Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein

Katalin F. Medzihradszky3, Daniel I.R. Spencer1,4, Surinder K. Sharma4, Jeetendra Bhatia4, R. Barbara Pedley4, David A. Read4, Richard H.J. Begent4 and Kerry A. Chester2,4

3 Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA 94143-0446; and 4 Cancer Research U.K. Targeting and Imaging Group, Department of Oncology, Royal Free and University College Medical School, University of London, Royal Free Campus, Rowland Hill Street, London, NW3 2PF, U.K.

Received on June 18, 2003; revised on August 31, 2003; accepted on August 31, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor. MFE-CP localizes to cancer deposits in vivo, but we propose that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris. Glycosylation could enhance clearance from healthy tissue and result in better tumor:normal tissue ratios. To test this, glycosylated MFE-CP was expressed and purified from P. pastoris. The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumor localization. This favorable glycosylation pattern was analyzed by tandem mass spectrometry. High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its nonglycosylated form. There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Nonglycosylated forms of Asn-442 and Asn-492 were not observed.

Key words: ADEPT / CID / mass spectrometry / N-glycosylation / Pichia pastoris


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
An ideal chemotherapeutic agent would be one that can be so specifically targeted that only cancerous cells are destroyed, leaving all healthy tissues unscathed. This can be achieved using antibody directed enzyme prodrug therapy (ADEPT) (Bagshawe, 1989Go, 1995Go). ADEPT is a two-phase system. In the first stage an antibody-enzyme conjugate is administered systemically and allowed to localize within the tumor and clear from normal tissues. When there is a high tumor to plasma ratio of enzyme, the second stage, a relatively nontoxic prodrug, is administered. The prodrug is catalyzed by the prelocalized enzyme to produce a potent cytotoxic agent selectively within the tumor. ADEPT has shown efficacy in several xenograft systems in nude mice (Sharma et al., 1991Go; Springer et al., 1991Go; Eccles et al., 1994Go; Pedley et al., 1999Go) and has also been tested in clinical trials (Bagshawe, 1995Go; Napier et al., 2000Go).

The ADEPT system we describe employs carboxypeptidase G2 (CPG2), a well-characterized, homodimeric, bacterial enzyme with no mammalian equivalent (Sherwood et al., 1985Go) linked chemically or genetically to antibodies reactive with carcinoembryonic antigen (CEA). CPG2 cleaves glutamic acid from a variety of prodrugs, releasing potent nitrogen mustard drugs within the tumor. Previous xenograft and clinical studies with this ADEPT system have shown that cytotoxic levels of active drug are generated in the tumor, but there is a need for accelerated clearance of the antibody-enzyme conjugate from blood and other normal tissues (Sharma et al., 1991Go, 1994Go; Bagshawe, 1995Go; Napier et al., 2000Go). Clearance can be achieved using a galactosylated monoclonal antibody directed at the active site of CPG2. The galactosylated antibody inactivates the conjugate and accelerates clearance via liver receptors (Sharma et al., 1990Go, 1991Go, 1994Go).

In clinical trials, the galactosylated antibody clearing system resulted in favorable tumor:normal tissue ratios of enzyme activity (Napier et al., 2000Go) showing that glycosylation is an effective means of removing protein therapeutics from circulation. However, the use of a clearing antibody would add the complexity of a third treatment stage for ADEPT. Seeking to optimize and simplify the system, we wanted to investigate whether glycosylation of the antibody-enzyme moiety itself would result in rapid clearance and favorable tumor:normal tissue ratios of enzyme activity.

The antibody-enzyme molecule designed for ADEPT was MFE-CP, a recombinant fusion protein of CPG2 with MFE-23, an anti-CEA single chain Fv antibody variable fragment (Chester et al., 1994Go; Begent et al., 1996Go; Michael et al., 1996Go). Tumor targeting potential of unglycosylated bacterially expressed MFE-CP has already been established in model systems (Bhatia et al., 2000Go). This work showed that MFE-CP could localize effectively to CEA-expressing tumors but did not clear rapidly from normal tissues.

We proposed that rapid clearance from the circulation may be obtained by adding branched mannose to MFE-CP, which would mediate uptake of MFE-CP by mannose receptors (Opanasopit et al., 2001Go). To achieve this, MFE-CP was obtained in glycosylated form by expression in the methylotropic yeast Pichia pastoris. P. pastoris typically glycosylates at N-glycosylation sequence motifs (Asn–Xaa–Ser/Thr) with branched mannose (Montesino et al., 1998Go; Bretthauer and Castellino, 1999Go). CPG2 features three consensus sequences for N-glycosylation, Asn-200, -242, and -250, corresponding to positions 442, 484, and 492 in the fusion protein, but it was not known how these sites would be occupied or how glycosylation would effect the behavior of MFE-CP in vivo.

This article establishes that glycosylation of MFE-CP does not destroy enzyme activity and leads to favorable biodistribution of MFE-CP in in vivo models of human tumor xenografts. The N-glycosylation pattern required to achieve this is subsequently analyzed in detail. The analysis was performed using a combination of reverse phase chromatography, electrospray ionization mass spectrometry (ESI-MS), and low-energy collision-induced dissociation (CID) analysis on a quadrupole-orthogonal-acceleration-time-of-flight tandem mass spectrometer.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In vivo distribution of MFE-CP
MFE-CP was purified from fermentation broth using affinity chromatography exploiting the recombinant C-terminal hexa-histidine tag. The purified protein was found to have CPG2 activity when tested using the methotrexate cleavage assay and was subsequently tested for clearance from the circulation and ability to localize to human tumor xenografts in nude mice. Results of these experiments are shown in Figures 1 and 2. The first experiment (Figure 1) demonstrates that MFE-CP enzyme activity cleared remarkably quickly from plasma. When 25 U of enzyme activity were administered to each mouse, the plasma concentration of enzyme activity was found to be reduced over 100-fold in the first 4 h. At this time point the CPG2 activity was 0.06 U/ml, an acceptable plasma level for prodrug administration (Sharma et al., 1990Go). Twenty-five units of nonglycosylated MFE-CP, administered at the same manner in previous experiments, did not reach this low plasma concentration until 72 h after injection (Bhatia et al., 2000Go).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Plasma clearance of CPG2 activity in nude mice bearing LS147T xenografts: groups of four mice received 25 U MFE-CP. Animals were sacrificed and catalytic activity in plasma assessed at 1, 2, 3, 4, 6, and 24 h after injection.

 



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2. Biodistribution of MFE-CP in nude mice bearing LS147T xenografts: one group of four mice received 25 U MFE-CP. Animals were sacrificed 4 h after injection and CPG2 catalytic activity assessed in plasma, liver, kidney, and tumor. (a) CPG2 concentrations; (b) tumor to normal tissue ratios.

 
Four hours was chosen as an appropriate time point to investigate whether the concentration of enzyme in the tumor was high enough to achieve therapy with P. pastoris– expressed MFE-CP. To test this, 25 U of MFE-CP was given by IV injection to nude mice bearing CEA-positive LS174T human colon adenocarcinoma xenografts. Animals were sacrificed at 4 h, and excised tissues were tested for enzyme activity. Results shown in Figure 2a demonstrated that over 1.5 U/g of CPG2 activity were present in the tumor at 4 h, which is in excess of the amount required for therapy. Furthermore, very little activity was detected in plasma, liver, or kidney at this time point, which resulted in favorable tumor to normal tissue ratios, as shown in Figure 2b.

The results shown in Figures 1 and 2 indicated that P. pastoris–expressed MFE-CP would have favorable characteristics for cancer therapy in comparison to nonglycosylated MFE-CP. The glycosylated MFE-CP presented herein gave a tumor to plasma ratio of over 50:1 at 4 h after administration (Figure 2b), whereas the highest tumor to plasma ratio for nonglycosylated MFE-CP was 19:1, and this was only achieved 48 h after administration (Bhatia et al., 2000Go). Furthermore, in a separate pilot experiment (Sharma et al., 2003Go), MFE-CP was shown to remain in the tumor when tested at 6 h after injection and in this instance gave 1.14 U/g of CPG2 in the tumor and a tumor:plasma ratio of 163:1. On the basis of these experiments, MFE-CP has been earmarked for clinical use and the c-myc tag has been removed from MFE-CP to avoid regulatory issues in potential clinic trials. This later version of P. pastoris–expressed MFE-CP has been evaluated for efficacy after prodrug administration in vivo. Preliminary results show growth delay or regressions in two xenograft models with minimum toxicity (Sharma et al., 2003Go).

However, detailed characterization of the MFE-CP glycoforms obtained in P. pastoris is essential to understand the biological behavior of glycosylated MFE-CP. For example, mammalian glycosylation of CPG2 results in loss of enzyme activity (Marais et al., 1997Go) and increased mannose may lead to slower clearance due to interactions with the serum mannose-binding protein (Opanasopit et al., 2001Go). Furthermore, because MFE-CP is destined for clinical use, a fuller knowledge of its structure is desirable for safety and documentation. We therefore determined the glycosylation of MFE-CP more accurately using MS.

Glycosylation analysis
Glycosylation patterns of P. pastoris expressed MFE-CP were analyzed in terms of size of the carbohydrates as well as site occupancy and heterogeneity. The fusion protein was initially digested with trypsin overnight (12 h) and subjected to reverse phase, microbore liquid chromatography (LC)/ESI-MS analysis. Because the flow had to be split to accommodate online mass detection, fractions were also collected manually at the split. These fractions were concentrated and used later for offline MS and CID analysis by nanospray sample introduction (see later discussion). Online mass detection was carried out using a "ping-pong" acquisition set up. Every regular MS acquisition was followed by data acquisition at increased nozzle potential in order to induce in-source fragmentation. Glycopeptides fragment readily under these conditions and produce diagnostic carbohydrate ions (Carr et al., 1993Go; Huddleston et al., 1993Go). All N-linked structures yield an abundant fragment at m/z 204.09, that is, the oxonium ion for N-acetylhexosamines (Domon and Costello, 1988Go). Thus potential glycopeptide-containing chromatographic peaks were identified by generating an extracted ion profile of m/z 204.1 ± 0.2 for all the data acquired under in-source fragmentation conditions.

As expected, the LC/MS chromatogram contained at least two distinct 204-positive fractions (data not shown). The corresponding low nozzle potential MS data for these 204-positive fractions were analyzed. We searched for multiple charged ion series yielding molecular masses 162 Da apart, that is, for signals reflecting the heterogeneity in the oligomannose structure. None were found. Similarly, a search for multiply charged ions representing predicted tryptic peptides with the consensus sites bearing Man8 or Man9 structures was not successful. No glycopeptides were identified. Unfortunately, peptides having a Gly-Lys C-terminus also may yield a fragment at m/z 204.1, a y2 ion. The fusion protein has Gly-Lys sequences at six different positions, and some of these legitimate tryptic peptides underwent in-source fragmentation and produced m/z 204.1 ions. After careful analysis of the complete LC/MS data, only consensus peptide, [483–489]—MH+ calculated: 879.47, measured: 879.43—was found, unglycosylated; the other consensus peptides were not identified in any form. At the same time numerous components yielding abundant ions did not match any of the predicted tryptic peptides.

Because there were so many unexplained peptides in the digest, contamination was suspected and the collected fractions were subjected to further mass analyses. Mastrix-assisted laser desorption ionization (MALDI)-postsource delay (PSD) results suggested that the components unaccounted for were the results of nonspecific cleavages. One of the reasons could be that the digestion time (12 h) was too long. At the same time long sequence stretches were detected containing "missed" cleavage sites. Thus the next digestion was performed in 2 M urea to denature the protein, but the incubation lasted for only 4 h.

This tryptic digest was also subjected to LC/MS analysis. To ensure the detection of glycopeptides, the sample was separated on a capillary column, and thus a more concentrated solution without a split was introduced in the mass spectrometer. The trifluoracetic acid (TFA)-containing mobile phase was also replaced by a more MS-friendly solvent system: formic acid was used as the ion-pairing reagent. Again in-source fragmentation was induced in alternative scans, and the extracted ion chromatogram for m/z 204.1 was generated (Figure 3). The profile of 204-ion time-distribution was essentially the same as in the previous analysis. However, careful examination of the "fraction" eluting at ~55 min data (Figure 4) revealed the presence of glycopeptides. This fraction contained multiple peptide components with MH+s at m/z 1020.51, 1107.48, 1184.66, 1630.8, 1657.71, 1777.84 and 2014.98 in form of doubly and triply charged ions. Only two of these components corresponded to predicted tryptic products: 1107.51 and 2015.12 are the calculated masses for peptides [31–38] and [1–19], respectively. This fraction also featured a series of ions that may represent consensus-peptide [490–508] bearing Man8 and Man9 structures (Figure 4, Table I).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. LC/ESI-MS chromatogram of a tryptic digest of the fusion protein acquired on an electrospray-orthogonal acceleration-time-of-flight mass spectrometer with a "ping-pong" acquisition setup, that is, with nozzle voltage switched to low and high values. Higher voltage induces in-source fragmentation. The upper panel shows the base peak ion (BPI) trace for the normal MS acquisitions; the lower panel shows the extracted ion chromatogram of the N-acetylhexosamine oxonium ion at m/z 204.1 from the high nozzle voltage acquisitions. Glycopeptides were identified only in the peak labeled (see Figure 4).

 


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. MS data for the glycopeptide-containing peak labeled in Figure 3. Summed low nozzle voltage scans corresponding to the 204+ peak are presented here. Only ions representing the most abundant peptide-component and the glycopeptides are labeled. The upper panel shows the full mass range monitored; the lower panel is a zoom-in on the glycopeptide ions. The inset shows the resolution afforded by the Mariner mass spectrometer.

 

View this table:
[in this window]
[in a new window]
 
Table I. Ions representing tryptic peptide [490–508] glycosylated in Figure 4

 
Interestingly, ammonia and Fe(III) adducts were detected for some of the glycopeptide ions as well as for some of the peptide components (Figure 4). We found that Fe(III) adducts are formed regularly when formic acid is used as the ion-pairing reagent, and ions of higher charge states usually display this adduct with higher intensity. No other glycopeptides were identified in this LC/ESI-MS experiment. Peptide [483–489] was consistently observed without modification. The third consensus peptide was not identified in any form, and most of the nonspecific cleavage products seemed to be identical to those observed in the first experiment as confirmed by CID data (data not shown).

To confirm these assignments, to determine the nature of the nonspecific cleavages, and to find the third consensus sequence modified or unmodified, the fractions collected during the first LC/MS experiments were concentrated and subjected to electrospray MS and low-energy CID experiments via nanospray sample introduction. During these analyses the molecular masses of the components were determined first. Species matching predicted tryptic peptides were usually not analyzed further. Potential glycopeptides were identified by a series of ions of the same charge state that represented molecular masses 162 Da apart. The most abundant ions of these series were subjected to low energy CID experiments.

Figure 5 shows the ESI-MS mass spectrum of a typical glycopeptide-containing fraction. The major component is a peptide (MH+ = 823.29), the product of nonspecific cleavages and its identity was not determined, a series of potential glycopeptide ions, 720.23(3+), 828.24(3+), 882.29(3+), and 1044.35(3+) were subjected to low-energy CID instead. These CID experiments yielded good quality spectra, as illustrated in Figure 6. This CID spectrum was acquired selecting m/z 828.25(3+) as the precursor ion. The spectrum features the expected oxonium ions at m/z 163 and 204 corresponding to hexose and N-acetylhexosamine, respectively. However, most other carbohydrate ions were observed as Na adducts, indicating that the precursor ion also must have contained Na+, most likely the nanospray capillary being the source of the metal ions. Fragments with charge retention at the nonreducing terminus such as m/z 347.08, 509.13, and 671.18 (Hex2–Hex4) and m/z 874.24, 1036.30, 1198.33, 1360.41, 1522.48, and 1684.6 (Hex4HexNAc–Hex9HexNAc) permitted "building up" the carbohydrate structure and revealed its identity as a Man9GlcNAc2-structure (Scheme 1).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5. Partial ESI-MS spectrum of a concentrated fraction of the first digest (retention time ~40 min), acquired by nanospray sample introduction. Some of the glycopeptide ions are presented in comparison to a coeluting peptide (MH+ m/z 823.29, not characterized, but not tryptic). The low relative intensity of glycopeptide ions, as demonstrated, was typical throughout the analysis of the collected fractions—because of the substoichiometric quantities of the glycopeptides due to carbohydrate heterogeneity as well as nonspecific proteolytic cleavages. In addition, the glycopeptides readily formed Na and K adducts. The inset shows the high resolution afforded by the QSTAR mass spectrometer.

 


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 6. Low-energy CID spectrum of precursor ion at m/z 828.25(3+) shown in Figure 5. This ion corresponds to [MHNa2]3+ of 440QVN(GlcNAc2Man9)IT444. The carbohydrate fragments labeled with their sugar composition are B-type oxonium ions or their Na adducts (as indicated): the glycosidic bond was cleaved, and the charge was retained at the nonreducing terminus. The Y-type fragments are formed also via glycosidic bond cleavages with charge retention at the reducing end. These fragments are labeled indicating the hexose losses from the full structure. The identity and the linkages of the hexoses and N-acetylhexosamines cannot be determined from these data.

 


View larger version (25K):
[in this window]
[in a new window]
 
Scheme 1. Fragments observed in the low-energy CID spectrum of [MH2Na]3+ of glycopeptide QV(Man9GlcNAc2)NIT (Figure 6).

 
Carbohydrate cleavages from the molecule were also detected (Figure 6, Scheme 1) in the form of doubly charged ions at m/z 1160.92, 1079.88, 998.86, 917.84, and 836.80, indicating 1–5 hexose losses, respectively. If the mass of the Man9GlcNAc Na fragment observed is subtracted from the singly charged molecular mass determined the result should correspond to the neutral peptide modified by a single N-acetylglucosamine: 2482 - 1684 = 798. A proton has to be added for ionization, and indeed, there is an abundant fragment at m/z 799.36 in the spectrum. Eliminating the 203-Da residue weight of the sugar unit, the MH+ value of the unmodified peptide must be ~596.3. However, no peptide containing any of the potential glycosylation sites of the fusion protein corresponds to this mass value. From MS data it was suspected that the precursor ion contained two Na+s, and one of them was retained with the peptide. Taken this into consideration, the MH+ for the unmodified peptide will be m/z 574.3 that corresponds to QVNIT, containing a consensus sequence indeed.

Interestingly, most of the abundant glycopeptide ions selected for CID analysis proved to be Na or Na2 adducts. However, this worked to our advantage: the carbohydrate structure retained the Na+, and via glycosidic bond cleavages it formed series of Y- and B-ions, with charge retention at the reducing or nonreducing end, respectively (Domon and Costello, 1988Go). No peptide fragmentation was observed in these experiments, and the peptides always retained one of the core N-acetylglucosamines. CID spectra of multiply protonated glycopeptides exhibited peptide fragmentation and less complete carbohydrate ion series. Interpretation of the CID spectra allowed the determination of the size of the carbohydrate as well as the identification of the peptide modified. There were instances when the overlapping ions of two different glycopeptides were selected as precursors for CID data acquisition (Table II). In these CID spectra two distinct N-acetylglucosamine-bearing peptides were detected, and thus the identity of the glycopeptides could be established. Some other glycopeptides eluting in the same or adjacent fractions were identified by mass measured, drawing conclusions from the mass differences in comparison to the species sequenced. Some peptide ions corresponding to molecules that did not fit the predicted tryptic digest were also subjected to low-energy CID analysis. Peptides lacking basic residues also frequently formed Na or K adducts. Interpretation of these CID data was complicated by the fact that not all of the fragments retained the metal ions (data not shown).


View this table:
[in this window]
[in a new window]
 
Table II. Glycoforms detected of Asn-442

 
In summary, low-energy CID experiments on masses previously unaccounted for confirmed that the protein was cleaved not only at tryptic and chymotryptic sites but practically at any amino acid indiscriminately, and in certain instances C-terminal ladder peptide series were formed. A likely explanation for this is that parallel to the tryptic digestion, a fusion-protein autolysis may have occurred. Eventually, all three consensus sequences were detected and characterized in these experiments, as follows.

Consensus site Asn-442 was found in multiple, nonspecifically cleaved peptides and was always glycosylated (Table II). This site was found to be modified with nine different oligomannose structures from Man5GlcNAc2 to Man13GlcNAc2.

Predicted tryptic peptide containing Asn-484 was observed in both digests unmodified. No glycopeptide representing this consensus site was detected in the nanospray experiments either. Furthermore, low-energy CID analysis of the peptide with MH+ at m/z 879.45 indeed confirmed its sequence as 483FNWTIAK489.

The third consensus site, Asn-492, was detected in the second LC/MS experiment, in a predicted tryptic peptide bearing Man8GlcNAc2- and Man9GlcNAc2-oligomannose structures. The same species were detected in one of the fractions by the nanoscale electrospray experiments (Table III). The identities of these molecules were confirmed by performing low-energy CID analyses selecting the triply charged ions of these molecules as precursor ions. Shorter peptides containing this site, formed by nonspecific enzyme cleavages, were also detected featuring the same carbohydrate structures. In addition, one of these molecules also was observed modified by a Man10GlcNAc2 structure.


View this table:
[in this window]
[in a new window]
 
Table III. Glycoforms detected of Asn-492

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Recombinant proteins have many advantages over the chemically modified fragments originally employed for ADEPT. They have the potential to be tailored to give optimal results and overcome hurdles such as immunogenicity and tumor penetration. They are readily defined and manipulated at the molecular level, and the complexity, expense, and difficulty of obtaining a reproducible product with chemical conjugation of the components may be avoided. Furthermore, they can be produced in organisms such as yeast, which allows high protein yields and posttranslational glycosylation but avoids the problem of contamination inherent with mammalian or viral DNA.

We originally expressed MFE-CP in unglycosylated format using a bacterial host (Michael et al., 1996Go; Bhatia et al., 2000Go). This unglycoslyated MFE-CP showed efficacy of localization, but we predicted that the therapeutic potential could be improved by accelerating clearance to give better tumor:normal tissue ratios. Theoretically, rapid clearance could be achieved by decorating the recombinant protein with branched mannose as may be obtained by expression in yeast P. pastoris, where the majority of glycoforms that have been identified on glycoproteins expressed in the organism are Man8GlcNAc2 and Man9GlcNAc2 structures. Shorter oligomannoside structures have also been reported in P. pastoris–expressed proteins and up to Man18GlcNAc2 found on aspartic protease and enterokinase expressed in this organism (Montesino et al., 1998Go; Bretthauer and Castellino, 1999Go). These glycosylated molecules can react with one of the pattern recognition mannose-binding proteins, which occur in soluble form in circulation or as cell surface receptors on macrophages, dendritic cells, and hepatic endothelial cells (Fraser et al., 1998Go; East and Isacke, 2002Go; Gordon, 2002Go). As the list of mannosyl receptors continues to grow and their ligands become better characterized, it is becoming evident that certain carbohydrate motifs will preferentially target particular receptors, resulting in a different biological fate, including the rate of clearance (Opanasopit et al., 2001Go; Roseman and Baenziger, 2001Go). Thus it is desirable to characterize expressed glycoproteins as extensively as possible to understand their biological behavior.

To test our hypothesis that mannose motifs of the P. pastoris type would target MFE-CP for rapid clearance and improve therapeutic ratios, we cloned and expressed MFE-CP in P. pastoris. After purification, we tested for function by measuring in vivo localization to human tumor xenografts in a mouse model. Critically for ADEPT the glycoforms should not only have a highly favorable effect on the clearance of the therapeutic from the bloodstream but also not impair biological function of the fusion protein, in particular its ability to localize enzyme to the tumor.

Characterization of the glycoforms obtained by P. pastoris expression of MFE-CP was important, and we used LC/MS as well as nanospray sample introduction of collected fractions for mass analysis and low-energy CID for this task, which exploited the presence of Na+ adducts. The results showed that high mass resolution and high detection sensitivity were essential to identify all the coeluting glycopeptides and to decipher their structures. Our data also suggest that the removal/lack of basic residues may lead to increased metal adduct formation. However, the presence of these metal ions seems to be beneficial for the analysis of glycopeptides—Na or Na2 adducts produce more comprehensive fragmentation than the corresponding "just" protonated ions. The results showed that only two of the three potential sites were occupied. Of these, Asn-442 was most heterogeneous, showing nine different oligomannose structures, from Man5GlcNAc2 to Man13GlcNAc2. Asn-492 was largely populated by Man8GlcNAc2 and Man9GlcNAc2, although one Man10GlcNAc2 structure was observed. MFE-CP is a homodimer, so this would result in four glycosylation motifs per molecule. The in vivo data obtained show clearly that this glycosylation pattern is appropriate to target the molecule for accelerated clearance. The glycosylated MFE-CP cleared to less than 1/1000 of its original plasma value by 6 h after administration, resulting in less than 0.04% the injected activity/ml. In contrast, unglycosylated MFE-CP showed over 15% of the injected activity/ml of plasma at this time point (Bhatia et al., 2000Go). Furthermore, the glycosylation pattern was not detrimental to efficacy as MFE-CP localized to its tumor target, despite its rapid clearance from circulation to give a tumor:plasma ratio in excess of 50:1, which has not previously been achieved (Bhatia et al., 2000Go). The results also showed that unglycosylated forms of Asn-442 and Asn-492 were not present, which indicates that all MFE-CP molecules will be targeted for clearance. This is important in a therapeutic setting because even low amounts of unglycosylated MFE-CP may persist in circulation and activate prodrug in normal tissues.

The observation that Asn-484 was always unglycosylated also gives insight into the biology of MFE-CP. It has been reported that the CPG2 is inactivated when all three potential glycosylation sites are occupied (Marais et al., 1997Go). Activity was regained by mutating all three sites to remove the N-glycosylation consensus sequence (Marais et al., 1997Go). Our results suggest that only glycosylation of Asn-484 (equivalent to position 264 in Marais et al., 1997Go) is responsible for enzyme inactivation as we obtained enzymatically active MFE-CP with the other positions glycosylated.

In conclusion, the work in this manuscript offers an improved targeting system to those previously reported for ADEPT with CPG2 and describes a method suitable to characterize the carbohydrate structures responsible for the improvement.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
MFE-CP
The gene fusion of MFE-23 with CPG2 and expression in bacteria has been described previously (Michael et al., 1996Go). For expression in P. pastoris, the MFE-CP construct from pPM331 (i.e., no linker between MFE-23 and CPG2) (Michael et al., 1996Go) was inserted into pPICZ{alpha}B (Invitrogen, Carlsbad, CA), as a SfiI–XbaI fragment. This was achieved using the existing pPM331 SfiI site at the 5' start of MFE-23 (Michael et al., 1996Go) and by creating a unique XbaI at the 3' end of GPG2. Transformants were selected on Zeocin according to the manufacturer's instructions. Integration of the MFE-CP/pPICZ{alpha}B construct into P. pastoris cells resulted in expression of MFE-CP with a C-terminal c-myc and 6xHis (histidine) tag. Fermentation of the MFE-CP/pPICZ{alpha}B transformed P. pastoris was kindly performed by Invitrogen, and the clarified supernatant was stored at -80°C prior to purification. MFE-CP fusion protein was purified from the cell-free supernatant using immobilized metal affinity chromatography employing the His 6 tag as previously described (Mayer et al., 1998Go).

CPG2 catalytic activity
CPG2 activity was measured in plasma by incubating 10 µl of sample in 1 ml assay buffer (100 mM Tris–HCl, pH 7.3, 0.2 mM ZnSO4, and 60 mM methotrexate [MTX]), at 37°C for 1 min. Change in absorbance of MTX after CPG2 hydrolysis was measured by spectrophotometry at 320 nm (Beckman DU-64 spectrophotometer, Soft Pac Module KINETICS Software Package, Beckman Instruments, Bucks, U.K.). Enzyme activity was expressed in units (U), where one unit is the amount of enzyme required to hydrolyze 1 mmol of MTX per min at 37°C.

CPG2 activity in excised tumor and mouse organs was estimated using an indirect high-performance liquid chromatography (HPLC) method described previously for homogenized solid tissues (Blakey et al., 1996Go; Bhatia et al., 2000Go).

In vivo distribution and pharmacokinetics
Biodistribution studies were performed in nude mice bearing LS174T colorectal xenografts. Twenty-five units of MFE-CP was administered into the tail vein at time 0, and one group of four mice was sacrificed at 4 h. Liver, kidney, and tumor were removed and homogenized. Enzyme activity was measured by the HPLC procedure already described. Enzyme activity in plasma was assessed at selected time points by spectrophotometry as described.

Tryptic digestions
For the first digestion 0.1 µg trypsin (porcine, side-chain-protected, Promega, Madison, WI) was added to 100 pmoles (~7 µg) of the fusion protein dissolved in 50 mM NH4HCO3 buffer and was incubated with it overnight at 37°C. The digestion volume was 35 µl. For the second digestion 20 µl of the original protein solution (~70 pmoles or ~5 µg) was mixed with 8 µl 8 M urea in 200 mM NH4HCO3 buffer, and 2 µl trypsin solution (0.05 µg/µl in 0.01% TFA) was added to it. This mixture was incubated at 37°C for 4 h.

LC/MS analysis
The tryptic digests were analyzed by reversed-phase HPLC directly coupled with electrospray MS. The reversed phase chromatography was carried out using an Applied Biosystems (Foster City, CA) 140B HPLC system. A Mariner (Applied Biosystems, Framingham, MA) ESI-orthogonal-acceleration-time-of-flight mass spectrometer served as the MS detector. The mass spectrometer was operated in a "ping-pong" set-up: in-source fragmentation was induced in every second scan by increasing the nozzle voltage to 320 V; in normal data acquisition mode the nozzle potential was set at 100 V. The mass range monitored was m/z 200–2000.

The first digest was separated on a microbore column (Vydac, Vesperia, CA, C-18, 1.0 x 150 mm) at a flow rate of 50 µl/min. Solvent A was 0.1% TFA in H2O. Solvent B was 0.08% TFA in acetonitrile. The column was equilibrated in 2% solvent B. The eluent was kept isocratic for 5 min after the injection, then solvent B concentration was linearly increased to 40% in 60 min. The eluting peptides were monitored by UV absorbance at 210 nm. The eluent was split postdetector at a ratio of ~1:4, with approximately 20% introduced into the mass spectrometer. Fractions were collected manually at the split.

The second digest was separated on a capillary column (LC-Packings, San Francisco, CA, C-18, 180 µm x 150 mm) at a flow rate of 1 µl/min. Solvent A was 0.1% formic acid in H2O. Solvent B was 0.05% formic acid in EtOH/PrOH 5:2. The gradient was developed as already described. All of the column eluent was introduced in the mass spectrometer, no fractions were collected this time.

MS of the fractions collected during the first LC/MS analysis
MALDI and MALDI-PSD analyses were carried out on a Voyager Elite (Applied Biosystems) MALDI-TOF mass spectrometer. ESI-MS and low-energy CID analyses of the collected fractions were carried out on a quadrupole-orthogonal-acceleration-time-of-flight hybrid tandem mass spectrometer, QSTAR (MDS Sciex, Toronto, Canada), equipped with a Protana (Odense, Denmark) nanospray source.


    Acknowledgements
 
We thank Tom Purcell and August Sick at Invitrogen for fermentation of MFE-CP, Al Burlingame and Mike Waterfield for support and advice, and David Blakey for expert reading of the manuscript. K.F.M. was supported by NIH NCRR 01614 and RR12961 grants (to the UCSF MS Facility). Other authors supported by Cancer Research U.K. and the Ronald Raven Cancer Trust.


    Footnotes
 
1 Present address: Ludger Ltd., Littlemore Park, Oxford, OX4 4SS, U.K. Back

2 To whom correspondence should be addressed; email: k.chester{at}ucl.ac.uk Back


    Abbreviations
 
ADEPT, antibody directed enzyme prodrug therapy; CEA, carcinoembryonic antigen; CID, collision-induced dissociation; CPG2, carboxypeptidase enzyme G2; ESI-MS, electrospray ionization mass spectrometry; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MTX, methotrexate; PSD, postsource decay; TFA, trifluoracetic acid


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Bagshawe, K.D. (1989) Towards generating cytotoxic agents at cancer sites. Br. J. Cancer, 60, 275–281.[ISI][Medline]

Bagshawe, K.D. (1995) Antibody-directed enzyme prodrug therapy: a review. Drug Dev. Res., 34, 220–230.[ISI]

Bagshawe, K.D., Sharma, S.K., Springer, C.J., and Antoniw, P. (1995) Antibody directed enzyme prodrug therapy: a pilot-scale clinical trial. Tumor Targeting, 1, 17–29.

Begent, R.H., Verhaar, M.J., Chester K.A., Casey, J.L., Green, A.J., Napier, M.P., Hope-Stone, L.D., Cushen, N., Keep, P.A., Johnson, C.J., and others. (1996) Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library. Nat. Med., 2, 979–984.[ISI][Medline]

Bhatia, J., Sharma, S.K., Chester, K.A., Pedley, R.B., Boden, R.W., Read, D.A., Boxer, G.M., Michael, N.P., and Begent, R.H.J. (2000) Catalytic activity of an in vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein. Int. J. Cancer, 85, 571–577.[CrossRef][ISI][Medline]

Blakey, D.C., Burke, P.J., Davies, D.H., Dowell, R.I., East, S.J., Eckersley, K.P., Fitton, J.E., McDaid, J., Melton, R.G., Niculescu-Duvaz, I., and others. (1996) ZD2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts. Cancer Res., 56, 3287–3292.[Abstract]

Bretthauer, R.K. and Castellino, F.J. (1999) Glycosylation of Pichia pastoris–derived proteins. Biotechnol. Appl. Biochem., 30, 193–200.[ISI][Medline]

Carr, S.A, Huddleston, M.J., and Bean, M.F. (1993) Selective identification and differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid chromatography-mass spectrometry. Protein Sci., 2, 183–196.[Abstract/Free Full Text]

Chester, K.A., Begent, R.H., Robson, L., Keep, P., Pedley, R.B., Boden, J.A., Boxer, G., Green, A., Winter, G., and Cochet, O. (1994) Phage libraries for generation of clinically useful antibodies. Lancet, 343, 455–456.[CrossRef][ISI][Medline]

Domon, B. and Costello, C.E. (1988) A systematic nomenclature for carbohydrate fragmentation in FAB-MS MS spectra of glycoconjugates. Glycoconj. J., 5, 397–409.[ISI]

East, L. and Isacke, C.M. (2002) The mannose receptor family. Biochim. Biophys. Acta, 1572, 364–386.[ISI][Medline]

Eccles, S.A., Court, W.J., Box, G.A., Dean, C.J., Melton, R.G., and Springer, C.J. (1994) Regression of established breast carcinoma xenografts with antibody-directed enzyme prodrug therapy against c-erbB2. Cancer Res., 54, 5171–5177.[Abstract]

Fraser, I.P., Koziel, H., and Ezekowitz, R.A. (1998) The serum mannose-binding protein and the macrophage mannose receptor are pattern recognition molecules that link innate and adaptive immunity. Semin. Immunol., 10, 363–372.[CrossRef][ISI][Medline]

Gordon, S. (2002) Pattern recognition receptors: doubling up for the innate immune response. Cell, 111, 927–930.[ISI][Medline]

Huddleston, M.J., Bean, M.F., and Carr, S.A. (1993) Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests. Anal. Chem., 65, 877–884.[ISI][Medline]

Marais, R., Spooner, R.A., Stribbling, S.M., Light, Y., Martin, J., and Springer, C.J. (1997) A cell surface tethered enzyme improves efficiency in gene-directed enzyme prodrug therapy. Nat. Biotechnol., 15, 1373–1377.[ISI][Medline]

Mayer, A., Chester, K.A., Bhatia, J., Pedley, R.B., Read, D.A., Boxer, G.M., and Begent, R.H.J. (1998) Exemplifying guidelines for preparation of recombinant DNA products in phase I trials in cancer: preparation of a genetically engineered anti-CEA single chain Fv antibody. Eur. J. Cancer, 34, 968–976.[CrossRef][ISI][Medline]

Michael, N.P., Chester, K.A., Melton, R.G., Robson, L., Nicholas, W., Boden, J.A., Pedley, R.B., Begent, R.H.J., Sherwood, F.R., and Minton, P.N. (1996) In vitro and in vivo characterization of a recombinant carboxypeptidase G2::anti-CEA scFv fusion protein. Immunotechnology, 2, 47–57.[CrossRef][ISI][Medline]

Montesino, R., Garcia, R., Quintero, O., and Cremata, J.A. (1998) Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris. Prot. Express. Purif., 14, 197–207.

Napier, M.P., Sharma, S.K., Springer, C.J., Bagshawe, K.D., Green, A.J., Martin, J., Stribbling, S.M., Cushen, N., O'Mally, D., and Begent, R.H.J. (2000) Antibody-directed enzyme prodrug therapy, efficacy and mechanism of action in colorectal carcinoma. Clin. Cancer Res., 6, 765–772.[Abstract/Free Full Text]

Opanasopit, P., Shirashi, K., Nishikawa, M., Yamashita, F., Takakura, Y., and Hashida, M. (2001) In vivo recognition of mannosylated proteins by hepatic mannose receptors and mannan-binding protein. Am. J. Physiol. Gastrointest. Liver Physiol., 280, G879–G889.[Abstract/Free Full Text]

Pedley, R.B., Sharma, S.K, Boxer, G.M., Boden, R., Stribbling, S.M., Davies, L., Springer, C.J., and Begent, R.H.J. (1999) Enhancement of antibody-directed enzyme prodrug therapy in colorectal xenografts by an antivascular agent. Cancer Res., 59, 3998–4003.[Abstract/Free Full Text]

Roseman, D.S. and Baenziger, J.U. (2001) The mannose/ N-acetylgalactosamine-4-SO4 receptor displays greater specificity for multivalent than monovalent ligands. J. Biol. Chem., 276, 17052–17057.[Abstract/Free Full Text]

Sharma, S.K., Bagshawe, K.D., Burke, P.J., Boden, R.W., and Roger, G.T. (1990) Inactivation and clearance of the anti-CEA carboxypeptidase G2 conjugate in blood after localisation in a xenograft model. Br. J. Cancer, 61, 659–662.[ISI][Medline]

Sharma, S.K., Bagshawe, K.D., Springer, C.J., Burke, P.J., Rogers, G.T., Boden, J.A., Antoniw, P., Melton, R.G., and Sherwood, R.F. (1991) Antibody directed enzyme prodrug therapy (ADEPT): a three phase system. Dis. Markers, 9, 225–231.[ISI][Medline]

Sharma, S.K., Bagshawe, K.D., Burke, P.J., Roger, G.T., Boden, J, A., Springer, C.J., Melton, R.G., and Sherwood, F.R. (1994) Galactosylated antibodies and antibody-enzyme conjugates in antibody-directed enzyme prodrug therapy. Cancer, 73, 1114–1120.[ISI][Medline]

Sharma, S.K., Pedley, R.B., Irwin, H.L., El-Emir, E., Boxer, G.M., Boden, R.W., Chester, K.A., and Begent, R.H.J. (2003). The effect of ADEPT, as a single or combined therapy, in two colorectal xenografts. Brit. J. Cancer, 88(suppl 1), S37

Sherwood, F.R., Melton, R.G., Alwan, S.M., and Hughes P. (1985) Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16, Use of a novel triazine dye affinity method. Eur. J. Biochem., 148, 447–453.[Abstract]

Springer, C.J., Bagshawe, K.D., Sharma, S.K., Searle, F., Boden, J.A., Antoniw, P., Burke, P.J., Rogers, G.T., Sherwood, R.F., and Melton, R.G. (1991) Ablation of human choriocarcinoma xenografts in nude mice by antibody-directed enzyme prodrug therapy (ADEPT) with three novel compounds. Eur. J. Cancer, 11, 1361–1366.