PEDS Advance Access originally published online on May 8, 2006
Protein Engineering Design and Selection 2006 19(7):317-324; doi:10.1093/protein/gzl015
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Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody
Department of Research, Biogen Idec, Inc. 12 Cambridge Center, Cambridge, MA 02142, USA
2To whom correspondence should be addressed. E-mail: ann.boriack-sjodin{at}biogenidec.com
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Abstract |
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Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 Å structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody–chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.
Keywords: Antibody engineering/monocyte chemoattractant proteins/protein-antibody complex/protein therapeutics/x-ray crystallography
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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Chemokines are proinflammatory molecules that coordinate the host immune response by promoting leukocyte migration to the site of infection or injury (Rossi and Zlotnik, 2000
; Zlotnik and Yoshie, 2000). These molecules are thought to bind to and cluster on glycosaminoglycans, resulting in a locally high concentration of chemokine which triggers recruitment of leukocytes bearing chemokine receptors (Rot, 1993; Chakravarty et al., 1998; Proudfoot et al., 2003). The chemokine superfamily is currently comprised of more than 50 members that can be classified based on the arrangement of their cysteine residues into four subfamilies: CXC, C, CX3C and CC (Rollins, 1997). Monocyte chemoattractant proteins (MCPs) are members of the CC family of chemokines with five isoforms in humans: MCP-1-4 and eotaxin. Human MCPs bind to G-protein coupled receptors CCR1, CCR2, CCR3, CCR5 and CCR10, with multiple MCPs binding to multiple CCRs.
The structures of many chemokines have been solved by protein crystallography and NMR spectroscopy, including MCP-1, MCP-2 and MCP-3 (Handel and Domaille, 1996; Kim et al., 1996; Lubkowski et al., 1997; Meunier et al., 1997; Blaszczyk et al., 2000). The structures are extremely similar to each other and share the same topology as related CC chemokines. The structures consist of an N-terminal loop region, a three-strand antiparallel ß sheet forming part of a ‘Greek key’ motif and a C-terminal 
helix which overlays the ß sheet. The structure also contains two disulfide bridges. The crystal structure of MCP-2 contains a pyroglutamic acid at the N-terminus which increases the chemotactic activity of the cytokine (Blaszczyk et al., 2000). A monomeric mutant of MCP-1 (Pro58
Ala) has also been solved in complex with M3, a herpesvirus decoy receptor (Alexander et al., 2002).
MCP-1 is one of the most studied members of the chemokine family and has been shown to be a potential intervention point for the treatment of a variety of diseases, including multiple sclerosis (Sorensen et al., 2004), rheumatoid arthritis (Hayashida et al., 2001), atherosclerosis (Kusano et al., 2004) and insulin-resistant diabetes (Sartipy and Loskutoff, 2003). The MCP pathway has been validated through numerous animal models of disease using MCP-1 knockouts (Gu et al., 1998; Lu et al., 1998), CCR2 knockouts (Kurihara et al., 1997; Boring et al., 1998; Dawson et al., 1999), or monoclonal antibodies against MCP-1 or CCR2 (Fujinaka et al., 1997; Karpus and Kennedy, 1997; Lloyd et al., 1997; Kimura et al., 1998; Campbell et al., 1999; Furukawa et al., 1999; Ono et al., 1999; Schneider et al., 1999). Because members of the MCP family bind and share multiple receptors, it has been speculated that an inhibitor that blocks two or more MCP isoforms would be a strong therapeutic candidate.
11K2 is a monoclonal antibody (mAb) raised against human MCP-1 (hMCP-1) that also binds with high affinity to hMCP-2 and to murine MCP-1 (mMCP-1) and mMCP-5. This mAb has proven effective in treating murine models of atherosclerosis (Lutgens et al., 2005) and ulcerative colitis (de Fougerolles, A.R., Mencarelli, A., Sprague, A.G., Cachero, T.G., Jarpe, M., Kotelianski, V.K., Rennert, P.J. and Fiorucci, S. in preparation). It is thought that the ability of this mAb to inhibit the activity of mMCP-1 and mMCP-5 contributes to its activity in these models. In order to determine the basis for the high affinity and broad selectivity of 11K2, we undertook a structural analysis of hMCP-1 with the Fab fragment of 11K2 (11K2Fab).
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Materials and methods |
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MCP-1 synthesis and purification
The 76 amino acid peptide of hMCP-1 was synthesized by established automated protocols on an Applied Biosystems 433A Peptide Synthesizer using Fmoc-protecting amino acids and HBTU/HOBt/DIEA-mediated activation in DMF (Fields et al., 1991). Peptide cleavage/deprotection was accomplished with mixture of 91% TFA, 3% H2O, 3% EDT, 3% TIS for 3.5 h at room temperature. Soluble peptide was precipitated, rinsed with cold ethyl ether, then dissolved in 95% aqueous acetic acid and lyophilized. The protein was purified by reverse-phase HPLC (>98% purity) on a Varian 210 HPLC. MALDI-MS of unfolded, purified protein was performed on an Applied Biosystems STR Voyager mass spectrophotometer. The purified MCP-1 synthetic peptide was lyophilized and then redissolved at 1 mg/ml in 2 M guanidine–HCl, 100 mM Tris, pH 8.0, containing 8 mM cysteine and 1 mM cystine. The protein was allowed to fold for 48 h at room temperature while rocking gently. The folded MCP-1 protein was then purified by reverse-phase HPLC, lyophilized and finally redissolved at 10 mg/ml in 25 mM Tris, pH 8.4. About 90–95% of the protein was refolded properly as measured by mass spectrometry and chromatographic analysis and the protein was tested in a chemotaxis assay and found to be active.
MCP expression and purification
In order to efficiently make small amounts of a large number of MCP mutants, an Escherichia coli expression system utilizing the refolding methods of the synthetic protein was used. The MCP mutants listed in Tables II and III were made by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, catalog no. 200518). Mutagenesis reactions were performed according to the manufacturers protocol using 50 ng of template and 150 ng each of oligonucleotide primers. Oligonucleotide primers used to generate the MCP mutants can be found in the Supplementary data available at PEDS online. Native hMCP-1, hMCP-2, hMCP-3 and MCP mutants were expressed in E.coli using the pET11 vector (Novagen) encoding the mature MCP sequence in either BL21 (DE3) or Rosetta (DE3) cells. For some mutants SM102, a plasmid encoding Arg-AAG/A tRNA, was used to facilitate expression. Cells were lysed and inclusion bodies were isolated by a differential centrifugation-detergent wash procedure. The washed inclusion bodies were solubilized in 8 M urea, 0.5 mM EDTA, 10 mM DTT and purified using reverse-phase chromatography. The resulting product was lyophilized and refolded according to the procedure above. Owing to expression difficulties, hMCP-3 Thr56Lys was synthesized (Cell Sciences) and then refolded.
Antibody mutation, expression and purification
11K2 was generated from hybridomas and purified by affinity chromatography using Protein A resin. The Fab fragment of 11K2 was generated by digestion with papain. Briefly, 200 mg of 11K2 monoclonal antibody were incubated in digestion buffer (20 mM sodium phosphate, pH 7.2, 10 mM EDTA and 20 mM Cysteine–HCl) in the presence of 5 ml of immobilized papain beads (Pierce) at 37°C for 18 h with shaking. The digested antibody was diluted with 4 vol of high salt binding buffer (1.5 M glycine, 3 M sodium chloride) to increase the affinity of murine IgG1 for Protein A. The solution was loaded onto a 10 ml Protein A column to deplete the digested Fc fragments. This process was performed three times. The Fab fragment of 11K2 antibody was identified by SDS–PAGE and dialyzed against phosphate-buffered saline. The purified 11K2 Fab was further characterized by mass spectrometry and found to be the expected mass.
Antibody mutants were designed using plasmids pCR060 (light chain) and pCR101 (heavy chain). Oligonucleotide primers used to generate the 11K2 mutants can be found in the Supplementary data available at PEDS online. Antibody mutants were expressed in HEK293e cells by co-transfection of plasmid DNAs encoding the heavy and light chains. Light chain mutants were co-expressed with wild-type heavy chain, and heavy chain mutants were co-expressed with the wild-type light chain. All antibodies were expressed as chimeric Fab fragments bearing murine variable regions fused to human constant (IgG1, kappa) regions. Transfections were performed in 10 cm dishes using Effectene reagent according to the manufacturer's protocol (Qiagen catalog no. 301427). Supernatants containing secreted antibodies were harvested 3 days post-transfection.
Crystallization and structure determination
Synthetic hMCP-1 and 11K2Fab were mixed in a 1:1 molar ratio and concentrated to 8 mg/ml. Two microliters of the protein was mixed with an equal amount of reservoir buffer containing 13% PEG 4000, 100 mM HEPES, pH 7.5, 30 mM glycyl-glycyl-glycine using vapor diffusion techniques. Crystals typically appeared after 1–2 days with microseeding and grew to full size (0.1 x 0.1 x 0.1 mm) in 1–2 weeks. Crystals were briefly incubated in 15% PEG 4000, 100 mM HEPES, pH 7.5, 30 mM glycyl-glycyl-glycine, and 25% glycerol and flash frozen in liquid nitrogen prior to data collection. Data were measured at 100°K at Brookhaven National Laboratory (beamline X4A) using a Quantum4 CCD detector (ADSC) at wavelength 0.97945 Å. Data processing was performed using Denzo, and data reduction was performed using Scalepack (Otwinowski and Minor, 1997). The crystals contain one MCP-1 monomer–11K2Fab complex per asymmetric unit and belong to space group C2221 (a = 86.4 Å, b = 89.1 Å, c = 176.2 Å). Molecular replacement was performed using AMoRe (Navaza, 1994) using a model of 11K2Fab generated by homology modeling with Modeller (Sali and Blundell, 1993) using the light chain of 184.1 [anti-OspA; PDB code 1OSP
[PDB]
(Li et al., 1997)] and the heavy chain of 5G9 [anti-human tissue factor; PDB code 1FGN (Huang et al., 1998)] as the templates. The top solution in both the rotation and translation searches was determined to be the correct solution after rigid body refinement of the individual Fab domains resulted in a 8.8% point drop in the Rfree value and electron density maps clearly revealed the position of the MCP-1 monomer. Model building was performed using O (Jones et al., 1991) iteratively with simulated annealing refinement in CNX (Brunger et al., 1998) with bulk solvent corrections and anisotropic B-factor scaling protocols to achieve the final structure. The final protein model has been refined at 2.5 Å resolution to a final R-value of 0.219 and free R-value of 0.277 (Table I). The Ramachandran plot shows that 85.6% of all residues are in the most favored regions and no residues are in disallowed regions. The coordinates have been deposited at the Protein Data Bank (2BDN).
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Affinity measurements
The solution phase equilibrium dissociation constants of 11K2 Fabs binding to MCPs were determined using the KinExA 3000 instrument (Sapidyne Instruments, Boise, ID, USA). MCP-1 human IgG1–Fc fusion protein was coated onto 98 µ polymethylmethacrylate beads. These beads were packed into a small glass column in the instrument, and purified 11K2Fab or supernatants from Fab expressing cells were flowed over the beads. The captured Fab was detected with a Cy-5 labeled anti-murine F (ab)'2 antibody (Jackson ImmunoResearch, West Grove, PA, USA). The signal detected was proportional to the amount of free Fab that flowed over the column.
To measure the solution phase binding affinity, Fab was incubated with varying concentrations of purified antigen. The mixtures were incubated at room temperature for 3 h to allow them to come to a steady state. The amount of free Fab was measured using the KinExA. The resulting data were fit to a quadratic equation using the KinExA software and the dissociation constants were determined as well as the percent error from the least squares fit.
First, the binding of purified wild-type 11K2Fab to purified wild-type and mutant MCPs was measured. However, it was unfeasible to purify the large number of Fabs expressed in mammalian cells in order to measure the affinity of mutant 11K2Fabs to MCPs. The KinExA method measures the loss of unbound Fab in solution as a function of the concentration of antigen. For this reason unpurified supernatants of mammalian cells expressing Fabs were able to be used rather than purified protein. To validate this approach the affinities of purified 11K2Fab and unpurified supernatant containing 11K2Fab to wild-type hMCP-1 were compared. The two values, 4.6 and 7.5 pM, respectively, are not significantly different.
Chemotaxis inhibition
THP-1 human monocytic cells were grown in RPMI, 10% FBS with 4 mM L-glutamine. To measure chemotaxis, ChemoTX plates from Neuroprobe were used. Chemokines and 11K2Fabs were incubated together for 30 min at 37°C to reach equilibrium. The chemokine, with or without Fab, was added to the bottom chamber of the plate. A filter was placed on top and 160 000 THP-1 cells were added to the top of the filter. The plates were incubated for 4 h at 37°C in a tissue culture incubator. After the incubation the cells and media were removed from the top of the plate, the plate was centrifuged briefly and the filter was removed. The cells that migrated into the lower chamber were quantified with Promega Cell Titer using a standard curve of THP-1 cells. The percent inhibition was calculated as the decrease in the number of cells migrated in the presence of Fab compared with the chemokine alone.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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Overall structure of MCP-1–11K2Fab
To understand the cross-reactivity of antibody–MCP binding, we determined the crystal structure of synthetic hMCP-1 complexed to the Fab fragment of the 11K2 mAb (Table I). The structure was solved using data from a single crystal by molecular replacement with a model of the Fab as the search model. The position, orientation and stoichiometry of MCP-1 were clear in the electron density maps calculated after rigid body and positional refinement of the Fab fragment. The asymmetric unit of the crystal contains one MCP monomer and one Fab molecule (Fig. 1A).
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In previous structures of MCP molecules, the chemokine is a dimer with the N-terminal residues of the protein forming the dimer interface. In this structure, the MCP-1 dimer is generated using crystallographic symmetry. Based on the structure, the MCP-1 dimer should bind to two 11K2 antibodies in solution, as the orientation of the Fab fragments does not allow for bivalent binding of a single antibody (Fig. 1B). This result is supported by light scattering studies in which two antibodies appear to bind one MCP-1 dimer, and by ELISA experiments showing that MCP-1 and MCP-2 can bridge two 11K2 mAbs together (data not shown).
Superposition of the complexed MCP-1 dimer with native structures [PDB codes 1DOL and 1DOK (Lubkowski et al., 1997)] shows that there are few changes to the overall structure of the MCP-1 monomer (CA RMSD = 0.93 and 0.96 Å over residues 4–70 for 1DOL and 1DOK, respectively), but the orientation of the monomers to each other has been slightly altered. Figure 1C shows that the orientation of the second MCP-1 monomer is within the variation seen with uncomplexed MCP-1 X-ray structures. 11K2 does not bind to the residues involved in dimer formation. Therefore, the changes in orientation of the second monomer relative to the first molecule are presumably owing to small local changes of the structures or effects of crystal packing rather than the binding of the antibody. The variability of dimer orientation in MCP-1 structures solved by X-ray and NMR has been commented on previously (Lubkowski et al., 1997).
11K2Fab binds to MCP-1 at a relatively flat region of the chemokine. The antibody binds to MCP-1 in a region consisting of the end of ß1, the ß1– ß2 loop, the beginning of ß2, the loop between ß3 and 1, and the 1 helix. The formation of the complex buries 
1550 Å2 of solvent exposed surface area and 15% of the MCP-1 surface is involved in the interface. Most of the contacts with MCP-1 are made through CDRs H1 and H3 of the heavy chain and CDRs L1 and L3 of the light chain. The center of the interface is dominated by hydrophobic interactions. Phe101 of the antibody heavy chain (Phe101H) is imbedded in a hydrophobic pocket of MCP-1 composed of Arg30, Thr32, Glu39, Val41, Pro55 and Met64 (Fig. 2A and B). The pocket is created by the movement of Arg30 about chi3 from its native conformation. Other than Arg30, very little movement of the MCP-1 residue side chains is needed to accommodate the insertion of the phenylalanine.
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. (C) Lys56 forms two hydrogen bonds with 11K2. (D) Negatively charged residues at the C-terminus of hMCP-1 interact with Arg32L of 11K2. (E) Glu39 interacts with 11K2 through polar interactions.The periphery of the interface is composed of hydrophilic salt bridge pairs and hydrogen bond interactions. Lys56 of MCP-1 is involved in a salt bridge–hydrogen bond interaction with Asp52 of the heavy chain (Asp52H) and makes an additional interaction with the Lys30H backbone carbonyl oxygen (Fig. 2C). Asp65 and Asp68 of the chemokine are forming hydrogen bonds with Arg32 of the light chain (Arg32L) (Fig. 2D). Glu39 makes a hydrogen bond with Thr32H and stacks against Arg98H (Fig. 2E). Multiple water-mediated and backbone hydrogen bonds are also made between the two molecules.
How 11K2 blocks CCR2 binding
The binding site of CCR2 on MCP-1 has been mapped to a region of the chemokine containing Tyr13, Arg24, Lys35, Lys38, Lys49 (Fig. 3) (Hemmerich et al., 1999). These residues form a discontinuous binding site that is mostly distinct from the binding site of 11K2. However, Lys38 is in van der Waals contact with the heavy chain of 11K2Fab. Given the close proximity of the 11K2 binding surface with the CCR2 binding surface, it is likely that the activity of 11K2 is owing to steric blockage of the MCP-1–CCR2 complex as opposed to directly overlapping binding sites.
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To confirm that the binding of 11K2 functionally blocks hMCP-1 and hMCP-2, we tested the effect on chemotaxis (Fig. 4). Human THP-1 monocytic cells migrated in response to hMCP-1, and less robustly to hMCP-2. Pre-incubating the chemokines with 11K2Fab resulted in the inhibition of chemotaxis. As expected the concentration of antibody at which 50% of the chemotaxis signal is inhibited is roughly equal to the concentration of chemokine used in the assay, since the concentrations of both chemokines that resulted in measurable chemotaxis are well above the KD for the binding interaction of the Fab with the chemokines.
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Mapping 11K2 specificity
The residues involved in the binding of Phe101H are identical between hMCP-1, hMCP-2 and hMCP-3; all residue differences between the three chemokines at the interface of the complex are located at the periphery of the interaction (Fig. 5). This was surprising, since hMCP-1 and hMCP-2 bind to 11K2Fab with subnanomolar affinity, while no binding is detected to hMCP-3. Therefore, peripheral residues are likely involved in the specificity of these interactions. Additionally, there are more residue differences between hMCP-1 and hMCP-2, the molecules that bind to 11K2 with high affinity, than between hMCP-1 and hMCP-3 or hMCP-2 and hMCP-3, which differ greatly in binding the antibody (Table II). This suggests that specific interactions by one or more residues of hMCP-1 and hMCP-2 with the antibody account for the tight binding of 11K2 to these chemokines. This hypothesis was tested by constructing a series of mutations to the MCP molecules and to 11K2Fab and then the affinity of the interactions was determined (Tables II and III). These mutation experiments are now described in sections labeled by the location in the chemokines or 11K2.
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Lysine 56 (MCP-1)
It was speculated that Lys56 of MCP-1 is important to 11K2 binding owing to the fact that most high-affinity MCP isoforms (hMCP-1, hMCP-2, mMCP-1 and mMCP-5) have a Lys at this position, whereas the isoforms with low affinity [hMCP-3, mMCP-2, rat MCP-1 (rMCP-1)] have different residue types (Thr, Thr and Asn, respectively). In this structure Lys56 forms two hydrogen bonds with the side chain of Asp52H and the backbone carbonyl of Lys30H of 11K2Fab. Mutation of Lys56 to Asn resulted in a 160-fold reduction in affinity, implying that this residue plays a key role in the selectivity profile of the antibody. Interestingly, mutation of Lys56 to Arg resulted in a 14-fold loss in activity (Table II). Therefore, the specific size and conformation of Lys, not just the positive charge, are important in this specific interaction.
Since the residue at position 56 of the chemokine is likely to be important in determining the selectivity against hMCP-3, this hypothesis was tested by mutating residue 56 on hMCP-3. As hypothesized, mutation of hMCP-3 Thr56 to lysine increased MCP-3 binding for 11K2Fab with an affinity of 1.7 nM for mutant hMCP-3 Thr56Lys versus no detectable binding to native hMCP-3. Therefore, while this result highlights the importance of Lys56 for the interaction with 11K2, it also underscores the importance of other interface residues, since the mutant MCP-3 (Thr56Lys) still has 4- and 350-fold lower affinity for 11K2 than hMCP-2 (430 pM) and hMCP-1 (4.6 pM), respectively.
Phenylalanine 101 (11K2 heavy chain)
At the center of the interface, Phe101H is inserted into a hydrophobic pocket of MCP-1. We mutated residues of MCP-1 and 11K2 to determine the importance of this phenylalanine and nearby residues that were expected to affect this interaction. Not surprisingly, mutation of Phe101H to arginine resulted in a complete loss of binding. Introducing a bulkier residue (Val100H Phe) near Phe101H also results in a 16 500-fold decrease in affinity (Table III).
To further explore this area of interaction we introduced several mutations in MCP-1. Replacing Glu39 with lysine disrupts the charge–charge interaction across the interface with Arg98H and introduces a longer side chain (Fig. 2E). Mutation of Val41 to lysine introduces a flexible and charged side chain into an area in the center of the hydrophobic pocket that is tightly packed and has no compensating negative charge. As a result these MCP-1 mutants have >1000-fold affinity losses (Glu39Lys, Val41Lys; Table II). An additional MCP-1 mutant, Pro55Phe, would not refold under the standard protocols indicating the stability of this variant protein was compromised. Therefore, the packing of this area is important for the stability of MCP-1 itself and is critical for the high affinity of the MCP-1–11K2Fab complex.
MCP-1 C-terminus
Residues involved in 11K2 binding at the C-terminus offer particularly interesting targets for mutagenesis. Some of these residues are different between hMCP-1 and hMCP-2, and they might explain the slight differences in affinity between hMCP-1 and hMCP-2 to 11K2. In hMCP-1, Asp65 and Asp68 form salt bridges with Arg32L of 11K2 (Fig. 2D). Interestingly, Asp65 is a lysine residue in hMCP-2 (and mMCP-1) and thus cannot form the same salt bridge. Mutation of Asp65 to lysine decreased affinity of this mutant by only 2.5-fold over wild-type. Because the side chain of Asp65 is solvent exposed, mutation to lysine may be relatively easily accommodated and results in a modest decrease on MCP-1 binding. Mutation of Lys65 to an aspartic acid in hMCP-2 increased affinity of hMCP-2 to 11K2 3.5-fold, which is consistent with the corresponding affinity decrease of the Asp65Lys mutation in hMCP-1 (Table II).
The salt bridge triad between Asp65 and Asp68 in hMCP-1 and Arg32L in 11K2 was further probed by making mutations in 11K2Fab. It was predicted that changing Arg32L to glutamic acid would decrease hMCP-1 binding because of a loss of the salt bridge, but perhaps increase hMCP-2 binding because a new salt bridge could be formed with Lys65. An Arg32L to tryptophan change was speculated to decrease 11K2Fab binding to both hMCP-1 and hMCP-2 because in addition to removing the salt bridge, a very bulky side chain was introduced. As predicted, the Arg32L Glu mutation had a very large effect on hMCP-1 binding (10 000-fold affinity reduction), but did not affect hMCP-2 binding dramatically (2.5-fold affinity reduction). Changing Arg32L to tryptophan decreased hMCP-1 binding, although only 6-fold, and surprisingly increased hMCP-2 binding 70-fold. The complex was able to accommodate the large tryptophan residue without severely disrupting binding, and in the case of hMCP-2 the introduced tryptophan picked up a significant interaction, perhaps with the side chain of Lys65.
Glutamate 39 (MCP-1)
The side chain of Glu39 in MCP-1 is inserted in a shallow pocket on the 11K2 heavy chain, and contacts the side chain of Arg98H on 11K2. It was hypothesized that reversing the charges on one or the other binding partner would destroy the binding, but that the charge-reversed mutants would bind one another with higher affinity than either mutant to the wild-type partner. Mutating the 11K2Fab residue Arg98H to aspartate caused a three order of magnitude loss in binding to wild-type hMCP-1 (Table III). Mutating the MCP-1 residue Glu39 to lysine decreased the binding to wild-type 11K2Fab by four orders of magnitude (Table II). However, much of the binding affinity was restored when these two mutant proteins were measured against one another (Table III; Fig. 6).
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Implications for therapeutic use
The 11K2 mAb has some unique properties that make it worthy of detailed structural study. Although it was raised in normal mice against hMCP-1, the mAb recognizes several members of the MCP family across species. Without any special efforts to break tolerance to self proteins, the immunized mouse produced a mAb that reacted against two mouse proteins with high affinity. 11K2Fab binds to mMCP-1 with an affinity of 300 pM and mMCP-5 with an affinity of 1.5 nM (Table II).
The chemokine system is characterized by redundancy and promiscuity, with multiple chemokines binding to the same receptors and individual chemokines capable of binding and activating multiple receptors. This redundancy may complicate chemokine blocking strategies from having clinical efficacy. One strategy to overcome this limitation is to design inhibitors that are specific for more than one receptor, or for more than one chemokine. In the ApoE–/– murine atherosclerosis model, transcript profiling identified both mMCP-1 and mMCP-5 as upregulated in diseased tissue. It was hypothesized that blocking both of these chemokines may be important in controlling disease (Lutgens et al., 2005). The unexpected modulation of binding affinity for hMCP-1 and hMCP-2 with the Arg32L Trp mutation of 11K2 illustrates the potential power of antibody engineering in modulating the binding of pan-antibodies to their target antigens.
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Footnotes |
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1Present address: Department of Research, Alnylam Pharmaceuticals, Inc., 300 Third Street, Cambridge, MA 02142, USA
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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We thank Charles R. Mackay for the initial 11K2 mAb, Rich Tizard and the Biogen Idec DNA sequencing lab for oligonucleotide synthesis and DNA sequencing, Ami Horne for assistance with crystallization, Randy Abramowitz and the staff at X4A for help in data collection, and Laura Silvian and Alan Corin for helpful discussions.
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Received November 29, 2005; revised March 2, 2006; accepted March 15, 2006.
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