Measurement of in Vivo Drug Load Distribution of Cysteine-Linked

Feb 27, 2014 - Seattle Genetics Inc., 21823 30th Drive Southeast, Bothell, ... (LC−MS) methods to measure the in vivo drug load distribution have be...
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Measurement of in Vivo Drug Load Distribution of Cysteine-Linked Antibody−Drug Conjugates Using Microscale Liquid Chromatography Mass Spectrometry Shawna Mae Hengel,* Russell Sanderson, John Valliere-Douglass, Nicole Nicholas, Chris Leiske, and Stephen C. Alley Seattle Genetics Inc., 21823 30th Drive Southeast, Bothell, Washington 98021, United States ABSTRACT: Analysis of samples containing intact antibody−drug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biological and chemical factors. Liquid chromatography−mass spectrometry (LC−MS) methods to measure the in vivo drug load distribution have been established for ADCs containing native disulfide bonds (lysine-linked or cysteinelinked). However, because of an IgG reduction step in conjugation processes, using LC−MS to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the analytical scale, to date, these methods have not been translated to a smaller scale that is required for animal or clinical doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC−MS methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.

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spectrometry (LC−MS) is typically used to obtain quantitative values of the small molecule. While ELISA can be used to detect the concentration of a circulating ADC, the data does not reflect the drug load. LC−ultraviolet (UV) methods are commonly used to determine the drug to antibody ratio of a pure ADC; however, these methods are limited based on lack of specificity and sensitivity. The use of mass spectrometry to characterize ADCs is gaining popularity due to the complex nature of ADCs. Recently Valliere-Douglass et al. developed a native LC−MS assay for the detection of cysteine linked ADCs.9 Because of conjugation methods, i.e., reduction of native disulfide bonds, for cysteine-linked ADCs all intact analyses must be conducted under native conditions in order to maintain noncovalent associations of heavy and light IgG chains. It was observed using the new LC−MS method in the native state that relative intensities of the deconvoluted species were proportional to the fractional contribution of the drug load distribution as characterized by orthogonal techniques.9 Designed for throughput and robustness, this method was developed for analysis of pure samples at the analytical scale. Additionally, Chen et al. demonstrated successful native static nanospray ionization of cysteine-linked ADCs after proteolytic drug removal.10

s new therapeutic molecules are developed and aimed toward preclinical and clinical studies, new analytical techniques are required to ensure thorough characterization of these new analytes both pre- and postadministration. Specifically, antibody−drug conjugates (ADCs) are a potent class of molecules that are gaining a strong presence in the clinic.1,2 ADCs are composed of three parts: an antibody that binds to a specific cell surface antigen, a therapeutic agent that is responsible for biological activity, and a chemical linker that covalently attaches the drug to the antibody backbone. Once the ADC binds to the antigen, the complex is internalized and trafficked to the lysosome and the small molecule drug is released upon protein turnover. ADCs are currently conjugated using three techniques based on amino acid functional group synthetic handles: at naturally occurring cysteine residues following reduction of interchain disulfide bonds (cysteine-linked ADCs), at lysine residues (lysine-linked ADCs), or engineered cysteine residues.3−6 In the case of cysteine and lysine-linked ADCs, the conjugation method results in a range of drug load distributions. In vivo, both clearance and deconjugation contribute to a changing drug load distribution. It has been previously demonstrated that higher loaded species are cleared faster compared to ADCs with fewer conjugated drug-linkers.7,8 Standard techniques for measuring ADCs from in vivo samples include enzyme-linked immunosorbent assays (ELISA) for total antibody, or ADC, concentrations from a range of biological matrices; where liquid chromatography−mass © 2014 American Chemical Society

Received: November 27, 2013 Accepted: February 27, 2014 Published: February 27, 2014 3420

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Because of the inherent complexity of in vivo samples, the large dynamic range of endogenous protein concentrations, and low levels of circulating ADCs, sample purification and concentration steps are required prior to using intact ADC LC−MS methods. Depending on the amino acid sequence of the ADC and any overlap between endogenous IgG in an in vivo study, it is possible to use species specific generic IgG capture reagents coupled to a solid phase. However, for clinical studies, or the cases where the ADC contains a significant sequence/structure overlap with endogenous IgG, custom affinity resins must be constructed. Coupling ADC target receptors or anti-idiotypes to a solid phase allows for specific capture of the ADC of interest. This has been previously optimized for lysine-linked and engineered cysteine ADCs where acidic conditions and organic solvents are used as washes and elution steps.11,12 When native protein conformation must remain intact, as is the case for cysteine-linked ADCs, limited sensitivity and available solvent modifiers complicate the analyses. This manuscript describes customized sample preparation for cysteine-linked ADCs from an in vivo source and the optimization of a native microscale LC−MS method for accurate drug load distribution determination. To our knowledge this represents the first native intact mass spectra of cysteine-linked ADCs from an in vivo study.

evaluated using individual pure loads and established mixes. Samples were processed as described above except following the elution step; beads were incubated with 2 × 400 μL of 8 M guanidine-HCl for 5 min at room temperature to remove any remaining 3H-ADC not recovered in the elution fractions. Samples were analyzed by liquid scintillation (Tri-Carb 2910 TR, Perkin-Elmer, Waltham, MA). LC−MS. For intact size-exclusion chromatography (SEC) methods, antibody−drug conjugates were deglycosylated overnight using 2 μL of PNGase F (New England Biolabs, Ipswich, MA) at 37 °C prior to LC−MS analysis. For intact LC−MS methods, ADC (0.5−1.5 μL, ranging from 5 μg to 50 ng) was injected onto a 0.3 mm × 150 mm (5 μm 300 Ǻ ) polyhydroxyethyl-A (PHEA) SEC column (PolyLC, Columbia, MD) and eluted into the mass spectrometer with an isocratic flow of 200 mM ammonium acetate at 1 μL/min. All mass spectral data were acquired using an Agilent 6510 Q-TOF mass spectrometer (Agilent, Santa Clara, CA) and deconvoluted using standard Agilent Mass Hunter software. The standard electrospray ionization (ESI) source equipped with a microspray adapter was used to accommodate low flow rates. Source parameters were set to 3000 V, 15 psi, 300 V, and 5.0 L/min for the source capillary voltage, nebulizer, skimmer, and sheath gas flow, respectively. MS spectra (approximately 15.5−17.5 min for intact ADC) were summed prior to deconvolution. Nonreduced separations were performed using a 2.1 mm × 50 mm (5 μm 4000 Ǻ ) polymeric reversed-phase (PLRP) column (Agilent Technologies, Santa Clara, CA), with a gradient of 20% to 50% acetonitrile, containing 0.035% TFA, in 50 min where 80% H2O containing 0.05% TFA were the starting conditions. Source settings were adapted to the higher flow rate of 500 μL/min, and the standard source probe was used. In Vitro Incubations. Cysteine conjugated monomethyl auristatin E (MMAE) ADCs were spiked into rat plasma at a range of concentrations from 0.3 mg/mL to 0.1 mg/mL and recovered using ADC specific capture reagents described above. In Vivo Study. Female Sprague−Dawley rats were administered a single 10 mg/kg ADC intravenous bolus injection, and whole blood was drawn at 30 min, 6 h, 24 h, and 7 days post dose. Samples were kept on ice and processed to plasma within 1 h of collection. All animals were treated in accordance with the Institutional Animal Care and Use Committee guidelines. Absolute ADC concentrations from in vivo samples were determined using previously described methods.14 Prior to affinity capture, 200 μL of plasma was diluted to 2 mL with PBS-T.

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EXPERIMENTAL SECTION Monoclonal antibodies were all expressed and purified inhouse; conjugation methods have been described elsewhere.5 ADC pure load isolation was performed as previously described.7 Anti-idiotype mAbs were prepared and biotinylated as described previously.13,14 ADC Specific Capture Reagents. Streptavidin coupled magnetic beads (Thermo Scientific, Waltham, MA), 1 mL at 1 mg/mL, were transferred to microcentrifuge tubes and a magnet was applied to separate beads from the storage solution. Beads were washed twice with 1 mL of PBS-T (PBS + 0.05% Tween-20). Biotinylated anti-idiotype mAbs, 100 μg in 1 mL of PBS-T, were added to the beads and rotated at 4 °C for 1 h. At the end of the incubation period, supernatant was removed and the resin was washed twice with 2 mL of PBS-T. Prior to affinity capture, samples were diluted in PBS-T to a total volume of 2 mL with a final ratio of 1:9 or 1:3 (v/v) plasma/PBS-T and final concentration range of 30 to 5 μg/mL of ADC. Once biotinylated anti-idiotypes were added to the beads and washed, diluted samples were added to the beads. Samples were incubated for 1 h at 4 °C with rotation. The matrix was removed, followed by one PBS-T wash and two PBS washes. Affinity captured ADC was eluted by incubating the beads with commercially available IgG elution buffer, a proprietary amine buffer pH 2.8 (Thermo Scientific, Waltham, MA), for 5 min at 4 °C. Eluent was neutralized with 1:10 (v/v) of 1 M Tris pH 8.0. Samples were concentrated to approximately 1 mg/mL using Amicon ultra centrifugal filters with a 30k MW cutoff (Millipore, Billerica, MA). Radioactivity. ADCs were labeled as described elsewhere.15 3 H-labeled ADC pure loads were prepared separately in rat plasma (Bioreclamation, Liverpool, NY) and diluted into PBST at a ratio of 1:9 or 1:3 (v/v). Prior to labeling, ADCs were thoroughly characterized for relative levels of each drug load species for both pure load samples (single fixed number of drugs per antibody backbone) and mixed samples (distribution of number of drugs per antibody) using methods previously described.7 Capture and elution of affinity reagents were

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RESULTS AND DISCUSSION ADC Specific Affinity Reagents. ADC specific affinity reagents using biotinylated anti-idiotype mAbs were constructed using magnetic beads conjugated to streptavidin. Anti-idiotypic mAbs were previously characterized and prepared/screened to bind at the unique ADC sequence domain allowing for specificity in the presence of endogenous IgG across species.14 When designing and testing the affinity reagents, four fractions were monitored using 3H-labeled ADCs. The amount of radioactivity was measured from the binding, wash, and elution steps in addition to the final guanidine-HCl incubation to determine if any ADC remained bound to the affinity reagents. To ensure accurate determination of the drug load distribution, equal capture, retention, and elution must be achieved across all drug load species. For 3421

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Figure 1. Binding (a) and elution (b) efficiency of pure load ADCs in the presence of 10% and 25% plasma. Binding across 2, 4, 6, or 8 drugs per antibody backbone was equivalent with a maximum of 10% detected in the flow through fraction by liquid scintillation counting. Elution efficiency was determined to be equivalent across 2−8 loaded 3H-ADCs with a minimum of 79% of total radioactivity recovered, detected by liquid scintillation counting.

the greatest sample recovery, both capture and elution of the ADC were optimized. ADC mixtures were used for initial reagent characterization, and after preliminary optimization, affinity reagents were evaluated using ADC pure load species. The largest observed differences between the pure load species was 3% and 6% for binding and elution, respectively (Figure 1). Because of the minimal observed variation between pure-load ADCs and high recovery of ADC mixtures, it was determined that all drug load species were captured and eluted equally using the ADC specific capture reagents. Additionally, control samples were evaluated using native LC−MS to ensure equivalent measured drug load distribution prior to, and after, affinity reagent capture/elution; see the discussion below. Microscale Native LC−MS. The analytical scale native SEC LC−MS method developed by Valliere-Douglass et al.9 was modified to operate at a microscale. In this context, microscale is referred to as low (90% of the bound ADC across all drug load levels. To

Figure 2. Deconvoluted mass spectrum of a cysteine-linked ADC, average drug to antibody ratio of 4, acquired using the microscale native SEC LC−MS platform with 2 μg on column. Measured mass accuracy is denoted for each drug load species in parts per million (ppm).

determine any effects of the elution buffer on ADC drug load distribution, microscale native SEC LC−MS was performed on ADCs exposed to the elution buffer alone, omitting the affinity capture step. ADCs were then neutralized after 5 min, the standard resin elution incubation time. Equivalent drug load distributions and overall signal intensities were detected between the mock elution conditions and control, indicating that elution conditions did not disrupt the native protein conformation. Plasma samples that did not contain ADC were also processed through the affinity capture/elution steps and analyzed using the microscale native LC−MS method; no signal over background was observed in the expected IgG range confirming specificity of the anti-idiotypes used in the affinity reagents. Additionally, control samples of ADCs in buffer were processed using the affinity reagents in parallel to the in vitro and in vivo studies to monitor the effect of the reagents/elution efficiency on the drug load distribution. In all cases noted in this manuscript, the control drug to antibody ratio before and after capture/elution were equivalent. In Vitro Studies. ADCs were spiked into rat plasma at 0.1 mg/mL and recovered using the ADC specific magnetic capture reagents described above. Following elution, neutralization, and 3422

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deglycosylation, ADCs were analyzed using the microscale native SEC LC−MS platform. LC−MS signal was compared between ADC purified using affinity resins from plasma, and ADC from the same stock solution that was not exposed to rat plasma or affinity capture (control). Equal drug load distributions were detected between all conditions, further confirming equivalent ADC binding and elution across drugload species (Figure 3).

Figure 4. Deconvoluted mass spectra of cysteine-linked ADCs from an in vivo study. Top panel, control ADC; middle panel, ADC from 30 min postdose; bottom panel, ADC from 6 h post dose. Dashed lines indicate theoretical mass of ADCs containing 0−8 drugs per antibody.

Interestingly, the 5- and 7-loaded species were not detected. One possible explanation is that the lower abundant 8- and 6loaded species clear faster than the rate of 6-load deconjugation. Additionally, the 6- and 8-loaded species are of much lower abundance than the 2 and 4-load, which would hamper the detection of the minor 5 and 7 loaded deconjugation products. These hypotheses could be addressed in an additional in vivo study where pure 6 or 8 loaded species were dosed to monitor specific degradation products (i.e., 5 loaded species) over an extended period of time. Upon further examination of the data, compared to controls, the deconvoluted mass spectra from in vivo samples were found to have greater asymmetry on the high mass side of peaks across all drug loaded species observed. Because no change in chromatography was observed, and deconvoluted peak shape for equivalent column load of control samples did not contain the observed asymmetry, one possible explanation is increasing sample heterogeneity over time. Each post-translational modification or degradation event, in addition to drug-linker stability/modifications, increases the ADC mass heterogeneity thus broadening the high mass side of the deconvoluted peak. Because these changes are much less than the mass of one druglinker and all drug loaded species elute at the same retention time using this method, no changes in chromatography would be expected for these samples. Additionally, while even loaded species were all within expected instrument mass accuracy specifications, odd loaded species detected containing one or three drugs per antibody were observed at a higher than expected mass by more than 100 Da in the deconvoluted data, consistent with previous observations of engineered-cysteine linked ADCs.11,17 To test the hypothesis of increased in vivo ADC heterogeneity over time, nonreduced reversed-phase LC-MS analyses of the same samples were completed to elucidate observed increased mass shifts and asymmetry. Expected peaks corresponding to light, heavy, and combinations of the two chains with variable drug loads were detected. Peaks that were observed to increase over time in intensity or shift in mass were

Figure 3. Deconvoluted mass spectra of cysteine-linked ADC from an in vitro study, acquired using the microscale native SEC LC−MS platform with 2 μg on column. Top panel, control ADC; bottom panel, ADC spiked into rat plasma isolated using affinity capture.

In Vivo Study. After ADCs recovered from spiked plasma were successfully analyzed using microscale native SEC LC− MS, samples from an in vivo source were tested. As described above, female Sprague−Dawley rats were administered a single 10 mg/kg ADC intravenous bolus injection and samples were collected at 30 min, 6 h and 24 h, and 7 days post dose. Using ELISA ADC concentration data, samples that contained ≥20 μg of ADC in no more than 200 μL of plasma were selected for affinity capture and subsequent LC−MS analyses. A volume of 200 μL was chosen for the maximum sample volume as it is a feasible amount of sample to request from an in vivo source and would result in a volume that could still be manipulated with ease once it had been diluted for the binding step. Several interesting features were noted in the data from the short time course of intact ADC LC−MS data, representing to our knowledge the first spectra of this kind (Figure 4). First, the higher drug loaded species were not observed shortly after dosing. At the 30 min time point, the predominant species is the ADC containing four drugs per antibody; however, at 6 h post dose the main species has shifted from four to two drugs per antibody. On the basis of previously published studies detailing the pharmacokinetic properties of highly loaded (>4 drugs per antibody) auristatin conjugated ADCs,7 the observed shift is likely due to a combination of both deconjugation and clearance of the higher loaded species. The shift in drug load distributions to lower drug loaded species in this study is consistent with other reports that contain LC−MS data using non-native conditions of lysine conjugated ADCs.12,16 Second, while a distribution of even drug loaded species (antibodies with 2, 4, 6, or 8 drugs) was dosed, over time odd drug loaded species were observed. This is likely due to deconjugation of the drug-linker, resulting in the generation of species that are not part of the original dosed drug load distribution profile. 3423

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of specific interest for our hypothesis, because of the indication of a potential modification. For example, light chain alone and heavy−heavy−light chain were both observed with a mass addition of 120 Da in data from samples collected at later time points (Figure 5). These peaks appeared at retention times

Figure 6. Nonreduced PLRP LC−MS deconvoluted spectra of heavy chain containing three drug-linkers (H3) with increasing hydrolysis over time in vivo. Dashed lines indicate the theoretical mass of H3, H3 plus one hydrolysis, and H3 plus hydrolysis at two sites. Figure 5. Nonreduced PLRP LC−MS deconvoluted spectra of light chain cysteinylation (left) and heavy−heavy−light chain cysteinylation (right) increasing in abundance over time in vivo. Dashed lines indicate theoretical mass of light chain plus the mass of cysteinylation and heavy−heavy−light chain plus the mass of one cysteinylation.

expected was attributed to modification at the deconjugated drug-linker sites, specifically cysteinylation, and observed asymmetry in the deconvoluted data over time is hypothesized to be a consequence of increased sample heterogeneity due to in vivo modifications, including hydrolysis (Figure 7). Presumably, other modifications increase over time in vivo and will be investigated in future studies.

unique from the conjugated drug precursor. It has been previously demonstrated that maleimide drug-linkers react with plasma proteins through a reverse Michael addition.18,19 The resulting free thiol could then react with free cysteine, which would result in the addition of approximately 120 Da.18,20,21 From our analysis and the previously published observations, we propose that both heavy−heavy−light and light chains were identified with a mass shift corresponding to cysteinylation. Additionally, this modification would lead to the increased mass of intact odd load species that were observed in the intact in vivo data. While the observed mass shift is consistent with cysteinylation, the absolute structural confirmation of this would require additional MS/MS studies for site localization that were out of the scope of this study. In further support of increasing ADC mass heterogeneity in vivo, several peaks appeared to split or shift over time with an increase of 18 Da. Each conjugated drug-linker provides a potential hydrolysis site at the succinimide ring. Over time this would lead to a range of zero to eight hydrolysis events across the drug load distribution, with many potential combinations. Specifically, heavy chains containing multiple drug-linkers in theory would contain an increasing number of hydrolysis sites. This was observed in the nonreduced reversed-phase LC−MS data. For example, dominant peaks in deconvoluted mass spectra of the heavy chain containing three conjugated druglinkers shifted from the nonhydrolyzed form at early time points to peaks increasing by 18 Da, indicating one or more hydrolysis events over time (Figure 6). A heterogeneous population of hydrolysis events at the succinimide ring would add to the overall mass heterogeneity in vivo and contribute to increasing asymmetry in deconvoluted intact LC−MS data. To summarize the unique observations identified in the in vivo data: detection of odd loaded species at masses higher than

Figure 7. Summary of mass shifts and peak asymmetry observed in cysteine-linked ADCs in vivo.

Analysis of intact cysteine-linked ADCs from either in vitro or in vivo samples using LC−MS is highly valuable. Assessment of deconjugation with the formation of odd loaded species (in vitro and in vivo) and clearance rates (in vivo) of each drug load species individually can only be accomplished at present using native MS. This type of analysis is of interest on both the linker-stability/technology side of early ADC development as well the later stage pharmacokinetic/clinical side of drug development. Measurement of the in vivo drug-load distribution provides information on the conjugated drug in circulation and subsequently available drug for internalization into targeted cells. Currently, pharmacokinetic modeling that seeks to 3424

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(9) Valliere-Douglass, J. F.; McFee, W. A.; Salas-Solano, O. Anal. Chem. 2012, 84, 2843−2849. (10) Chen, J.; Yin, S.; Wu, Y.; Ouyang, J. Anal. Chem. 2013, 85, 1699−1704. (11) Xu, K.; Liu, L.; Saad, O. M.; Baudys, J.; Williams, L.; Leipold, D.; Shen, B.; Raab, H.; Junutula, J. R.; Kim, A.; Kaur, S. Anal. Biochem. 2011, 412, 56−66. (12) Xu, K.; Liu, L.; Dere, R.; Mai, E.; Erickson, R.; Hendricks, A.; Lin, K.; Junutula, J. R.; Kaur, S. Bioanalysis 2013, 5, 1057−1071. (13) Hermanson, G. T. Bioconjugate Techniques; Elsevier Science: Amsterdam, The Netherlands, 2008. (14) Sanderson, R. J.; Hering, M. A.; James, S. F.; Sun, M. M.; Doronina, S. O.; Siadak, A. W.; Senter, P. D.; Wahl, A. F. Clin. Cancer Res. 2005, 11, 843−852. (15) Alley, S. C.; Zhang, X.; Okeley, N. M.; Anderson, M.; Law, C. L.; Senter, P. D.; Benjamin, D. R. J. Pharmacol. Exp. Ther. 2009, 330, 932−938. (16) Kaur, S.; Xu, K.; Saad, O. M.; Dere, R. C.; Carrasco-Triguero, M. Bioanalysis 2013, 5, 201−226. (17) Shen, B. Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.; Yu, S. F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.; Eigenbrot, C.; Nguyen, T.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel, D.; Spencer, S. D.; Khawli, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.; Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R. Nat. Biotechnol. 2012, 30, 184−189. (18) Alley, S. C.; Benjamin, D. R.; Jeffrey, S. C.; Okeley, N. M.; Meyer, D. L.; Sanderson, R. J.; Senter, P. D. Bioconjugate Chem. 2008, 19, 759−765. (19) Baldwin, A. D.; Kiick, K. L. Bioconjugate Chem. 2011, 22, 1946− 1953. (20) Mansoor, M. A.; Svardal, A. M.; Ueland, P. M. Anal. Biochem. 1992, 200, 218−229. (21) Rossi, R.; Giustarini, D.; Milzani, A.; Dalle-Donne, I. J. Cell. Mol. Med. 2009, 13, 3131−3140.

optimize dosing and understand the nature of these molecules in vivo is limited when the drug load distribution is known only at the time of dosing. Ideally the drug load distribution would be monitored over time in vivo, and each drug load species could be treated as a separate entity in PK modeling in order to determine dosing regimens with optimal activity.

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CONCLUSIONS In conclusion, cysteine-linked ADCs were affinity purified and analyzed using native LC−MS to determine the drug load distribution in vivo. This body of work required the development of ADC specific affinity capture reagents, and existing LC−MS methods were modified to accommodate limited sample availability from an in vivo source. Observed drug load distributions were consistent with previously established analytical scale methods, while capture and elution efficiencies were confirmed to be consistent across all drug loaded species as not to skew the drug load distribution through sample processing. This method provides insight into the changing drug load distribution in vivo and will be an important tool for pharmacokinetic modeling and drug-linker stability assessments. This study serves to demonstrate proof of concept and feasibility of in vivo sample analysis, and the increasing sample complexity of cysteine-linked drug load distributions in vivo.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (425) 527-4802. Fax: (425) 527-4109. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank W. McFee, J. Jones, J. Setter, and S. Henry for thoughtful discussion and Cassandra Baker Lee for generating the ELISA data.

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REFERENCES

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