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
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˚ 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 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-
and 1 mol/L NaCl. The affinity values were calculated from the kon/koff ratios, Competition assays which were directly obtained from the kinetics experiments. The data were ELISA. Solid phase ELISA was performed using 96-well polystyrene evaluated with the Langmuir 1:1 binding model. The EGF binding inhibition A microtiter plates (High binding, Costar) coated with 5 g/mL of the eEGFR experiment for Nimotuzumab and eEGFR was carried out following the j (produced at CIM) at 37 C for 1 h. After washing with PBS containing 0.05% protocol described by Li and colleagues (9). Tween 20 (PBS-T), the plates were blocked with PBS containing 1% bovine serum albumin for 1 h at 37jC and washed again. The biotinylated Docking simulations of the Nimotuzumab/eEGFR-dIII complex Nimotuzumab and the nontagged Cetuximab (Merck) antibodies were Several docking simulations involving the variable region (Fv) of coincubated for 1 h at 37jC. The concentration of Nimotuzumab was kept Nimotuzumab and a fragment of eEGFR, composed of amino acids 310 to constant at 5 Ag/mL, whereas the concentration of Cetuximab was grad- 501, were run using the program RosettaDock (36). The structure of the ually lowered to Nimotuzumab/Cetuximab concentration ratios ranging eEGFR fragment was extracted from PDB entry 1IVO (4). Each run (in from 10 1 to 104. Alkaline phosphatase-conjugated streptavidin (Serotec) perturbation mode) started from a different orientation of the Fv fragment was added at a dilution of 1:200 and incubated for 1 h at 37jC. The with respect to eEGFR-dIII. A grid of 28 points was created over a selected reaction was developed by adding 1 mg/mL of p-nitrophenyl phosphate region of eEGFR-dIII to anchor each of these orientations. In addition to the
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Nimotuzumab Blocks the EGFR by a Novel Mechanism crystal geometry, two alternative conformations of the flexible CDR H3, A second synthetic gene, called chEGFR-dIII-siteC, is a mutated version obtained from a 10-ns molecular dynamics (MD) simulation performed for of the dIII segment of the murine EGFR, in which seven codons cor- the Nimotuzumab Fv fragment, were used in the docking runs. More than responding to amino acids located within the epitope recognized by 300,000 decoys were produced in total, in several docking rounds. Finally, we Cetuximab were replaced by their human counterparts. This gene was used obtained a small funnel (in the energy versus RMSD landscape) of Fv to substitute the dIII in the chEGFR-III construction, obtaining the chimera orientations, which is a sign of a possible true solution (36). named chEGFR-siteC. Both the chEGFR-III and chEGFR-siteC chimera were cloned into MD simulations for the Fv/eEGFR-dIII complex the expression vector pcDNA3 (Invitrogen). BHK-21 cells (ATCC CCL-10) Aiming to improve the stereochemistry of the final docking decoys, we were transfected using polyethylenimine. Transfected cells were stained ran a 10-ns MD simulation, taking the structure of the lowest energy with Nimotuzumab and binding was revealed with a FITC-goat anti- complex as starting geometry. The simulations were performed with the human IgG antibody (Sigma-Aldrich). The analysis was performed complex embedded in a water box, using the program NAMD (37). Ap- using a fluorescence-activated cell sorting (FACS) scan flowcytometer proximately after the first nanosecond, the Nimotuzumab Fv fragment was and the CellQuest program (Becton Dickinson). CT-26 cells (ATCC accommodated in a position slightly displaced from the starting geometry, CRL-2638) were used as a control for the negative binding of Nimotuzumab where it remained for the rest of the simulation. The fluctuations of the to the murine EGFR. A polyclonal anti-mouse EGFR serum (produced whole Fv fragment around the average binding position were very small, at CIM) and a FITC-goat anti-mouse immunoglobulin antibody (Jackson) with RMSDs for the Ca carbons in the order of 1.5 to 3.0 A˚. More sig- were used to confirm the presence of the murine EGFR on the surface of nificantly, CDR H3, which plays the most important role in the interaction these cells. with eEGFR-dIII (as detailed below), remained practically fixed after the first nanosecond, with deviations in the order of 1 A˚. Overall, the whole binding Antibody numbering scheme interface between the two molecules showed a remarkable stability. As the We have adopted the Kabat numbering scheme for the antibody variable final model, we selected a representative frame from the MD trajectory. regions, which for this particular antibody introduced insertion letters after positions 52 (52A), 82 (82A–82C), and 100 (100A–100F) in the heavy chain Construction, expression, and analysis of EGFR mutants sequence, and after position 27 (27A–27E) in the light chain sequence. The genes coding for EGFR mutants were chemically synthesized by the company Geneart (Geneart AG). The gene called chEGFR-III is a chimeric version of the murine EGFR (Genbank accession code: AK004944.1), in Results which the nucleotide sequence coding for domain III was replaced by the corresponding sequence from the human EGFR (Genbank: AK290352.1). A Crystal structure of the Nimotuzumab Fab. The crystal fewsubstitutions were introduced to keep five murine amino acids (at structure of the Nimotuzumab Fab fragment was determined at ˚ positions 324, 337, 340, 369, and 390). As a result, 16 amino acids were 2.5 A resolution and refined to crystallographic R/Rfree factors of mutated to their human counterparts (at positions 353, 359, 366, 388, 418, 443, 21.5% and 28.8%, respectively, with good stereochemistry and no 460, 461, 467, 468, 471, 473, 474, and 478–480). outliers in the Ramachandran plot. For the Fv region of the Fab, the
Figure 1. Crystal structure of the Nimotuzumab Fab fragment. Both panels show a cartoon representation of the antibody, with the light chain colored in wheat and the heavy chain in green. The helix at the base of CDR H1 is highlighted in magenta. A, overall view of the Fab fragment. B, VH domain of Nimotuzumab superimposed onto VH domains of 15 other antibodies (blue ribbons), which include the VH reference used for superposition (PDB entry 1MJU) and 14 structures randomly chosen from the list of 984 PDB VH domains. The conserved Trp H103 is shown explicitly for the 15 PDB structures (slate blue). For Nimotuzumab, this position is occupied by Phe H100F (red), whereas Trp H103 (red) has been displaced downwards. A distorted helix in this part of the backbone compensates for the amino acid shift. www.aacrjournals.org 5853 Cancer Res 2009; 69: (14). July 15, 2009
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Cancer Research electron density is very well-defined for a structure determined to Nimotuzumab and Cetuximab Fab fragments to the immobilized 2.5 A˚ resolution. In the constant domains, close to the C-terminal eEGFR by surface plasmon resonance (SPR). The values obtained regions of both the heavy and light chains, three loops were dif- for the dissociation constant (KD) and for the association and dis- ficult to trace due to discontinuities in the electron density map. sociation rate constants (kon and koff, respectively) for Nimotuzumab 8 4 1 The variable domains are involved in a number of crystal contacts, were as follows: KD =2.1 10 mol/L, kon =5.2 10 (s mol/L) , 3 1 9 whereas only few symmetry contacts were found for the constant koff =1.1 10 s ; whereas for Cetuximab: KD =1.8 10 mol/L, 6 1 3 1 domains. Two PEG fragments from the crystallization solution kon =3.1 10 (s mol/L) , koff =5.8 10 s . The KD value were found to be bound to the Nimotuzumab Fab in the crystal obtained for the Cetuximab Fab is practically the same as the value 9 structure (data not shown). reported previously (2.3 10 mol/L; ref. 9). The KD of Distinct structural features of Nimotuzumab. The overall Nimotuzumab is one order of magnitude higher due mainly to its crystal structure of the Nimotuzumab Fab is similar to the struc- much lower kon, whereas its koff is only slightly lower. tures of other Fab fragments, except for two distinct features, both In addition, we performed SPR/Biacore experiments in which in the variable domain of the heavy chain (VH). An evident pe- EGF was immobilized on a CM5 chip and different concentrations culiarity concerns CDR H1, which was expected to adopt one of the of either Nimotuzumab or Cetuximab were applied together with a canonical conformations described for this loop (38), but exhibits constant concentration of eEGFR. Figure 2B shows the inhibition instead a short a-helix involving residues H25-H32 (see Fig. 1A). A curves obtained for both antibodies, revealing a difference in their search among all the immunoglobulin VH domain structures inhibition properties that is in agreement with their different deposited in the Protein Data Bank yielded only two antibodies dissociation constants. having a similar helix at the base of CDR H1 [PDB entries 1B2W (39) and 2CMR (40)]. The second unusual feature of the Nimotuzumab structure concerns Trp H103, a residue that plays an important structural role in antibodies. This residue is shifted by three amino acids toward the C-terminus of the VH domain (Fig. 1B), whereas its structurally conserved position is instead occupied by Phe H100F. This change has several important consequences for the structure of the entire variable region. First, the CDR H3 loop is ‘‘pulled down’’ from its C-terminal end, so that its length in effect is shortened by three amino acid residues. This displacement is compensated for by an unusual distorted helix involving amino acids H101-H105, which allows the last seven residues of VH (H107- H113) to keep their conserved positions in the structure. As shown in Fig. 1B, Trp H103 in Nimotuzumab (colored red in the figure) is situated in the middle of this helix, with the tip of its side chain pointing toward the interface between the variable and the constant domains. As a consequence of the distorted helix created around Trp H103, the conformation of the VH framework 1 is severely affected. Specifically, the side chain of residue Gln H6 is pushed away from its conserved position, whereas the five N- terminal residues are not visible in the electron density map. Superimposition of all the crystal structures of the VH domains found in the PDB (984, from 680 entries) revealed that only one antibody [PDB code 1FH5 (41)] has the Trp H103 residue placed differently, toward the interior of the VH domain (see Fig. 1B; single side chain colored slate blue). Also in this case, the conformation of framework 1 is disrupted and the structural role of Trp H103 is taken over by Tyr H102. Binding studies. In a previous report (14), we showed that Nimotuzumab competes with EGF for binding to the EGFR. More recently, the crystal structure of Cetuximab in complex with the eEGFR (9) revealed that this antibody, which also blocks EGF binding, interacts exclusively with domain III. We therefore decided to test whether Nimotuzumab competes with Cetuximab for binding to eEGFR, as a first step in locating its binding epitope. The first test was carried out by ELISA, coating Figure 2. Competition experiments performed for Nimotuzumab. A, ELISA the plate with eEGFR and adding different dilutions of the plates were coated with 5 Ag/mL eEGFR and incubated with different mixtures containing a constant concentration of biotinylated Nimotuzumab Cetuximab Fab fragment while keeping constant the concentration Fab fragment and decreasing concentrations of the Cetuximab Fab fragment. of the Nimotuzumab Fab. Figure 2A shows that Nimotuzumab OD, optical density. B, SPR/Biacore experiments showing the inhibition of EGF binding decreases as the concentration of the Cetuximab Fab binding to eEGFR with increasing amounts of Nimotuzumab (!) or Cetuximab (.). Each antibody was added in molar excess to a constant concentration increases. of 600 nmol/L of eEGFR and flowed over the EGF molecules immobilized to a We also performed kinetic measurements of the binding of level of 500 response units (RU) on a CM5 chip.
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Nimotuzumab Blocks the EGFR by a Novel Mechanism
Figure 3. Structure of the predicted Nimotuzumab/eEGFR complex. A, overall view of the model, showing also the strong overlap in binding positions between Nimotuzumab and Cetuximab. The figure was made by superimposing the EGFR-dIII fragment of the computer model onto the eEGFR-dIII domain of the Cetuximab/ eEGFR complex (PDB entry 1YY9), in which the eEGFR adopts an inactive conformation. Nimotuzumab is shown in a cartoon representation with transparent molecular surface (VL in wheat and VH in green), whereas Cetuximab is represented as a red cartoon. B, detailed representation of the molecular interface between Nimotuzumab and eEGFR. The antibody is positioned in the front, with VH (CDRs H1 and H3) in green and VL (CDRs L1, L2) in wheat. eEGFR is in the back, in light blue. The side chains at the interface are shown as sticks. Labels in black capital letters correspond to Nimotuzumab residues, whereas those in magenta concern eEGFR. eEGFR residues that differ between the human and murine versions of the EGF receptor are marked in orange. Black dashed lines, direct intermolecular hydrogen bonds; pink dashed lines, water-mediated hydrogen bonds. C, blocking of EGF binding to EGFR by Nimotuzumab, while permitting the active receptor conformation. Our computer model was overlaid onto the EGF/eEGFR complex (PDB entry 1IVO) by superimposing the eEGFR-dIII fragments of the two structures. eEGFR in its active conformation is shown in blue, with its domains marked by roman numerals. EGF is in red and Nimotuzumab’s VH and VL domains are in green and wheat, respectively. The figure shows the C-terminus of EGF bumping into the light chain of Nimotuzumab, whereas domain I of the EGFR approaches the antibody, without clashing. D, Cetuximab (light blue and yellow) blocks the binding of EGF (red) and clashes with domain I, preventing the EGFR (blue)from adopting its active conformation. The figure was prepared based on PDB entries 1YY9 and 1IVO.
The model of the Nimotuzumab-EGFR complex: correspon- agreement with our experimental data, the Nimotuzumab binding dence with the experimental data. We decided to construct a epitope strongly overlaps with the binding surface recognized by computer model of the Nimotuzumab-eEGFR-dIII complex, in Cetuximab, as shown in Fig. 3A. The model shows a remarkable parallel to ongoing efforts to obtain the crystal structure of this geometric and chemical complementarity between Nimotuzumab molecular complex. According to the obtained model, and in and the EGFR, with a high number of hydrogen bonds, as www.aacrjournals.org 5855 Cancer Res 2009; 69: (14). July 15, 2009
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Figure 4. Recognition of the EGFR chimera by Nimotuzumab, measured by FACS. In the dot plots shown in the first three panels, the Y axis corresponds to size scattering. A, no recognition of BHK-21 cells, used for expression of the EGFR chimera. B, recognition of cells transfected with the chEGFR-III chimera, in which 16 of the 21 amino acids that differ between the murine and human versions of EGFR-dIII were humanized. C, recognition of cells transfected with the chEGFR-siteC chimera, where seven residues located in the Nimotuzumab binding epitope were humanized. D, bar graph showing the recognition level of control and transfected cells by Nimotuzumab (in percent of marked cells). The CT-26 cells constitutively express the murine EGFR (mEGFR+). illustrated in Fig. 3B. A total area of 1,584 A˚ 2 becomes buried upon H2, in contrast, stays far from the EGFR surface. In the light chain, Nimotuzumab binding to eEGFR, slightly less than the area a total of seven residues interact with the EGFR, two from CDR L1 buried in the Cetuximab-EGFR complex (1,770 A˚ 2;ref.9),which (Asn L28 and Tyr L32), four from CDR L2 (Tyr L49, Lys L50, Phe fits well with the higher affinity of Cetuximab. The overall shape L55, and Ser L56) and Leu L46 at the base of CDR L2. In addition, complementarity parameter (42) calculated for our model, there are three water molecules buried in the binding interface, however, is better than the one reported for the Cetuximab- mediating indirect hydrogen bonds between the antibody and the EGFR complex (0.78 versus 0.71; ref. 9). receptor. Most of the contacts of Nimotuzumab with eEGFR domain III The epitope recognized by Nimotuzumab on EGFR domain III, are accounted for by the heavy chain. CDR H1 contributes to according to our model, involves 20 residues comprised within amino binding with only two residues: Tyr H27 and Tyr H32, the first of acids 441 and 477 (i.e., within the last 40 residues of domain III, which them being positioned in the helix turn at the base of CDR H1. It is spans amino acids 310–481). All the amino acids within the sequence CDR H3 which contributes the majority of the interactions, with stretch from residues 462 to 474 are involved in contacts to Nimo- seven consecutive residues (from H98 to H100D) being highly tuzumab, whereas the rest of the interacting residues correspond to involved both in hydrophobic contacts and in hydrogen bonds with sequence positions 441, 443, 449, 450, 453, 457, and 477. None of the the receptor. Further contacts with the EGFR are provided by Asp saccharide chains in any of the reported EGFR crystal structures is H101 and Arg H94 (at the base of CDR H3; data not shown). CDR close to this epitope.
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Nimotuzumab Blocks the EGFR by a Novel Mechanism
One important experimental fact to take into account in In our model, 5 of the 20 amino acids constituting the Nimotuzumab analyzing the model is that Nimotuzumab practically does not epitope correspond to sequence variations between the human and recognize the murine EGFR (43), despite the high sequence simi- murine EGF receptors: I467M, S468N, G471A, N473K, and S474D larity with the human receptor (88% sequence identity for domain III). (Fig. 3B). Notably, these five amino acids also belong to the epitope
Figure 5. Nimotuzumab and Cetuximab inhibit EGFR-mediated signaling by different mechanisms. Cetuximab binding to the EGFR completely abrogates EGFR signal transduction by inhibiting both ligand-dependent and ligand-independent EGFR dimerization, by blocking EGF binding and sterically interfering with the active, untethered receptor conformation (the clash between Cetuximab and the EGFR in its active conformation is marked with a red circle). Nimotuzumab, on the other hand, binds to the EGFR and blocks EGF binding, but allows the EGFR to adopt its active conformation. By interfering only with ligand-dependent EGFR activation, Nimotuzumab strongly reduces EGFR signaling to a basal, ligand-independent level. www.aacrjournals.org 5857 Cancer Res 2009; 69: (14). July 15, 2009
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Cancer Research recognized by Cetuximab. The most critical mutations, according to C-terminus of eEGFR domain III, compared with the Cetuximab the model, are the substitutions Ser 468!Asn (because there is no binding site (Fig. 3A). In this position, Nimotuzumab sterically room for the slightly larger side chain of asparagine), and Gly 471!Ala interferes with EGF binding, while permitting eEGFR domain I to (because the a carbon of glycine is situated within a very tight approach domain III and adopt the active conformation (Fig. 3C). environment). The prediction that Nimotuzumab may inhibit EGF binding Mutagenesis studies supporting the modeling results. Two without precluding the active conformation of the receptor in- human/murine EGFR chimera were constructed, in which different troduces an important and unexpected element to the mechanistic groups of amino acids in domain III were mutated to their human study of this antibody, and provides a rationale for several results counterparts (were ‘‘humanized’’). In one of the chimera, called obtained in the clinics. As explained in the Introduction, the other chEGFR-III, 16 of the 21 amino acids that differ between the murine anti-EGFR antibodies for which the molecular inhibition mecha- and human versions of EGFR-dIII were humanized. The five un- nisms are known create steric impediments, either to the for- changed residues were either located on a side opposite to the mation of the active receptor conformation needed for dimerization Cetuximab epitope, or shielded by the carbohydrate chains. In the [e.g., Cetuximab (9) and Matuzumab (12)] or directly for the di- second chimera, called chEGFR-siteC, only the epitope recognized merization event [e.g., mAb 806 (13)]. Nimotuzumab, on the other by Cetuximab was humanized (mutated positions: 418, 443, 467, hand, is able to maintain the existing balance between the tethered 468, 471, 473, and 474). From these seven amino acids, the last five and extended EGFR conformations. belong to the Nimotuzumab epitope, according to our computer As pointed out above, it has been estimated that without ligand- model (Fig. 3B). induced activation, only a very small fraction (<15%) of the EGFR The constructed EGFR chimera were cloned for transient ex- molecules adopts the energetically less favorable active conformation pression in BHK-21 cells, and the transfected cells were then (7, 8). On the other hand, there are several reports demonstrating the tested by FACS for recognition of Nimotuzumab. As shown in existence of a ligand-independent level of EGFR activation, both in Fig. 4, cells expressing the chEGFR-III chimera were recognized by tumor and normal cells (44, 45). Nimotuzumab would not interfere Nimotuzumab. The antibody also bound, with similar intensity, to with this basal level of EGFR signaling, which is needed for the the cells expressing the chEGFR-siteC mutant (Fig. 4C), in which the survival of normal epithelial cells (46), because its binding is com- humanized surface patch allowing Nimotuzumab binding is more patible with the active conformation of the receptor. A comparison restricted. between the inhibition mechanism followed by Cetuximab and the Nimotuzumab blocks EGF binding without inhibiting the mechanism proposed here for Nimotuzumab is depicted in Fig. 5. active eEGFR conformation. As shown in a previous report (14) The lowdegree of adverse effects observed for Nimotuzumab and also in this work, Nimotuzumab and EGF compete for binding in the clinics has been attributed to its intermediate affinity, to the EGFR. To confirm that our computer model complies with compared with other anti-EGFR antibodies, e.g., Cetuximab (15). this experimental fact, we superimposed the eEGFR structure in Another possible explanation given by our model is that complex with EGF (PDB 1IVO; ref. 4) onto our model. The result is Nimotuzumab is less toxic for normal epithelial cells because it illustrated in Fig. 3C. Intriguingly, only the four C-terminal residues does not disrupt the basal level of EGFR signaling. This reasoning of EGF (of which the last two are not included in the crystal structure further suggests that an antibody having a higher affinity than of the EGFR-EGF complex) overlap with the Nimotuzumab Fv Nimotuzumab, but acting by the same mechanism, might also fragment, bumping into CDR L2. Even more remarkable is the fact showa lowtoxicity. A simple inspection of the structure of the that, in contrast to Cetuximab (see Fig. 3D), the binding of Nimo- EGFR-EGF complex reveals that a similar mechanism of action tuzumab is compatible with the extended, active conformation of could be exploited by binding to domain I of the EGFR. EGFR. As shown in Fig. 3C, the Nimotuzumab variable light chain Although the final validation of the model requires additional (i.e., CDR L1) approaches domain I of the EGFR in the superimposed experiments, its remarkable geometric and chemical complemen- structures, without clashing into it. tarity, the stability shown along extensive MD simulations and, more importantly, the correspondence with the available experi- mental data support its reliability. Most significantly, the proposed Discussion model has opened the door to newexperiments, designed not only Nimotuzumab is one of the very fewanti-EGFR monoclonal to validate its atomic details but foremost directed to corroborate antibodies that have been approved for therapeutic use in cancer the predicted mechanism of action of Nimotuzumab. treatment. Clinical trials with this antibody, involving >2,000 patients worldwide, have shown that the classic side effects Disclosure of Potential Conflicts of Interest observed for antibodies and drugs that inhibit the EGFR signaling pathway (19) are negligible in the case of Nimotuzumab. This No potential conflicts of interest were disclosed. unique safety profile allows a longer term treatment and provides a better quality of life to patients. In this work, by combining Acknowledgments several experimental and computational techniques, we have provided newmechanistic evidence that may explain the low Received 12/3/08; revised 3/27/09; accepted 5/8/09; published OnlineFirst 7/7/09. Grant support: Center of Molecular Immunology, Chalmers University of toxicity observed for Nimotuzumab. Technology, the University of Oslo as well as by grants from the Glycoconjugates in We have shown that Nimotuzumab binds to domain III of the Biological Systems program from the Swedish National Foundation for Strategic extracellular region of the EGFR, as is the case for several other Research (research position of Ute Krengel) and from the Norwegian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page known anti-EGFR antibodies. Moreover, we have confirmed by site- charges. This article must therefore be hereby marked advertisement in accordance directed mutagenesis that the Nimotuzumab epitope strongly with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Yngve Cerenius for support at the MAXII synchrotron in Lund, as well as overlaps with the Cetuximab binding site. According to our model, Magnar Bjøra˚s for his support at the Center for Molecular and Behavioral the epitope recognized by Nimotuzumab is displaced toward the Neuroscience, at Rikshospitalet in Oslo.
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Nimotuzumab Blocks the EGFR by a Novel Mechanism
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www.aacrjournals.org 5859 Cancer Res 2009; 69: (14). July 15, 2009
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Correction: Article on Nimotuzumab Blocks the EGFR by a Novel Mechanism
In the article on how Nimotuzumab blocks the EGFR by a novel mechanism in the July 15, 2009issue of Cancer Research (1), the last footnote in Table 1 should read as follows: "kRMSD, root mean square deviation from ideal geometry (47)."
1. Talavera A, Friemann R, Go´mez-Puerta S, Martinez-Fleites C, Garrido G, Rabasa A, Lo´pez-Requena A, Pupo A, Johansen RF, Sa´nchez O, Krengel U, Moreno E. Nimotuzumab, an antitumor antibody that targets the epidermal growth factor receptor, blocks ligand binding while permitting the active receptor conformation. Cancer Res 2009;69:5851–9.
I2009American Association for Cancer Research. doi:10.1158/0008-5472.CAN-69-16-COR3
Cancer Res 2009; 69: (16). August 15, 2009 6758 www.aacrjournals.org Published OnlineFirst July 7, 2009; DOI: 10.1158/0008-5472.CAN-08-4518
Nimotuzumab, an Antitumor Antibody that Targets the Epidermal Growth Factor Receptor, Blocks Ligand Binding while Permitting the Active Receptor Conformation
Ariel Talavera, Rosmarie Friemann, Silvia Gómez-Puerta, et al.
Cancer Res 2009;69:5851-5859. Published OnlineFirst July 7, 2009.
Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-08-4518
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