EMD Lexigen Research Center Corp., 45A Middlesex Turnpike, Billerica, MA 01821, USA
1 To whom correspondence should be addressed. E-mail: jway{at}emdlexigen.com
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Abstract |
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Keywords: erythropoietin/disulfide bond engineering/pharmacokinetics/protein design
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Introduction |
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To enhance the serum half-life of protein drugs, several strategies have been developed. One strategy is to increase the effective molecular weight of a protein and thereby reduce renal clearance. PEGylation involves the chemical modification of proteins with large, inert polymers (Molineux, 2003) and Schering-Plough's PEG-Intron, a PEGylated interferon-alpha, is an example of a recently approved protein using this approach. Alternatively, a protein's effective molecular weight may be increased by fusion to a carrier protein, such as to albumin or the Fc region of an antibody (Capon et al., 1989
; Yeh et al., 1992
). In general, the construction of an FcX fusion protein using an X with a molecular weight of less than about 40 kDa results in a dramatic extension of serum half-life (e.g. Lo et al., 2000
).
Other strategies modulate the biological activity of the protein. In one approach, extra N-linked oligosaccharides are incorporated into a protein: This has been used with Epo to generate darbepoietin and has recently been generalized to other cytokines such as leptin (Elliott et al., 2003). Additional N-linked glycosylation on Epo increases the protein's overall negative charge and therefore reduces the on-rate for the receptor, which is significantly charge-driven (Strickland, 1999
; Egrie and Browne, 2001
). As a result of reducing the on-rate, receptor-mediated endocytosis of the mutant Epo is reduced and the protein has an extended serum half-life.
Another strategy has been to modify the fate of proteins within the endosome. The fusion of therapeutic proteins to albumin or the Fc region of an antibody, in addition to increasing the effective molecular weight of the protein, promotes recycling out of the cell upon endocytosis via the Fc and albumin recycling receptors, so these strategies are particularly useful for enhancing serum half-life (Capon et al., 1989; Yeh et al., 1992
). Sarkar et al. (2002)
described mutations in G-CSF that enhance dissociation from G-CSF receptor in the low-pH endosomal compartment, allowing the mutant G-CSF to recycle out of the endosome. Gillies et al. (2002)
introduced mutations at the protease-sensitive junction in an antibody-IL2 fusion protein to reduce proteolysis in the endosomal compartment, so that the fusion protein could be recycled out of the cell via the recycling receptor FcRn.
Degradation of hormones and cytokines upon internalization may require that these proteins have structural weak points. The ability to denature easily and be degraded upon endocytosis may reflect a physiological adaptation of Epo. Kato et al. (1997) examined the pharmacokinetic behavior of Epo in rats and found a saturable component due to receptor-mediated endocytosis and non-saturable component due to renal clearance. Other possible clearance mechanisms, such as proteolysis in the serum, did not appear to play a significant role in Epo disappearance. Thus, alterations that promote Epo survival in the endosome may extend its serum half-life.
In this study, we analyzed Epo for potential structural weak points and investigated a set of mutations in Epo that affect protein structure (Figure 1). In the context of an FcEpo fusion protein, these mutations improved the folding characteristics and also the stability of the protein (Figure 2), which translated into a molecule with significantly enhanced serum half-life and potency (Figure 3).
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Materials and methods |
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A codon-optimized nucleic acid molecule for mammalian expression of the mature human Epo gene was generated by de novo synthesis. Oligonucletides of about 50 bases covering the coding and non-coding strands were designed such that complementary oligoncleotides were off-set by 45 bases to generate 5' overhangs. These were phosphorylated, annealed to one another and ligated, resulting in a molecule with an XmaI-compatible and a XhoI-compatible overhang at its 5' and its 3' ends, respectively. This fragment was ligated to a XmaI/XhoI-digested pdCs-Fc vector that encoded an Fc region from human IgG2 (Lo et al., 1998), generating pdCsFcEpo in which the Epo coding sequence was cloned directly downstream of the final codon in Fc. The Fc moiety also contained a mutation of the C-terminal lysine to an alanine (Gillies et al., 2002
). The mutations H32G, C33P, W88C and P90A in the Epo moiety of FcEpo(NDS) were introduced by total gene synthesis as described above.
Expression, purification and detection of FcEpo fusion proteins
Mouse myeloma NS/0 cells were transfected with pdCs-FcEpo or pdCs-FcEpo(NDS) DNA by electroporation and stable clones were selected by resistance to methotrexate as described (Lo et al., 1998). FcEpo proteins were purified from conditioned cell-culture supernatants based on the affinity of the Fc protein moiety for Protein A. The supernatants were loaded on to a pre-equilibrated Fast-Flow Protein A Sepharose column (Pharmacia), the column was washed extensively in sodium phosphate buffer (150 mM sodium phosphate, 100 mM NaCl at neutral pH), bound protein was eluted with pH 3 sodium phosphate buffer of like composition and the peak fractions of the eluate were neutralized. A 5 µg amount of the FcEpo proteins from the pooled peak fractions was analyzed by SDS-PAGE on a 412% Bis-Tris gel.
Protein concentrations for pharmacokinetics were determined using an Epo ELISA kit (R&D Systems). Intactness of the fusion proteins in serum was monitored by a PK-Western wherein serum samples were purified by Protein A, analyzed by SDSPAGE, blotted to filter membranes and probed with an anti huFc antibody to reveal the presence of full-length and clipped species of the fusion protein; no evidence of in vivo cleavage was seen.
Biochemical analysis of FcEpo fusion proteins
A peptide map analysis to verify the disulfide bonding pattern was performed as follows. FcEpo proteins (10 mg/ml) were treated with PNGase F to remove carbohydrate moieties and trypsinized (10 µg/ml trypsin final for 4 h at 37°C) under non-reducing conditions to preserve disulfide-linked peptides. A 20 µl sample was injected into an HPLC system, peptide fragments were resolved on a reversed-phase column (Phenomenex Synergi 4 µ Hydro RP) and the mass of the ionized fragments was determined by mass spectrometry, detecting mass ranges between 250 and 2800.
For analytical size-exclusion chromatography (SEC), 15 µl samples from the peak fraction were resolved by HPLCSEC (TosoHaas Super 3000 SW) in a 15 min run at a flow rate of 0.35 ml/min.
To test for sensitivity to removal of N-glycans, FcEpo and FcEpo(NDS) at concentrations of 0.5 mg/ml were incubated with 30 mU PNGaseF (New England Biolabs) for 0, 1, 2, 4 and 21 h at 37°C and the reaction was terminated by freezing at 20°C. A 1 h incubation was sufficient to remove N-linked oligosaccharides completely from the fusion proteins.
Characterization of Epo receptor binding
A kinetic analysis of the binding of the FcEpo variants to the Epo receptor was performed using BIAcore (Biacore International). The extracellular moiety of the Epo receptor fused to Fc (sEPOr-Fc, R&D Systems) was diluted in 10 mM sodium acetate pH 4.5 and immobilized to CM5 biosensor microchips using N-hydroxysuccinimide (NHS) and N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC). About 500700 RUs per flow cell were immobilized on the flow cells and one remaining flow cell served as a reference for non-specific binding. Microsensor chips were conditioned with four cycles of one injection each of 20 µl of 100 nM FcEpo and 10 µl of 100 mM HCl, followed by two injections of 10 µl of 100 mM H3PO4. FcEpo samples were diluted with HBS-EP buffer (10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA and 0.05% surfactant P20) to create concentrations of 250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 nM. These FcEpo samples were then passed over the chip and binding data were collected. An injection of HBS-EP was used as a blank/negative control for binding. Microchip sensor surfaces were regenerated with one injection of 10 µl of 100 mM HCl followed by two injections of 100 mM H3PO4 prior to each new FcEpo injection. Binding data weres analyzed using curve-fitting software from Biacore (BIAnalysis).
In vitro and in vivo analysis of FcEpo fusion proteins
Proliferation assays were performed with the cell line TF-1, measuring levels of tritiated thymidine incorporation as a function of Epo activity (Kitamura et al., 1989; Hammerling et al., 1996
). In brief, exponentially growing TF-1 cells were washed twice, plated at about 104 cells/well in microtiter plates and incubated in basal medium with a titrated dilution series of Epo (Procrit), darbepoietin (Aranesp), FcEpo or FcEpo(NDS) for 48 h. Ten hours before assaying cell proliferation, 0.3 µCi of [3H]thymidine (Dupont-NEN-027) was added to the wells and tritiated thymidine incorporation was measured as total TCA-precipitable counts. An ED50 value for each FcEpo protein variant was obtained from plotting a doseresponse curve and identifying the protein concentration that resulted in half-maximal response.
The pharmacokinetics of Epo (Procrit), FcEpo and FcEpo(NDS) molecules were tested as follows. Three CD1 mice were injected i.v. with FcEpo or FcEpo(NDS) at 200 µg/kg and blood samples were collected at 0, 0.5, 1, 2, 4, 8, 24 and 48 h. Serum was tested for human Epo or FcEpo levels using an R&D Systems Epo ELISA kit. Mouse Epo is not detected in this assay.
Hematocrits were determined as follows. Mice were administered a single intra-peritoneal injection of equimolar amounts of Epo (Procrit; 25 µg/kg), FcEpo or FcEpo(NDS) (42 µg/kg). On days 0, 4, 7, 11 and 14, blood was withdrawn from anesthetized mice retro-orbitally with a heparin-coated capillary tube, capped with clay, centrifuged for 5 min in an IEC IM-3411centrifuge with a Model 275 rotor and red blood cell volume was measured using a circular microcapillary tube reader according to standard procedure.
Molecular modeling and dynamics of Epo homologues and Epo(NDS)
A structural model of native human Epo was built, from which the structural models of Epo(NDS) and Epo of other species were derived. The native human Epo model was built with the Swiss PDBViewer molecular visualization and modeling software, using structural coordinates of human Epo from the Protein Data Base (PDB; available online at www.rcsb.org) determined by NMR [PDB entry 1BUY (Cheetham et al., 1998)] and X-ray crystallography [PDB entry 1EER (Syed et al., 1998
)], respectively. The biomolecular simulation package NAMD (Humphrey et al., 1996
) was used to produce a final low-energy structure by energy minimization, followed by the equilibration of the protein in a solvent bath and 1 ps of molecular dynamics at 300 K. The mutations necessary to generate the structural models of Epo(NDS), mouse, pig and bovine Epo were made using the Swiss PDBViewer (Guex and Peitsch, 1997
) and low-energy structures of these models were also generated by energy minimization and solvated molecular dynamics at 300 K, with NAMD.
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Results |
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Design of an Epo moiety with a novel disulfide bonding pattern
To improve the properties of FcEpo, we examined the structure of Epo for possible weak points that could lead to poor assembly and to degradation in vivo. Two lines of evidence pointed to the region around Cys29, His32, Cys33 and Trp88, which encompasses loops connecting -helices of the four-helix bundle (Figure 1C). First, a comparison of the NMR structure 1BUY and the Epo in the X-ray crystal structure 1EER revealed significant differences in this area (Cheetham et al., 1998
; Syed et al., 1998
). In the NMR structure, the His32 side chain is buried next to Trp88 at the edge of the hydrophobic core of the protein. In contrast, the His32 side chain in the 1EER structure is flipped out towards the solvent, resulting in a cavity in this end of the protein relative to the 1BUY structure.
Second, an alignment of human Epo with other mammalian Epos revealed that the human protein is unique in having His32 at this position and in having a pair of disulfide-bonded cysteines at positions 29 and 33, but not at spatially nearby positions 88 or 139 (Wen et al., 1994) (Figure 1A and 1B). In contrast, rodent Epo has a pair of cysteines at positions 29 and 139, forming an alternative disulfide bond. Most mammals, however, have an odd number of cysteines, at positions 29, 33 and 139, which suggests that most mammalian Epos may be present as a mixture of isoforms with disulfides at either Cys29Cys33 or Cys29Cys139 (and possibly Cys33Cys139). Similarly, ungulates (cows and sheep) have cysteines at position 29, 33 and 88. (All mammalian Epos have a further cysteine pair at positions 7 and 161, which form a disulfide bond at the opposite end of the protein.) In addition, thrombopoietin, which is distantly related to Epo, has cysteines corresponding to positions 29 and 88 (Foster, 1994
).
To investigate the structure around these cysteine residues within Epo from different species, we constructed molecular models of Epo from mouse, pig and cow (Figure 1C). The models were energy-minimized and underwent 1 ps of molecular dynamics to allow the region around the modeled disulfide bonds to assume a reasonable conformation. In the structure for pig Epo, the Cys29Cys139 isomer was modeled, while for bovine Epo, the Cys29Cys88 isomer was modeled. As is evident in the models,, the beginning of the AB loop and the BC loop were covalently attached in the bovine Cys29Cys88 structure, whereas in the other structures, these portions of Epo were held together by hydrophobic contacts that were not shielded from the solvent (Figure 1C).
Based on these observations, we inferred that a novel disulfide bond could be introduced into human Epo between positions 29 and 88 without disrupting the protein's structure and that alteration of His32 might also stabilize the Epo structure. Accordingly, a model of Epo with the substitutions His32Gly, Cys33Pro, Trp88Cys and Pro90Ala, Epo(NDS) (New DiSulfide), was constructed as described. For position 32, glycine was chosen as it is present at position 32 in rodents (which lack Cys33) and in ungulates (which have Cys88). Proline was chosen for position 33, as this is the corresponding amino acid in rodents. Pro90 was replaced by alanine, which is present at this position in ungulates, since the formation of the Cys29Cys88 disulfide bond was expected to pull on the loop containing Cys88 and a non-proline residue at this position should relieve the resulting strain in this loop. The modeled Epo(NDS) structure was similar to the modeled bovine structure with the Cys29Cys88 bond.
Molecular dynamics simulations of Epo and Epo(NDS)
To gain insight into how the mutations in Epo(NDS) might stabilize the Epo structure, we compared wild-type Epo and Epo(NDS) by molecular dynamics simulations. Three molecules were analyzed: wild-type Epo with His32 in a neutral state, as would be expected at pH 7.4 in the extracellular space; wild-type Epo with His32 in a protonated state, as would be expected at pH 6 and below in the endosome; and Epo(NDS). The molecular models were constructed primarily using the 1BUY NMR structure, in which His32 is buried; in the wild-type models, the proton on the ND1 nitrogen of His32 formed a hydrogen bond with the amide carboxyl group of Pro87. Multiple, independent 30 ps molecular dynamics simulations of wild-type Epo with His32 in both the protonated and unprotonated states were carried out. For both the His32-protonated and His32-unprotonated states of Epo, in about two-thirds of the simulations a water molecule entered this region of the protein and formed a hydrogen bond with an N-H of His32, often disrupting the H-bond with Pro87 (data not shown).
In contrast, there was essentially no change in this region of Epo(NDS), even during long dynamics simulations. In addition, water molecules did not enter other regions of wild-type Epo or Epo(NDS) during any of the molecular dynamics simulations (data not shown). These results support the idea that solvent molecules can enter human Epo in this region, consistent with the idea that unfolding may be initiated at this end of erythropoietin.
Improved folding and stability of FcEpo(NDS)
An FcEpo(NDS) fusion protein was constructed, purified from NS/0 cells and compared with an otherwise identical FcEpo protein. First, the presence of the new disulfide bond Cys29Cys88 was confirmed by peptide mapping (Figure 2A). Peptides from trypsin-digested, non-reduced FcEpo(NDS) and FcEpo proteins (pretreated with PNGaseF to remove the N-linked oligosaccharides) were separated by HPLC and analyzed by mass spectrometry. Peptides P1 (Epo amino acids 2145) with a mass of 2687 and P2 (Epo amino acids 7797) with a mass of 2359 were present in the FcEpo sample, while corresponding peptides were absent in the FcEpo(NDS) sample. Conversely, peptide P1'+P2' [Epo(NDS) amino acids 2145/7797] of mass 4851 was identified uniquely in the FcEpo(NDS) sample, demonstrating the presence of the novel disulfide bond (Figure 2A).
To assess the purity of FcEpo and FcEpo(NDS) protein after Protein A purification, 5 µg of each protein were separated by denaturing, reducing SDSPAGE (Figure 2B). FcEpo and FcEpo(NDS) were found to migrate substantially as single bands with a molecular weight of 60 kDa, although faint additional bands were visible in the FcEpo lane, indicating that only trace amounts of additional proteins were co-purifiying with FcEpo (Figure 2B).
The proteins were further evaluated by SEC to assess the uniformity of the protein product. Figure 2C shows a typical SEC profile of these proteins. For FcEpo, 65% of the protein eluted at an apparent molecular weight of 100 kDa, corresponding to the FcEpo unit dimer, while the remaining material eluted with a much higher molecular weight, corresponding to FcEpo aggregates (left panel). In contrast, about 93% of the FcEpo(NDS) eluted with an apparent molecular weight of
100 kDa and only 7% of the protein appeared to be higher molecular weight aggregates (right panel). Thus FcEpo(NDS) was secreted from NS/0 cells in a much less aggregated state than FcEpo and the amount of high molecular weight material seen in FcEpo was not accounted for by the trace contamination seen in the SDSPAGE gel, suggesting superior folding properties of FcEpo(NDS).
To examine the role of the His32Gly and Pro90Ala mutation in FcEpo(NDS) folding, FcEpo proteins with the mutation sets (His32+ Cys33Pro Trp88Cys Pro90Ala), (His32Gly Cys33Pro Trp88Cys Pro90+) and (His32+ Cys33Pro Trp88Cys Pro90+) were expressed in NS/0 cells. The proteins containing Pro90+ were expressed as >70% high molecular weight aggregates and were not characterized further. The protein containing His32+ and Pro90Ala was expressed primarily in a non-aggregated state, similarly to FcEpo(NDS).
Next, we used a bioactivity assay to assess whether the NDS mutations stabilized the Epo structure (Table I). Normally, enzymatic removal of N-linked oligosaccharides destabilizes the Epo structure. Takeuchi et al. (1990) found that removal of N-linked oligosaccharides with PNGase F abolished the activity of Epo, as assayed by its ability to promote TF-1 cell proliferation. We found that the activity of PNGase F-treated FcEpo was also rapidly lost (1% activity after 2 h), but that the biological activity of PNGaseF-treated FcEpo NDS was lost only gradually (20% activity after 2 h). The rate of removal of N-linked sugars, assayed by SDSPAGE, was the same for FcEpo and FcEpo(NDS) and was complete within 1 h (data not shown). Hence, by this assay, FcEpo(NDS) was significantly more stable than FcEpo.
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To test the pharmacokinetic effect of the NDS mutation on FcEpo, CD1 mice were injected intravenously with Epo, FcEpo or FcEpo(NDS) and serum concentrations of the proteins were determined in a 48 h time course (Figure 3B). We found that FcEpo disappeared rapidly, even significantly more rapidly than Epo produced in CHO cells, reaching 0.3% of its initial concentration after 24 h. In contrast, FcEpo(NDS) was present at 3% of its initial concentration after 24 h. To rule out that in vivo clipping of the fusion proteins could account for the difference, serum samples were also processed by a PK-Western, but no evidence of in vivo cleavage was seen (data not shown). Similar results were obtained in rats (data not shown). Hence FcEpo(NDS) had significantly improved pharmacokinetics compared with FcEpo, most likely due to increased recycling of FcEpo(NDS) back into the blood stream.
The contribution of the His32Gly mutation to the improved pharmacokinetics of FcEpo(NDS) was tested by comparing FcEpo(NDS) molecules that were either His32+ or had the His32Gly mutation. Both molecules were expressed and secreted from NS/0 cells with equally low levels of aggregated protein. The pharmacokinetic profile in mice of FcEpo(NDS-His32+) was compared with those of other FcEpo proteins and found to be intermediate between those of FcEpo and FcEpo(NDS) (data not shown). This result suggests that both the novel disulfide and the His32Gly mutation contribute to the improved pharmacokinetics of FcEpo(NDS).
Lastly, we determined whether the significant increase in serum half-life of FcEpo(NDS) translated into an enhanced pharmacological activity. Mice were injected with a single injection of equimolar amounts of Epo, FcEpo or FcEpo(NDS) and hematocrit (red blood cells as a percentage of total blood volume) was determined over a period of 2 weeks (Figure 3C). Only a slight increase in hematocrit was observed in mice treated with Epo or FcEpo, which returned to baseline levels by day 11. In contrast, treatment with FcEpo(NDS) resulted in a hematocrit increase of 10% of blood volume; the hematocrit remained high at day 7 and returned to baseline levels by day 14. Hence FcEpo(NDS) also had enhanced pharmacological activity compared with FcEpo and these results suggested that less frequent dosing could be required with FcEpo(NDS).
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Discussion |
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Epo is a highly conserved protein: in pairwise alignments of Epo from mammals, 80% of amino acid are identical (Wen et al., 1994
) and Epo from humans, for example, functions in mice and rats and dogs. Surprisingly, the cysteine residues and disulfide bonding pattern of Epo varies among mammals, with all of the variation occuring in the neighborhood of Cys29 and Cys33 in the Epo three-dimensional structure. We took advantage of the polymorphism in Epo disulfide bonds to engineer a particular bond, Cys29Cys88, into human Epo. This bond is probably present in Epo from cows and sheep and in the related cytokine thrombopoietin. We also designed the mutations His32Gly and Pro90Ala to compensate for the rearranged structure near the novel disulfide bond; His32Gly was also expected to eliminate a possible conformational change that could occur upon acidification in the endosome.
The resulting quadruple-mutant protein, with His32Gly Cys33Pro Trp88Cys Pro90Ala in human Epo, Epo(NDS), was expressed as an FcEpo fusion protein and compared with the corresponding wild-type FcEpo protein. About 35% of the wild-type FcEpo protein was secreted as a high molecular weight aggregate, whereas at least 90% of the FcEpo(NDS) protein was secreted as an non-aggregated unit dimer.
The structural stability of Epo within FcEpo and FcEpo(NDS) was compared in several assays. Biological activity of the Epo moiety in FcEpo disappears upon enzymatic deglycosylation, most likely because the protein denatures [similarly to Epo itself (Takeuchi et al., 1990)]. In FcEpo(NDS), Epo biological activity is much more stable upon enzymatic removal of the N-linked sugars. Molecular modeling suggested that water molecules could enter the wild-type Epo structure and form hydrogen bonds with His32, whereas the NDS mutant protein appeared to be water-tight and resistant to the entry of water molecules into the protein core.
FcEpo expressed in NS/0 cells was an ideal molecule for the study of mutations that might enhance Epo pharmacokinetics by stabilizing the protein after receptor-mediated endocytosis. First, FcEpo has a molecular weight of 100 kDa, so that renal clearance is blocked. Second, since the IgG2 isotype of Fc was used, binding to Fc receptor was expected to play little role in clearance of FcEpo. Third, FcEpo [and also FcEpo(NDS)] expressed in NS/0 cells was only partially sialylated, containing only about five sialic acids per Epo monomer [data not shown; Epo from CHO cells, such as commercial Procrit, has
1012 sialic acids per monomer (Strickland, 1999
)]. Since the on-rate of Epo for its receptor, and therefore Epo clearance, is partly charge-driven, clearance increases with decreasing numbers of sialic acid residues on the N- and O-linked oligosaccharides of Epo (Strickland, 1999
; Egrie and Browne, 2001
). Fourth, the relatively low sialic acid content of FcEpo was expected to lead to increased uptake of FcEpo by the asialoglycoprotein receptor. Hence, compared with a commerical erythropoietin such as Procrit, FcEpo expressed from NS/0 cells would be expected to show enhanced receptor-mediated endocytosis, but no renal clearance. Indeed, FcEpo protein expressed in NS/0 cells had poor pharmacokinetics, disappearing from the circulation even faster than monomeric Epo from CHO cells. In the context of an FcEpo fusion protein, the NDS mutations have a significant effect on pharmacokinetics and biological activity. Whereas FcEpo had a terminal half-life of about 8 h, FcEpo(NDS) had a terminal half-life of about 16 h. As a result, the biological activity of FcEpo (NDS) was also significantly improved compared with FcEpo.
In conclusion, the results described here suggest that the region around Cys29, His32, Cys33 and Trp88 is a structural weak point in human Epo. Mutations in this region that enhance the structural stability of human Epo by altering the disulfide bonding pattern also contribute to the enhanced pharmacokinetics of FcEpo(NDS). We envision that FcEpo(NDS) may interact with FcRn and be actively recycled out of at least some cells that may take it up, such as Kupffer cells expressing the asialoglycoprotein receptor. In this situation, mutations that stabilize the structure of Epo may prevent proteolysis of FcEpo as it transits through endosomal compartments.
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Acknowledgements |
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References |
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Received February 10, 2004; revised February 27, 2005; accepted March 9, 2005.
Edited by Mark Zoller
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