Published OnlineFirst July 7, 2009; DOI: 10.1158/0008-5472.CAN-08-4518 Experimental Therapeutics, Molecular Targets, and Chemical Biology Nimotuzumab, an Antitumor Antibody that Targets the Epidermal Growth Factor Receptor, Blocks Ligand Binding while Permitting the Active Receptor Conformation Ariel Talavera,1,2 Rosmarie Friemann,2,3 Silvia Go´mez-Puerta,1 Carlos Martinez-Fleites,4 Greta Garrido,1 Ailem Rabasa,1 Alejandro Lo´pez-Requena,1 Amaury Pupo,1 Rune F. Johansen,3 Oliberto Sa´nchez,5 Ute Krengel,2 and Ernesto Moreno1 1Center of Molecular Immunology, Havana, Cuba; 2Department of Chemistry, University of Oslo, Oslo, Norway; and 3Center for Molecular and Behavioral Neuroscience, Institute of Medical Microbiology, University of Oslo, Rikshospitalet HF, Oslo, Norway; 4Department of Chemistry, University of York, Heslington, York, United Kingdom; and 5Center for Genetic Engineering and Biotechnology, Havana, Cuba Abstract region of the EGFR (eEGFR), leaving the dimerization ‘‘arm’’ in Overexpression of the epidermal growth factor (EGF) receptor domain II ready for binding a second monomer (4, 5). It has been (EGFR) in cancer cells correlates with tumor malignancy and shown that the eEGFR adopts a ‘‘tethered’’ or inactive conformation poor prognosis for cancer patients. For this reason, the EGFR in the absence of EGF (6). In this characteristic conformation, has become one of the main targets of anticancer therapies. the dimerization arm is hidden by interactions with domain IV, Structural data obtained in the last few years have revealed whereas domains I and III remain separated. Thus, to adopt the the molecular mechanism for ligand-induced EGFR dimeriza- ‘‘extended’’ or active conformation observed in the crystal structure tion and subsequent signal transduction, and also how this of the complex with EGF (4), the receptor must undergo a major signal is blocked by either monoclonal antibodies or small conformational change that brings together domains I and III (6). molecules. Nimotuzumab (also known as h-R3) is a humanized On the cell surface, the tethered and extended conformations are in antibody that targets the EGFR and has been successful in the equilibrium, and it has been estimated that >85% of the EGFR clinics. In this work, we report the crystal structure of the Fab molecules adopt the more energetically favorable tethered or in- fragment of Nimotuzumab, revealing some unique structural active conformation (7, 8). EGF as well as other EGFR ligands shift features in the heavy variable domain. Furthermore, compe- this equilibrium in favor of the active conformation. tition assays show that Nimotuzumab binds to domain III of In the latest years, newstructural data have shownhowdifferent the extracellular region of the EGFR, within an area that antibodies inhibit the signal transduction via EGFR by different overlaps with both the surface patch recognized by Cetuximab mechanisms. Cetuximab, IMC-11F8, and Zalutumumab bind to (another anti-EGFR antibody) and the binding site for EGF. A domain III and act by blocking the binding of EGF and by sterically computer model of the Nimotuzumab-EGFR complex, con- interfering with the active conformation (9–11). Matuzumab, which structed by docking and molecular dynamics simulations and also binds to domain III, does not block EGF binding, but it does supported by mutagenesis studies, unveils a novel mechanism sterically interfere with the domain rearrangement needed for of action, with Nimotuzumab blocking EGF binding while receptor dimerization (12). Monoclonal antibody (mAb) 806 follows still allowing the receptor to adopt its active conformation, a different inhibition mechanism, binding directly to the dimer- hence warranting a basal level of signaling. [Cancer Res ization domain (13). 2009;69(14):5851–9] Nimotuzumab is a humanized mAb that binds to the extra- cellular domain of the EGFR and inhibits EGF binding (14). Nimotuzumab has been approved in several countries for the Introduction treatment of head and neck tumors (15) and glioma (16), and is in Overexpression of the epidermal growth factor (EGF) receptor clinical trials for various tumor types including colorectal, glioma (EGFR) has been shown to correlate with tumor malignancy and (pediatric and adult), pancreatic, prostate, non–small cell lung, poor prognosis for cancer patients (1). Therefore, different strat- esophageal, cervical, and breast cancer (17). One striking outcome egies have been developed to inhibit the aberrant EGFR-associated from the use of Nimotuzumab in the clinic is the absence of severe signal transduction cascade. The most important therapeutic ap- adverse effects. In particular, no serious skin rash has been reported proaches include small-molecule tyrosine kinase inhibitors (2), (18). This is in contrast to most of the other drugs targeting the which act by interfering with ATP binding to the receptor, and EGFR, either monoclonal antibodies or small molecules, for which monoclonal antibodies (3), which bind specifically to the extra- the severe skin rash they provoke has been assumed as an un- cellular region of the receptor, inhibiting its dimerization and avoidable negative side effect and, moreover, as a surrogate marker autophosphorylation. of antitumor effect (19, 20). The reason for the lowtoxicity of EGFR activation is induced by ligand binding. EGF binds to a Nimotuzumab might lie in its intermediate affinity, as proposed in cleft formed by domains I (dI) and III (dIII) of the extracellular ref. 15, but it might as well result from a different inhibition mechanism, determined by the way in which the antibody binds to the eEGFR. Requests for reprints: Ute Krengel, University of Oslo, Department of Chemistry, P.O. Box 1033 Blindern, Oslo, NO-0315, Norway. E-mail: [email protected] and In this work, we aimed at identifying the epitope recognized by Ernesto Moreno, Center of Molecular Immunology, P.O. Box 16040, Playa, Havana, Nimotuzumab on EGFR, by combining X-ray crystallography, 11600 Cuba. Phone: 53-7-2717933; Fax: 53-7-2720644; E-mail: [email protected]. I2009 American Association for Cancer Research. binding experiments, and molecular modeling. We report here the doi:10.1158/0008-5472.CAN-08-4518 crystal structure of the unliganded Fab fragment of Nimotuzumab at www.aacrjournals.org 5851 Cancer Res 2009; 69: (14). July 15, 2009 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst July 7, 2009; DOI: 10.1158/0008-5472.CAN-08-4518 Cancer Research ˚ 2.5 A resolution, which shows some unique features, untypical for Table 1. Data collection and refinement statistics antibody structures. This structure, together with key binding data, provided the starting point to construct a model of the Nimotuzumab- Data collection statistics EGFR complex, supported by mutagenesis studies. The obtained model indicates that Nimotuzumab has a very peculiar way of Beamline Max II, I711 inhibiting EGFR-mediated signaling, which may explain the unique Resolution (A˚) 20.0–2.5 (2.64–2.5)* clinical profile of this molecule. Space group P43212 Unit cell parameters a=b(A˚) 84.2 ˚ Materials and Methods c(A) 138.5 Total observations 122,255 (17,096) Unique observations 17,556 (2,510) Purification of Nimotuzumab (h-R3) Fab fragments c Fab fragments were prepared from purified mAb (produced at the Center Rmerge 12.5 (48.9) of Molecular Immunology) by papain digestion (21). Further purification of Redundancy 7.0 (6.8) the isoforms was performed as described by Krengel and colleagues (21). Mean (I/j(I)) 16.7 (3.6) The pH gradient was created in this case by mixing 50 mmol/L DEtA (pH 9.6) Completeness (%) 98.5 (98.1) 2 and 50 mmol/L DEtA (pH 8.6). The largest peak was collected and the buffer Wilson B (A˚ ) 37.5 exchanged by dialysis against 25 mmol/L Tris (pH 7.0). Finally, the protein Refinement statistics was concentrated to 12 mg/mL. b Rfactor/Rfree 21.5/28.8 Crystallization and data collection r.s.c.c.x 0.86 Initial crystallization screening was carried out with sparse matrix screens Average B factor (A˚2) 16.6 using the hanging drop vapor diffusion technique at room temperature. Model Typically, 1 to 2 AL of the precipitant solution were pipetted onto 1 to 2 ALof the protein solution (12 mg/mL) and equilibrated against 0.7 to 1 mL of the No. of residues 389 j precipitant solution. Highest diffracting crystals were obtained at 20 Cin No. of protein atoms 3079 25% polyethylene glycol (PEG) 1,000 and 0.1M MES (pH 6.75), within 10 d. The No. of solvent molecules 187 Â Â 3 square crystals (0.1 0.1 0.1 mm ) belong to space group P43212, contain No. of PEG atoms (2 chains) 23 a b ˚ k onemoleculeperasymmetricunitandhavecellaxesof = =84.2Aand RMSD angles (j) 1.40 c ˚ ˚ 3 =138.5A. The Matthews parameter is 2.5 A /Da, which corresponds to a RMSD bond lengths (A˚) 0.01 solvent content of 50% (22). PDB code 3GKW Diffraction data to 2.5 A˚ resolution were collected at cryogenic tem- perature (100K) at beamline 711 at MaxII, Lund, Sweden, using a MARCCD detector (180 frames, at 1j oscillation). As a cryoprotectant, a reservoir *Numbers in parentheses refer to high-resolution shell. c solution supplemented with 20% PEG 400 was used. The data were indexed, Rmerge = AhAi|Ihi-<Ih>|/AhAi<Ih> where <Ih> is the average intensity integrated, scaled, and merged using Mosflm (23, 24) and Scala (24, 25). over symmetry-related measurements. b A A Scaling statistics are summarized in Table 1. Rfactor = ||FO|-|FC||/ |FO| where FO and FC are the observed and calculated structure factors, respectively. Structure determination and refinement xr.s.c.c., real space correlation coefficient. The structure was solved by Molecular Replacement with the program kRMSD, root mean square deviation from ideal geometry. MOLREP (24, 26), using the coordinates of Fab 4G2 (PDB entry: 1UYW) with truncated complementarity determining regions (CDR) as a search model.