Bioconjugate Chem. 2004, 15, 1389−1391
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COMMUNICATIONS Converting Weak Binders into Infinite Binders Todd M. Corneillie, Paul A. Whetstone, Kelvin C. Lee, Jeremy P. Wong, and Claude F. Meares* Department of Chemistry, University of California, Davis, California 95616. Received July 22, 2004
Monoclonal antibody 2D12.5 binds DOTA chelates of all the rare earths with Kd ≈ 10-8 M, making it useful for the capture of probe molecules with a variety of properties. To make 2D12.5 even more useful for biological applications, we have engineered a single cysteine residue at position 54 of the heavy chain, a site proximal to the protein’s binding site, so that weakly electrophilic metal complexes of (S)-2-(4-acrylamidobenzyl)-DOTA (AABD) may bind and form permanent linkages. At 37 °C, pH 7.5, all of the rare earth-AABD complexes bind permanently to the 2D12.5 G54C mutant within 5 min, in yields that correlate with their relative binding affinities. Surprisingly, indium-AABD also binds permanently in >50% yield within 5 min, despite the fact that changing the metal to indium reduces the affinity ≈100×; even copper-AABD, which has ≈10 000× lower binding affinity than the rare earths, binds permanently in >70% yield within 2 h. However, acrylamido compounds with no measurable affinity do not bind permanently. The important practical implication is that the G54C mutant of 2D12.5 may be used for applications that include not only the rare earths, but also an unexpected range of other elements as well. This infinite binding system can exhibit selective and permanent attachment with a remarkable range of structurally related ligands, albeit at slower rates as affinities decrease.
A ligand-receptor system that permanently forms a specific complex may have a number of applications in the delivery or sequestration of the ligand. The purposeful development of such systems is a new endeavor, and a number of basic questions remain to be answered. So far, the most promising results in combining specificity with permanent attachment have involved engineering a reactive cysteine side chain at a position proximal to the ligand-binding site of a protein, accompanied by synthesis of a ligand bearing an appropriate complementary reactive group such as acryl (1-3). The reactivity of the protein side chain is modulated by its surface accessibility and location, while the reactivity of the ligand may be tuned with the tools of synthetic chemistry; attachment can occur within minutes, so that complexes form permanently in good yield when ligand and receptor first associate (1). Two acryl compounds that irreversibly target specific cysteines on human epidermal growth factor receptors are now in clinical trials (4). Recently Chen et al. explored ligands that bind permanently to a specific histidine (5), and Pollack et al. previously exploited reversible binding to attach a nucleophile that introduced catalytic activity near an antibody binding site (6); both attachments occurred at slower rates. Monoclonal antibody 2D12.5 binds DOTA chelates of all the rare earths with Kd ≈ 10-8 M, making it useful for the capture of probe molecules for imaging and therapy (7-9). To make 2D12.5 even more useful for biological applications, we have engineered single cysteine residues near the protein’s binding site so that * Address correspondence to this author. Telephone: 530-7520936. Fax: 530-752-8938. E-mail:
[email protected].
acryl-DOTA chelates may bind and form permanent linkages (10). Three consecutive glycine residues that do not contact the ligand are located in a loop of the heavy chain, near the ligand p-substituent (Figure 1). We anticipated that mutating a single glycine to cysteine would not alter the loop conformation drastically: the other glycines could flexibly adjust their conformation to maintain the geometry required for binding. We prepared the three mutants and selected G54C for more extensive study because it had the best expression level and activity. The G54C mutant binds reversible ligands such as Y-NBD1 with affinities comparable to the native protein (Supporting Information). Formation of a permanent link between the engineered ligand and receptor requires specific binding, which increases the effective local concentration, accelerating an otherwise slow bond-forming reaction. We chose a weak Michael acceptor for the ligand so that permanent attachment is specific even in mixtures containing a variety of competing nucleophiles (1-3). We synthesized Y-AABD (Figure 2) as a candidate irreversible ligand and found that this yttrium complex bound reversibly to native 2D12.5 with Kd ≈ 4 × 10-9 M, comparable to the reversible ligand Y-NBD. We prepared 90Y-AABD and tested it for permanent binding to the G54C mutant. The results in Figure 2B show that at 37 °C, pH 7.5, 90Y-AABD permanently and specifically attaches to the G54C mutant. Under these conditions, the half-time for attachment is ≈13 min. 1 Abbreviations: AABD, (S)-2-(4-acrylamidobenzyl)-DOTA, see Figure 2; HETD, (S)-2-(4-(2-(2-hydroxyethylthio)acetamido)benzyl)-DOTA; NBD, (S)-2-(4-nitrobenzyl)-DOTA.
10.1021/bc049825e CCC: $27.50 © 2004 American Chemical Society Published on Web 10/06/2004
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Figure 1. Site-directed cysteine mutations were designed (10) using the crystal structure of 2D12.5 Fab bound to the Y-DOTA derivative, Y-HETD1, which was modified in silico to the electrophilic acryl derivative, Y-AABD (the p-substituent does not contact the protein). The native glycine residues 54, 55, and 56 in complementarity determining region 2 of the heavy chain appeared to be best suited for replacement with cysteine. The G54C mutant (cysteine sulfur colored purple) is shown (CR's for G55 and G56 are colored red).
Figure 2. (A) Scheme describing the permanent binding pair. (B) Permanent binding is observed not only for 90Y-AABD but also for 111In-AABD, assayed by SDS-PAGE under denaturing conditions where only permanently bound complexes remain attached to the antibody. Increasing the concentration of unlabeled Y-NBD, a reversibly binding competitor, increasingly inhibits permanent attachment by preventing 90Y-AABD or 111In-AABD from accessing the binding site.
In addition, we discovered a new aspect of infinite binding: the reversible In-NBD chelate binds only weakly, but the acryl ligand 111In-AABD binds permanently to the G54C mutant. Figure 2B shows that 111InAABD binds specifically to G54C, with a yield similar to the stronger ligand 90Y-AABD. An important practical implication is that the G54C mutant of 2D12.5 may be used for applications that include not only radiotherapy with 90Y, but also imaging with 111In. The range of metals with useful probe properties thus extends beyond the rare earths (Kd ≈ 10-8 M for reversible binders) to scandium and indium, whose complexes do not have useful affinities as reversible binders (Kd ≈ 10-6 M), and perhaps beyond. We were curious to see if an even weaker binder such as a copper chelate (Kd ≈ 10-4 M for Cu-NBD) would
Corneillie et al.
Figure 3. The G54C mutant was preincubated in triplicate with 10 µM AABD complexes of Y3+, In3+, or Cu2+, a negative control N-(1-carbamoyl-2-(4-nitrophenyl)ethyl)acrylamide or N-(4carboxymethylphenyl)acrylamide, or just buffer, for 5, 20, or 120 min at 37 °C, pH 7.5. At the stated times, 90Y-AABD (1 µM) was added to each solution to compete for free G54C. Permanent binding was assayed by measuring band intensities after SDSPAGE. (A) Phosphorimage of gel. The fainter the bands, the greater the permanent binding by the unlabeled ligand. (B) Crystal structures of Y-DOTA, In-DOTA-mono(p-aminoanilide) (some atoms removed for clarity) and Cu-DOTA.
Figure 4. (A) Metal-AABD (10 µM) complexes were preincubated separately with aliquots of G54C for 5 min, followed by addition of 1 µM 90Y-AABD, which competes for free G54C, and SDS-PAGE analysis. (B) From quantitative phosphorimaging, the highest-affinity AABD complexes form permanent bonds with G54C with higher yields than more weakly binding rare earth-AABD complexes whose ionic radii are slightly smaller (Lu3+, Yb3+) or larger (Ce3+, La3+) than ideal.
bind irreversibly under ordinary conditions. We tested this by comparing the permanent binding of Y3+-, In3+-, and Cu2+-AABD, using competitive 90Y-AABD attachment as an indicator (Figure 3). The positive control Y-AABD does not allow a significant amount of 90YAABD to attach to the G54C: as expected, 90Y-AABD is blocked from >90% of the sites after preincubation with Y-AABD. Weaker binding In-AABD blocks >50% of the sites after 5 min preincubation and even more at later times. Very weakly binding Cu-AABD initially occupies only about 10% of the G54C sites under these conditions, but it has permanently attached to G54C in good yield after 2 h, blocking >70% of the sites. In accord with their Kd values, evidence from crystal structures shows significant differences in preferred coordination geometry between the DOTA complexes of Y, In, and Cu (Figure 3B) (11-15). Control experiments with two acrylamido
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compounds having no measurable affinity for 2D12.5 show no permanent attachment to G54C, indicating that ligand binding is required for the covalent bond-forming reaction to occur. DOTA complexes of all the trivalent rare earth ions bind to 2D12.5 with similar affinities (7). Upon careful examination, the free energies of binding the rare earthDOTA complexes to the antibody show a parabolic dependence on the ionic radius of the metal, with Gd at the bottom and La and Lu at the extremes. Figure 4 shows that the rare earth-AABD complexes bind permanently to G54C in yields that correlate with their relative affinities. These results demonstrate that an infinite binding system can exhibit selective and permanent attachment with a surprising range of structurally related ligands, albeit at slower rates as affinities decrease. Separately engineering the reactivities of both ligand and receptor provides a direct route to the capture of a set of similar molecules, with unsurpassed affinity. ACKNOWLEDGMENT
(6) Pollack, S. J., Nakayama, G. R., and Schultz, P. G. (1988) Introduction of nucleophiles and spectroscopic probes into antibody combining sites. Science 242, 1038-1040. (7) Corneillie, T. M., Whetstone, P. A., Fisher, A. J., and Meares, C. F. (2003) A rare earth-DOTA-binding antibody: probe properties and binding affinity across the lanthanide series. J. Am. Chem. Soc. 125, 3436-3437. (8) Corneillie, T. M., Fisher, A. J., and Meares, C. F. (2003) Crystal structures of two complexes of the rare-earth-DOTAbinding antibody 2D12.5: ligand generality from a chiral system. J. Am. Chem. Soc. 125, 15039-15048. (9) Goodwin, D. A., Meares, C. F., Watanabe, N., McTigue, M., Chaovapong, W., Ransone, C. M., Renn, O., Greiner, D. P., Kukis, D. L., and Kronenberger, S. I. (1994) Pharmacokinetics of pretargeted monoclonal antibody 2D12.5 and 88Y-Janus2-(p-nitrobenzyl)-1,4, 7,10-tetraazacyclododecanetetraacetic acid (DOTA) in BALB/c mice with KHJJ mouse adenocarcinoma: a model for 90Y radioimmunotherapy. Cancer Res. 54, 5937-5946. (10) Corneillie, T. M., Lee, K. L., Whetstone, P. A., Wong, J. P., and Meares, C. F. (2004) Irreversible engineering of the multielement-binding antibody 2D12.5 and its complementary ligands. Bioconjugate Chem. 15, 1392-1402.
We thank Nathan Butlin, Sean Gay, Mark McCoy, and Susan Lee for helpful discussions, and NIH Research Grant CA16861 (C.F.M.) for support.
(11) Liu, S., He, Z., Hsieh, W. Y., and Fanwick, P. E. (2003) Synthesis, characterization, and X-ray crystal structure of In(DOTA-AA) (AA ) p-aminoanilide): a model for 111In-labeled DOTA-biomolecule conjugates. Inorg. Chem. 42, 8831-8837.
Supporting Information Available: Syntheses and additional experimental details and results. This material is available free of charge via the Internet at http:// pubs.acs.org.
(12) Chang, C. A., Francesconi, L. C., Malley, M. F., Kumar, K., Gougoutas, J. Z., Tweedle, M. F., Lee, D. W., and Wilson, L. J. (1993) Synthesis, characterization, and crystal structures of M(DO3A) (M ) Fe, Gd) and Na[M(DOTA)] (M ) Fe, Y, Gd). Inorg. Chem. 32, 3501-3508.
LITERATURE CITED
(13) Parker, D., Pulukkody, K., Smith, F. C., Batsanov, A., and Howard, J. A. K. (1994) Structures of the yttrium complexes of 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (H-4 DOTA) and N, N′′-bis(benzylcarbamoylmethyl)diethylene-triamine-N,N′,N′′-triacetic acid and the solution structure of a zirconium complex of H-4 DOTA. J. Chem. Soc., Dalton Trans. 689-693.
(1) Chmura, A. J., Orton, M. S., and Meares, C. F. (2001) Antibodies with infinite affinity. Proc. Natl. Acad. Sci. U.S.A. 98, 8480-8484. (2) Chmura, A. J., Schmidt, B. D., Corson, D. T., Traviglia, S. L., and Meares, C. F. (2002) Electrophilic chelating agents for irreversible binding of metal chelates to engineered antibodies. J. Controlled Release 78, 249-258. (3) Levitsky, K., Ciolli, C. J., and Belshaw, P. J. (2003) Selective inhibition of engineered receptors via proximity-accelerated alkylation. Org. Lett. 5, 693-696. (4) Grunwald, V., and Hidalgo, M. (2003) Developing inhibitors of the epidermal growth factor receptor for cancer treatment. J. Natl. Cancer Inst. 95, 851-867. (5) Chen, G., Heim, A., Riether, D., Yee, D., Milgrom, Y., Gawinowicz, M. A., and Sames, D. (2003) Reactivity of functional groups on the protein surface: development of epoxide probes for protein labeling. J. Am. Chem. Soc. 125, 8130-8133.
(14) Riesen, A., Zehnder, M., and Kaden, T. A. (1986) Metal complexes of macrocyclic ligands. Part XXIII. Synthesis, properties, and structures of mononuclear complexes with 12and 14-membered tetraazamacrocycle-N,N′,N′′,N′′-tetraacetic acids. Helv. Chim. Acta 69, 2067-2073. (15) Cosentino, U., Villa, A., Pitea, D., Moro, G., Barone, V., and Maiocchi, A. (2002) Conformational characterization of lanthanide(III)-DOTA complexes by ab initio investigation in vacuo and in aqueous solution. J. Am. Chem. Soc. 124, 4901-4909.
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