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1 CSIRO Health Sciences and Nutrition, Parkville, Victoria 3052, Australia
2 CRC for Diagnostics, Parkville, Victoria 3052, Australia
3 Tumor Targeting Program, Ludwig Institute for Cancer Research, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia
Reprint requests to: Barbara E. Power, CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, VIC 3052, Australia; e-mail: barbara.power{at}csiro.au; fax: 61-3-9662-7314.
(RECEIVED August 14, 2002; FINAL REVISION December 10, 2002; ACCEPTED December 10, 2002)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0228503.
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Abstract |
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Keywords: scFv multimers; diabody; triabody; trimer; tetrabody; anti-Lewisy antibody; hu3S193
Abbreviations: scFvs, single-chain variable fragments • VH, variable heavy-chain domain • VL, variable light-chain domain • V
, kappa light chain • Fv, variable fragment consisting of VH and VL domains
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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The use of scFvs as immunodiagnostic and immunotherapeutic reagents is gaining momentum (Wu et al. 1996; Adams et al. 1998a; Pavlinkova et al. 1999; Yazaki et al. 2001). ScFv multimers such as diabodies (55 kD) and minibodies (80 kD) are significantly larger than scFv monomers and have in vivo application advantages of slower clearance from the circulation, good tumor accumulation, and show an increase in binding avidity (Wu et al. 1996; Adams et al. 1998b, Adams and Schier 1999). Recent studies using radiolabeled diabodies in vivo showed these molecules have a significant advantage over monomeric scFvs as indicated by tumor:blood ratios and better performance than disulphide-linked scFv monomers and F(ab')2 (Adams et al. 1998b, 1999).
The maximum peak tumor uptake of diabodies (~55 kD) is within the range of 5–15% injected dose per gram (%ID/g), which is unlikely to deliver sufficient of a radiolabeled ß-emitter for therapeutic efficacy. Diabodies will therefore require tagging with a toxic agent that is effective at low doses (Bera et al. 1999) or alternatively labeled with a strong 
-emitter (Adams et al. 2000; McDevitt et al. 2001) to achieve effective tumor killing. scFv Trimers (~80 kD) are likely to provide higher %ID/g tumor uptake because of the increased avidity providing increased accumulation of scFv at the tumor site and therefore are more likely to provide therapeutic levels of reagent for tumor killing. Hu et al. (1996) have shown that anti-CEA T84.66 scFv minibody (80 kD) exhibited pharmacokinetic properties more therapeutically effective than the smaller diabodies.
We have previously shown that an scFv of anti-Lewisy hu3S193 humanized monoclonal antibody (hu3S193; Scott et al. 2000), with a five residue linker formed a bivalent diabody (Power et al. 2001). Radiolabeling of this diabody with 111Indium via the bifunctional metal chelator, CHX-A''-DTPA (Tahtis et al. 2001), demonstrated a blood clearance rate similar to other diabodies (Adams et al. 1998a; Wu et al. 1999). Biodistribution and tumor localization of hu3S193 diabody was compared to labeled parent F(ab')2 with a tumor:blood ratio of 20:1 for the diabody and 5:1 for the F(ab')2 at 8 h post injection (Tahtis et al. 2001). To investigate the properties and efficacy of a hu3S193 scFv trimer as an imaging and therapeutic reagent, we undertook the construction of a series of scFvs with different linker lengths to define the optimum conditions for the formation of a trimer. Here we characterize the hu3S193 scFv multimers formed with linkers of +2 residues to (-2) residues and their solution properties.
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Results |
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. To investigate the effect of linker length upon the multimer produced and to define the switch between dimer and trimer for the hu3S193 scFv in the VH-VL orientation, the following constructs were made where the V domains were joined with either +2 residues or +1 residues, directly linking the scFv domains, and a VH domain lacking one of the C-terminal serine residues to produce scFv (-1), and scFv (-2) lacking both C-terminal Ser residues (Fig. 1A,B). Three scFv direct-linked constructs were made. In the first construct (Fig. 1A), the VH-0-VL were directly linked. In the second construct, VH-0-VL, where the VL domain C-terminal threonine was replaced with basic residues KR to give VH-VLKR variant. In the construction of the third construct, VL-0-VH were directly linked in the reverse orientation with the VL domain first, (containing the C-terminal KR residues) linked directly to the N-terminal glutamic acid residue of the VH domain to give VL-0-VH KR variant (Fig. 1C). The rationale for these KR variants arose from the observation that the sequence alignment of the light-chain V of hu3S193 with other V sequences showed that the C-terminal residue was a threonine rather than a lysine or arginine as observed in many V sequences (Johnson and Wu 2001). The C-terminal threonine in the parent hu3S193 V was replaced by residues KR, to introduce a basic C terminus as in other VL domains, and to enhance the solubility of the protein and to provide an additional lysine residue for possible attachment of a metal ion chelator for radiolabeling studies (Fig. 1C). All constructs contain a C terminal FLAG affinity purification tail (DYKDDDDK).
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) and Western transfer of duplicate gel (Fig. 2B). The induced protein is clearly visible as a major 32-kD band. Minor levels of cleaved scFv, as indicated by the faint 19-kD band, are also present in some of the crude extracts.
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).
Characterization of hu3S193 scFv multimers by size exclusion chromatography
The multimeric state of affinity purified hu3S193 scFvs with the different linker lengths was analyzed by gel filtration on a calibrated Superose 12 column. The hu3S193 scFv +15 monomer (VH -VL) with Mr 26.8 kD and hu3S193 scFv +5 dimer with Mr 53.4 kD have been previously characterized (Power et al. 2001). The hu3S193 scFvs with +2 and +1 residue linkers both produced a major peak eluting with an apparent molecular mass of 54 kD (Fig. 3) for the soluble and urea-solubilized scFvs preparations as previously found for the hu3S193 scFv +5 diabody molecule (Power et al. 2001). Both fractions of scFv +2 and +1 contained a small amount of higher molecular mass multimers, and some preparation runs also contained a lower molecular mass peak of ~15 kD (Fig. 3B). Peak purified scFv +2 dimer from the urea-soluble fraction was stable at 4°C for >4 wk (Fig. 3C). The ~15 kD peak (Fig. 3B) isolated by gel filtration gave a single protein band of Mr ~18 kD on SDS-PAGE that did not react with the anti-FLAG antibody on Western blot analysis. N-terminal sequence analysis revealed the sequence of the VH domain indicating that this peak was a fragment of the linked VH-VL domains arising from proteolytic cleavage within the VL domain.
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) with elution times consistent with trimeric (Mr ~81 kD) and monomeric (Mr ~26.5 kD) protein. However, the monomer peak from this (VH-0-VL) preparation elutes just prior to the time observed for the scFv +15 monomer peak (previously characterized), suggesting that the scFv monomer and trimer are in equilibrium, and their elution profile reflects that it may be a reaction boundary, which is likewise true for all of the elution profiles in Figures 3–5. Isolation of both the scFv (VH-0-VL) trimer and monomer peaks by gel filtration, concentration, and subsequent rechromatography showed that both species rapidly reequilibrated to the trimer/monomer mixture.
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) as well as higher-molecular-mass multimers.
The soluble fraction of the scFv (VH-0-VL) variant with KR residues at the C terminus of the VL domain yielded a series of peaks consistent with a mixture of scFv monomer, trimer, and higher Mr multimers (possibly a hexamer). In addition, the scFv-VL domain cleavage fragment (Rt = 32.13 min) was found in appreciable amounts in soluble scFv (VH-0-VL) preparations (Fig. 4C), which seems to increase with the age of the sample. The urea-solubilized hu3S193 scFv (VH-0-VL) KR variant yielded a similar elution profile, but the trimer and higher-sized multimers were poorly resolved (Fig. 4D).
In contrast, reverse VL-0-VH on gel filtration showed that the affinity-isolated soluble scFv fraction contained a mixture of predominantly trimer (Fig. 4E), whereas the urea-solubilized scFv fraction contained a mixture of trimer and tetramer (Fig. 4F).
The two scFv constructs in which the C-terminal serine, scFv (-1), and the C-terminal and penultimate serine, scFv (-2), of the VH domain were removed and directly linked to the VL domain, expressed active scFv products that were isolated by affinity chromatography. Soluble and urea solubilized hu3S193 scFv (-1) yielded a major peak, eluting with an apparent molecular mass of a trimer (~81 kD) with only a trace of the monomer peak that was observed for the hu3S193 scFv (VH-0-VL) construct. Both affinity purified scFvs from the soluble and urea-solubilized fractions contained multimers larger than the trimer, but these were not resolved into discrete multimer peaks on the Superose 12 column (Fig. 5). Peak-purified hu3S193 scFv (-1) trimer from the urea-soluble fraction eluted largely as a symmetrical peak on rechromatography and was stable at 4°C for ~1 wk (Fig. 5C). After 2 wk at 4°C, tetramer, monomer, and the VL cleavage fragment appeared indicating that the trimer is in equilibrium with these other multimeric forms and additionally, some proteolytic cleavage occurred on storage (Fig. 5D). Gel filtration of hu3S193 scFv (-2), soluble and urea solubilized preparations, yielded a mixture of multimers that was not resolved into discrete peaks (data not shown), and further characterization was not pursued.
Formation of hu3S193 scFv-Fab complexes to determine valency of multimers
Two anti-hu3S193 antibodies that compete with the binding of the Ley tetrasaccharide antigen to antibody hu3S193 were used to investigate the valency of the scFv multimers by measuring the sizes of the complexes formed. The two antibodies were cleaved with papain as described previously (Gruen et al. 1993). Antibody F2–3A produced a Fab fragment as expected, but antibody F3–27 yielded an F(ab')2 fragment that was not reduced to Fab' with the standard mercaptoethylamine reduction protocol.
The anti-idiotype F2–3A Fab formed a stable complex with hu3S193 Fab resulting in an elution time consistent with the formation of a 1:1 complex with a molecular mass of ~100 kD. The interaction between F2–3A Fab and hu3S193 IgG resulted in a complex with a Mr of ~250 kD consistent with two Fabs binding to each arm of the IgG. Similarly, the hu3S193 scFv +15 monomer formed a stable complex with F2–3A Fab with the Mr of ~77 kD expected for a 1:1 complex. Surprisingly, the hu3S193 scFv +2 dimer and hu3S193 scFv (-1) trimer yielded complexes eluting with molecular masses of ~104 kD and ~131 kD, respectively, consistent with the binding of only one Fab per mole of dimer and trimer (Fig. 6). Similar results were obtained with scFvs isolated from either the soluble or urea-solubilized factions. These results suggested that (i) only one arm of the diabody and triabody was active, or (ii) the binding of one F2–3A precludes the binding of subsequent Fabs. However, BIAcore binding data clearly show that both hu3S193 scFv dimer and trimer exhibit multivalent binding to immobilized F2–3A antibody consistent with two and three active antigen binding sites per molecule expected for correctly assembled diabodies and triabodies.
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A plot of the calculated molecular masses of the hu3S193 scFv multimers, hu3S193 IgG, and various complexes with anti-idiotype antibody fragments against elution times showed a linear relationship for this family of proteins (Fig. 6). This linear relationship provides confidence in the molecular masses assigned to the hu3S193 scFv multimers based on their elution times on Superose 12.
Binding activity of hu3S193 scFv multimers
Flow cytometry binding analysis
The binding activity of peak purified hu3S193 scFv multimers to native Ley tetrasaccharide antigen expressed on a cell surface was determined by flow cytometry binding analysis using the breast cancer cell line MCF-7 (Fig. 7). To overcome detection problems as a result of epitope accessibility and recognition by a secondary-labeled antibody, the hu3S193 scFv multimers were labeled with Alexa Fluor 488 fluorescent dye for direct detection. The labeling procedure incorporated between two and four dye molecules per mole of hu3S193 scFv multimer, and the labeled scFv multimers retained between 30–70% binding activity compared to unlabeled hu3S193 scFv. Directly labeled hu3S193 scFv monomer, dimer, trimer, tetramer, and labeled Fab specifically bound to the native antigen on the MCF-7 cells (Fig. 7), with good yields of mean fluorescent intensity for all species. No interaction was observed with the antigen-negative SK-MEL-28 cells and irrelevant labeled NC10 scFv-0 trimer on MCF-7 cells used as a control.
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). The dissociation rate constants for hu3S193 scFv +1 and hu3S193 scFv +2 dimers (kd = 8.0 x 10-3 s-1) are essentially the same as those for the hu3S193 scFv +5 dimer (kd = 7.8 x 10-3 s-1) and parent IgG (kd = 8.4 x 10-3 s-1) (Power et al. 2001).
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). These results show that (i) the hu3S193 trimer and tetramer contain at least two active antigen-binding regions per molecule, and (ii) the spatial arrangement of the antigen-binding regions in these hu3S193 scFv multimers is such that the binding regions can bind to two or more Ley tetrasaccharide antigen molecules coupled to BSA and immobilized on the sensor surface. This is in contrast to the bivalent dimers (hu3S193 scFv +5, +2, +1) and parent IgG, which do not interact with the synthetic Ley tetrasaccharide-BSA complex on the sensor surface in a bivalent manner, as previously reported in Power et al. (2001). A comparison of the binding interaction of hu3S193 Fab and IgG with immobilized hu3S193 anti-idiotype antibody F2–3A showed that the dissociation rate for the IgG (~10-5 s-1) was significantly slower than for the monomeric Fab fragment consistent with an increase in avidity as a result of the bivalent binding by the parent hu3S193 IgG.
The binding of hu3S193 scFv +5, +2, and +1 dimers, direct-linked (VH-0-VL) trimers and reverse (VL-0-VH) trimer/tetramer showed dissociation rates slower than those for the hu3S193 scFv +15 monomer consistent with multivalent binding to immobilized hu3S193 anti-idiotype antibody F2–3A (Fig. 9). The binding data shows that the two antigen-binding regions of the hu3S193 scFv dimers are both active and can cross-link two antigen molecules when the steric orientation of the antigens on the sensor surface is favorable for such an interaction to occur. A comparison of the biosensor data for the two immobilized antigens, Ley tetrasaccharide-BSA and hu3S193 anti-idiotype antibody F2–3A indicates that binding data obtained with synthetic immobilized antigens may not necessarily reflect the valency of the molecule or its activity in solution toward native antigen.
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Discussion |
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The directly linked hu3S193 scFv (VH-0-VL) and (VH-0-VL-KR) variant constructs form predominantly a trimer (Mr 81 kD) that is in a rapid equilibrium with a monomeric form (Mr 27 kD); in addition, higher molecular mass multimers are also found (Fig. 4B). Rechromatography of trimer and monomer peak fractions isolated by size exclusion chromatography showed that the trimer-monomer equilibrium was rapidly reestablished. The gel filtration data shows that in solution, direct linked (VH-0-VL) occurs as a monomer that is presumably inactive in binding antigen as the linked V domains cannot associate to a Fv module, which reversibly associates with other V domains (monomer) to form active trimers and higher Mr multimers. In contrast, NC10 scFv-0 (VH-0-VL) yielded a stable trimer with no evidence of a monomeric VH-0-VL.
Linking the hu3S193 V domains directly in the reverse orientation VL-0-VH, using the VL KR variant domain, yielded an equilibrium mixture of trimer and tetramer as reported previously for NC10 scFv-0 (VL-0-VH) (Dolezal et al. 2000). The hu3S193 scFv (-1) construct predominantly formed a trimer as well as a mixture of poorly resolved higher Mr multimers (Fig. 3C). Peak purified hu3S193 scFv (-1) trimer (Fig. 3C) on storage at 4°C showed conversion to an equilibrium mixture of trimer and tetramer with a trace of monomer evident indicating that the multimeric forms of this construct were not structurally stable. Reengineering the VH-VL interface in these short-linker scFv species could make them more stable and is something to consider for future work.
This study provides further support for the paradigm that scFvs in which the V domains linked with +3 residues or more associate to form noncovalent dimers and those linked with a shorter linker or directly linked, form a trimer/tetramer mixture. The exception is hu3S193 scFv +1, which formed a dimer. While hu3S193 scFvs with +2 to +5 residue linkers formed stable dimers, scFvs with directly linked V domains did not form a single stable multimer but rather associated to form an equilibrium mixture of predominantly trimer and tetramer. Studies on the effect of linker length on the assembly of NC10 multimers showed that linking VH-0-VL directly yielded a stable trimer (Kortt et al. 1997) while in the reverse orientation directly linked VL-0-VH domains yielded an equilibrium mixture of trimer and tetramer (Dolezal et al. 2000). Thus, while the size of noncovalent multimer (dimer or trimer/tetramer) formed is governed mainly by the linker length, the stability of the trimer/tetramer in the equilibrium is dependent upon the affinity of the interaction of the V domains to associate into the Fv module. The direct-linked hu3S193 VH-0-VL and VL-0-VH constructs generate trimeric and tetrameric units that provide increased avidity, which will make them interesting reagents for use in imaging studies depending on their clearance rate, or possibly for therapeutic use as they may be able to achieve a greater accumulation %ID/g at the tumor-targeting site (than the hu3S193 scFv dimers) a required property for therapeutic use. The most favorable constructs for further development are hu3S193 scFv(-1) VH VL for the trimer work and the direct-linked hu3S193 VL-0-VH scFv for tetrameric studies.
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Materials and methods |
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Construction of hu3S193 scFv genes with various linker lengths
Genes encoding the VH and VL domains genes were isolated by PCR from the hybridoma cell line expressing hu3S193 as previously described (Power et al. 2001). The scFv gene constructs with different linker lengths joining the VH and VL domains of hu3S193 ranging from +2 residues down to (-2) residues (residues removed from the C terminus of the VH domain) are shown in Figure 1. Sequences of the oligonucleotide primers used to assemble these constructs are listed in Table 1. A BamHI site was engineered into the (Gly4Ser)3 linker region of hu3S193 scFv +5 (Power et al. 2001) to assist in the construction of scFvs with various linker lengths. The VH domain was cloned as NcoI-BamHI and the VL domain as BamHI-EcoRI encoding either FLAG purification tag or a KR variant that replaces the VL C-terminal threonine with a lysine and arginine before the FLAG tag (Fig. 1).
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) to replace the region between the BstEII site and the BamHI site, using the +5 residue construct as a template. The +1 residue construct was made in a similar way utilizing the BstEII site and the BamHI sites and restored the residues spanning these two sites ligating a dsDNA linker N5536 and N5535 (Table 1). The C terminus of the VH domain was shortened by one residue (Ser) to make the (-1) construct using a pair of oligonucleotide primers N5911 and N5912 utilizing the QuikChange (Stratagene, Integrated Sciences) site-directed mutagenesis strategy. The VH domain was further shortened by another Ser residue to make the (-2) construct, using the same PCR strategy as for the direct linkage construct. The VH domain was digested with BstEII and the VL domain was PCR amplified with a new primer N5196, which removed the two C-terminal serine residues.
The hu3S193 scFv gene with the domains in the reverse orientation (VL-0-VH) with a direct linkage as shown in Figure 1, was constructed by utilizing the SfiI site for cloning into pPOW3 vector and the PflMI site at the N terminus of the VH domain for joining the two domains. The VL domain gene in the VL-VH construct was PCR amplified with primer N5183 containing the SfiI site and N5181, which contains the VL domain with the lysine and arginine residues at the C terminus and the first few residues of the N-terminal VH domain sequence and the PflMI site. The VH domain gene was made with the primers N4793 and N5182 (Table 1).
PCR amplification was used to synthesize the VH and VL gene fragments incorporating restriction endonuclease sites required for the cloning of the scFv fragments. PCR products were digested with restriction enzymes and fragment sizes were confirmed by electrophoresis on 8% polyacrylamide gels and the DNA fragments were excised and eluted from the gels. The scFv genes were cloned into pPOW3 bacterial expression vector for protein production (Power and Hudson 2000). Oligonucleotide primers that bind to the pPOW3 vector N2175 and N2357 were used for colony screening and dsDNA sequencing within the pPOW3 vector. Colony screening with these two oligonucleotides generated a band of 215 bp in addition to the expected size of the cloned gene. The scFv constructs contain a C-terminal extension FLAG octapeptide affinity tag (DYKDDDDK). The DNA constructs were confirmed by nucleotide sequencing using internal primers N4905 VH domain forward primer binding to the sequence FCARGT, N5205 VH domain reverse primer and N4796 VL domain reverse primer.
Hu3S193 scFv expression and purification
Cultures of E. coli TOP 10 (Invitrogen, Silenus Labs) transformed with pPOW3 vector encoding the appropriate scFv gene were grown and induced to express protein as described (Power and Hudson 2000). After initial, small-scale expression in 1-L shake flasks to establish that the correct product was synthesized, 10-L fermenter runs were performed as described previously (Bayly et al. 2002). ScFv protein was extracted from the bacterial cell pellet to yield soluble and urea solubilized protein fractions as described (Bayly et al. 2002). The scFvs were isolated from these fractions by affinity chromatography on anti-FLAG (KM5–1C7–8–5) antibody resin by a batch procedure as described previously (Bayly et al. 2002). The affinity-purified protein in 1 X TBS (Tris-buffered Saline) pH 8.0 was stored frozen at -20°C. The purity of the affinity-isolated scFvs was monitored by SDS-PAGE and Western blot analysis (Kortt et al. 1994).
Hu3S193 scFv multimer characterization by size exclusion chromatography
The multimeric state of each affinity purified hu3S193 scFv was evaluated by size exclusion chromatography on a calibrated Superose 12 HR 30/10 column (Amersham Pharmacia) in PBS pH 7.4, at a flow rate of 0.5mL/min. The apparent molecular mass of each hu3S193 scFv multimer was estimated from a calibration curve (Fig. 6) derived for this family of proteins, with the molecular mass of hu3S193 scFv +5 dimer whose molecular mass was determined by ultracentrifugation (Power et al. 2001) as an internal standard for the calibration. The concentrations of the scFvs were determined spectrophotometrically using an extinction coefficient for a 0.1% (w/v) solution at 280 nm of 1.68 calculated from the protein sequence (Gill and Hippel 1989).
Formation of hu3S193 scFv multimer-anti-idiotype Fab complexes
The anti-Ids were generated by Ludwig Institute of Cancer Research and were Protein A purified in the Biological Production Facility, LICR. Fab fragments of anti-idiotype hu3S193 antibodies F2–3A and F3–27 were prepared as described in Kortt et al. (1997). Purified hu3S193 scFv multimers were mixed with a small molar excess of the anti-idiotype Fab and the complexes formed were analyzed by size exclusion chromatography on Superose 12 as described above. The apparent molecular mass estimated for each complex was used to calculate the size and valency of each multimer.
Flow cytometry binding analysis
The binding of various hu3S193 scFv multimers to Ley tetrasaccharide antigen expressed on breast carcinoma cells was demonstrated by flow cytometry. The fluorescent dye Alexa FluorTM 488 (Molecular Probes) was conjugated to each hu3S193 scFv multimer according to manufacturer’s directions. The breast carcinoma cell line, MCF-7 (ATCC HTB-22), which expresses high quantities of the Ley tetrasaccharide on its membrane surface was used for the binding studies. SK-MEL-28 melanoma (ATCC HTB-72) cells were used as a negative control line. The cells were maintained in RPMI 1640 (supplemented with 100 U/mL penicillin, 100µg/mL streptomycin and 10% heat-inactivated fetal bovine serum (FBS) (all from Invitrogen) at 37°C in 5% CO2 in humidified air.
For binding studies, cells were harvested at about 75% confluence, treated with trypsin-EDTA buffer (defined as trypsin at 0.25 mg/mL diluted 1 in 10 into PBS containing 0.02% EDTA) and washed in at least 10 volumes of growth medium. 1 x 105 cells in 100 µL of growth medium were incubated with 5 µg of hu3S193 scFv multimer-Alexa Fluor 488 conjugate for 30 min on ice. Trypsin does not cleave the Ley antigen from the surface of the cells (data not shown). NC10 scFv-0 (synthesized and purified from the same bacterial expression vector)-Alexa Fluor 488 conjugate was used as a negative control. After washing with cold wash buffer (2 mL PBS +2% FBS +0.1% sodium azide), the cells were resuspended in 200 µL wash buffer containing 2 µL propidium iodide (1 mg/mL) (Sigma) to detect dead cells. Measurements of relative fluorescence of stained viable cells were performed with a flow cytometer (Epics Elite, Beckman-Coulter, NSW) with a 488 nm argon laser. Data analysis was performed on WinMDI software (Scripps). Green fluorescence was estimated on propidium iodide negative cells gated on forward and side scatter.
Biosensor binding analysis
The binding properties of the hu3S193 scFv multimers to immobilized Ley tetrasaccharide antigen and to anti-hu3S193 anti-idiotype antibody F2–3A, were measured using a BIAcore biosensor (BIACORE AB).
Synthetic Ley tetrasaccharide ligand coupled to BSA (at a ratio of 30:1) (Alberta Research Council) was immobilized to a CM5 BIAsensor chip at a concentration of 155 µg/mL in 10 mM sodium acetate buffer pH 3.0, using standard amide coupling reactions (N-hydroxysuccinimide and N-ethyl-N'-dimethylaminopropyl-carbodimide). The immobilization was carried out at 25°C at a flow rate of 5 µL/min and on injecting 20 µL of the antigen ~2340 RU was coupled to the sensor chip surface. Anti-idioytpe antibody F2–3A was also immobilized on a CM-5 chip at at concentration of 20 µg/mL in 10 mM sodium acetate buffer, pH 4.5 using standard amine coupling chemistry as previously described (Kortt et al. 1999). Injection of 40 µL of IgG at a flow rate of 5 µL/min resulted in the immobilization of 2480 RU of F2–3A. The total concentration of immobilized protein may be lower than initially calculated, which would not alter the overall shape of the curve but only alter the scale of the RU units.
Binding experiments were performed in HBS buffer (10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4) at a constant flow rate of 5 µL/min. Aliquots (30–50 µL) of each hu3S193 scFv multimer and hu3S193 Fab were injected over the sensor chip surfaces to measure the association and dissociation reactions. The three sensor surfaces were regenerated with a 10 µL aliquot of 100-mM HCl with negligible loss of binding activity.
The hu3S193 scFv multimers and hu3S193 IgG and Fab samples for binding analyses were peak purified by gel filtration on Superose 12 to remove any higher molecular mass oligomers that may have formed on storage following freezing and thawing. The binding data, when appropriate, was evaluated using the BIAevaluation 3.0.2 software as described previously (Kortt et al. 1999).
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References |
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Adams, G.P., Schier, R., McCall, A.M., Crawford, R.S., Wolf, E.J., Weiner, L.M., and Marks, J.D. 1998a. Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu. Br. J. Cancer 77: 1405–1412.[Medline]
Adams, G.P., Schier, R., Marshall, K., Wolf, E.J., McCall, A.M., Marks, J.D., and Weiner, L.M. 1998b. Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies. Cancer Res. 58: 485–490.
Adams, G.P., Shaller, C.C., Chappell, L.L., Wu, C., Horak, E.M., Simmons, H.H., Litwin, S., Marks, J.D., Weiner, L.M., and Brechbiel, M.W. 2000. Delivery of the -emitting radioisotope bismuth-213 to solid tumors via single-chain Fv and diabody molecules. Nucl. Med. Biol. 27: 339–346.[CrossRef][Medline]
Atwell, J.L., Breheney, K.A., Lawrence, L.J., McCoy, A.J., Kortt, A.A., and Hudson, P.J. 1999. scFv multimers of the anti-neuraminidase antibody NC10: Length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies. Protein Eng. 12: 597–604.
Bayly, A.M., Kortt, A.A., Hudson, P.J., and Power, B.E. 2002. Large-scale bacterial fermentation and isolation of scFv multimers using a heat-inducible bacterial expression vector. J. Immunol. Methods 262: 217–227.[CrossRef][Medline]
Bera, T.K., Viner, J., Brinkmann, E., and Pastan, I. 1999. Pharmacokinetics and antitumor activity of a bivalent disulfide-stabilized Fv immunotoxin with improved antigen binding to erbB2. Cancer Res. 59: 4018–4022.
Dolezal, O., Pearce, L.A., Lawrence, L.J., McCoy, A.J., Hudson, P.J., and Kortt, A.A. 2000. ScFv multimers of the anti-neuraminidase antibody NC10: Shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers. Protein Eng. 13: 565–574.
Gill, S.C. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182: 319–326.[CrossRef][Medline]
Griffiths, A.D., Malmqvist, M., Marks, J.D., Bye, J.M., Embleton, M.J., McCafferty, J., Baier, M., Holliger, K.P., Gorick, B.D., Hughes-Jones, N.C., et al. 1993. Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 12: 725–734.[Medline]
Gruen, L.C., Kortt, A.A., and Nice, E. 1993. Determination of relative binding affinity of influenza virus N9 sialidases with the Fab fragment of monoclonal antibody NC41 using biosensor technology. Eur. J. Biochem. 217: 319–325.[Medline]
Holliger, P., Prospero, T., and Winter, G. 1993. "Diabodies": Small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. 90: 6444–6448.
Hu, S., Shively, L., Raubitschek, A., Sherman, M., Williams, L.E., Wong, J.Y., Shively, J.E., and Wu, A.M. 1996. Minibody: A novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts. Cancer Res. 56: 3055–3061.
Huston, J.S., Mudgett-Hunter, M., Tai, M.S., McCartney, J., Warren, F., Haber, E., and Oppermann, H. 1991. Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol. 203: 46–88.[Medline]
Iliades, P., Kortt, A.A., and Hudson, P.J. 1997. Triabodies: Single chain Fv fragments without a linker form trivalent trimers. FEBS Lett. 409: 437–441.[CrossRef][Medline]
Johnson, G. and Wu, T.T. 2001. Kabat database and its applications: Future directions. Nucleic Acids Res. 29: 205–206.
Kortt, A.A., Malby, R.L., Caldwell, J.B., Gruen, L.C., Ivancic, N., Lawrence, M.C., Howlett, G.J., Webster, R.G., Hudson, P.J., and Colman, P.M. 1994. Recombinant anti-sialidase single-chain variable fragment antibody. Characterization, formation of dimer and higher-molecular-mass multimers and the solution of the crystal structure of the single-chain variable fragment/sialidase complex. Eur. J. Biochem. 221: 151–157.[Medline]
Kortt, A.A., Lah, M., Oddie, G.W., Gruen, C.L., Burns, J.E., Pearce, L.A., Atwell, J.L., McCoy, A.J., Howlett, G.J., Metzger, D.W., et al. 1997. Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero- residue linker a trimer. Protein Eng. 10: 423–433.
Kortt, A.A., Nice, E., and Gruen, L.C. 1999. Analysis of the binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase from tern and whale using the BIAcore biosensor: Effect of immobilization level and flow rate on kinetic analysis. Anal. Biochem. 273: 133–141.[CrossRef][Medline]
Kortt, A.A., Dolezal, O., Power, B.E., and Hudson, P.J. 2001. Dimeric and trimeric antibodies: High avidity scFvs for cancer targeting. Biomolecular Engineering 18: 95–108.[CrossRef][Medline]
Le Gall, F., Kipriyanov, S.M., Moldenhauer, G., and Little, M. 1999. Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: Effect of valency on cell binding. FEBS Lett. 453: 164–168.[CrossRef][Medline]
Malby, R.L., Caldwell, J.B., Gruen, L.C., Harley, V.R., Ivancic, N., Kortt, A.A., Lilley, G.G., Power, B.E., Webster, R.G., Colman, P.M., et al. 1993. Recombinant anti-neuraminidase single chain antibody: Expression, characterization, and crystallization in complex with antigen. Proteins 16: 57–63.[CrossRef][Medline]
McDevitt, M.R., Ma, D., Lai, L.T., Simon, J., Borchardt, P., Frank, R.K., Wu, K., Pellegrini, V., Curcio, M.J., Miederer, M., et al. 2001. Tumor therapy with targeted atomic nanogenerators. Science 294: 1537–1540.
Pavlinkova, G., Booth, B.J., Batra, S.K., and Colcher, D. 1999. Radioimmunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody construct. Clin. Cancer Res. 5: 2613–2619.
Pei, X.Y., Holliger, P., Murzin, A.G., and Williams, R.L. 1997. The 2.0-Å resolution crystal structure of a trimeric antibody fragment with noncognate VH-VL domain pairs shows a rearrangement of VH CDR3. Proc. Natl. Acad. Sci. 94: 9637–9642.
Power, B.E. and Hudson, P.J. 2000. Synthesis of high avidity antibody fragments (scFv multimers) for cancer imaging. J. Immunol. Methods 242: 193–204.[CrossRef][Medline]
Power, B.E., Caine, J.M., Burns, J.E., Shapira, D.R., Hattarki, M.K., Tahtis, K., Lee, F.-T., Smyth, F.E., Scott, A.M., Kortt, A.A., et al. 2001. Construction, expression and characterisation of a single-chain diabody derived from a humanised anti-Lewis Y cancer targeting antibody using a heat-inducible bacterial secretion vector. Cancer Immunol. Immunother. 50: 241–250.[CrossRef][Medline]
Scott, A.M., Geleick, D., Rubira, M., Clarke, K., Nice, E.C., Smyth, F.E., Stockert, E., Richards, E.C., Carr, F.J., Harris, W.J., et al. 2000. Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors. Cancer Res. 60: 3254–3261.
Tahtis, K., Lee, F.T., Smyth, F.E., Power, B.E., Renner, C., Brechbiel, M.W., Old, L.J., Hudson, P.J., and Scott, A.M. 2001. Biodistribution properties of (111)indium-labeled C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid humanized 3S193 diabody and F(ab')(2) constructs in a breast carcinoma xenograft model. Clin. Cancer Res. 7: 1061–1072.
Whitlow, M., Filpula, D., Rollence, M.L., Feng, S.L., and Wood, J.F. 1994. Multivalent Fvs: Characterization of single-chain Fv oligomers and preparation of a bispecific Fv. Protein Eng. 7: 1017–1026.
Willuda, J., Kubetzko, S., Waibel, R., Schubiger, P.A., Zangemeister-Wittke, U., and Pluckthun, A. 2001. Tumor targeting of mono-, di-, and tetravalent anti-p185(HER-2) miniantibodies multimerized by self-associating peptides. J. Biol. Chem. 276: 14385–14392.
Wu, A.M., Chen, W., Raubitschek, A., Williams, L.E., Neumaier, M., Fischer, R., Hu, S.Z., Odom-Maryon, T., Wong, J.Y., and Shively, J.E. 1996. Tumor localization of anti-CEA single-chain Fvs: Improved targeting by non-covalent dimers. Immunotechnology 2: 21–36.[CrossRef][Medline]
Wu, A.M., Williams, L.E., Zieran, L., Padma, A., Sherman, M., Bebb, G.G., Odom-Maryon, T., Wong, J.Y.C., Shively, J.E., and Raubitschek, A.A. 1999. Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting 4: 47–58.
Yazaki, P.J., Wu, A.M., Tsai, S.W., Williams, L.E., Ikler, D.N., Wong, J.Y., Shively, J.E., and Raubitschek, A.A. 2001. Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and T84.66 minibody: Comparison to radioiodinated fragments. Bioconjug. Chem. 12: 220–228.[CrossRef][Medline]
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