L2). Taken together, these paratope–epitope interactions mediated a high level of specificity to amivantamab binding to MET. Amivantamab Fab binding to MET stabilized the Sema loop 1 to 2 in a conformation slightly different from other MET structures (Protein Data Bank [PDB] codes 1SHY, 4K3J, 2UZX, and 2UZY) (20Merchant M. Ma X. Maun H.R. Zheng Z. Peng J. Romero M. Huang A. Yang N.Y. Nishimura M. Greve J. Santell L. Zhang Y.W. Su Y. Kaufman D.W. Billeci K.L. et al.Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E2987-E2996Crossref PubMed Scopus (157) Google Scholar, 35Niemann H.H. Jager V. Butler P.J. van den Heuvel J. Schmidt S. Ferraris D. Gherardi E. Heinz D.W. Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB.Cell. 2007; 130: 235-246Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 36Stamos J. Lazarus R.A. Yao X. Kirchhofer D. Wiesmann C. Crystal structure of the HGF beta-chain in
Referência(s)