A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities

Z. Wang, A. Mathias, S. Stavrou and D.M. Neville, Jr1

Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Building 10, 36 Convent Drive, Bethesda, MD 28092-4034, USA

1 To whom correspondence should be addressed. E-mail: davidn{at}mail.nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3{varepsilon} scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH2 terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3{varepsilon}{gamma} was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH2 terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3{varepsilon}{gamma}. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3{varepsilon}{gamma} increased ~3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand–scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements.

Keywords: CD3/FN18/pYD5/scFv/yeast display


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recombinant single-chain antibodies have wide utility as diagnostic and therapeutic reagents. The affinity of these scFvs is an important variable and must exceed a certain threshold to be useful. Over the past 8 years techniques have been developed to increase the affinity and stability of scFv with random mutagenesis coupled with yeast display technology utilizing sorting flow cytometry and magnetic bead selection methodologies (Colby et al., 2004Go). The available commercial yeast display vector pYD1 (Invitrogen) is based on pCT202 developed by Boder and Wittrup (1997)Go. These vectors display the protein of interest flanked on the N-terminus by mating protein Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1 (Boder and Wittrup, 1997Go). On the C-terminus a small peptide marker epitope is present. Our interest in this area arose from an attempt to affinity mature the anti-monkey anti-CD3{varepsilon}{gamma} scFv derived from FN18 for use in an anti-monkey anti-T cell immunotoxin fusion protein. These anti-T cell immunotoxins can induce long term transplantation tolerance in non-human primate models and recombinant fusion immunotoxins are needed for pre-clinical studies (Thomas et al., 2000Go; Contreras et al., 2003Go). A successful human analogue of this fusion immunotoxin has been generated from the non-cross-reacting antibody UCHT1 (Thompson et al., 2001Go). However, preliminary data indicated that the affinity of scFv FN18 was considerably less than that of the UCHT1 scFv (Ma et al., 1997Go). We attempted to affinity mature the scFv of FN18 but found that the affinity of the scFv of FN18 for its ligand, the ectodomain of the CD3{varepsilon}{gamma} complex, was too low to measure. Previously we had noted that the scFv of UCHT1 suffered a 50- to 100-fold loss of affinity compared with the parental antibody when expressed as a fusion protein when the scFv was placed at the C-terminus of either truncated diphtheria toxin or human serum albumin (Thompson et al., 2001Go). However, when the truncated toxin was placed distal to the scFv the loss of affinity was only 3-fold compared with the parental antibody (Hexham et al., 2001Go). We reasoned that the very low observed affinity of the pYD1 displayed scFv of FN18 for its ligand might be compounded by the sensitivity of this scFv to steric constraints when the NH2 terminus of this scFv was used in tethering the scFv to the Aga2 protein. Consequently, we rebuilt this yeast display vector so that the NH2 terminus of the displayed protein of interest was free. We then compared the binding affinities of two anti-CD3{varepsilon}{gamma} scFvs for their ligands in the original pYD1 vector and the new vector pYD5 in order to evaluate positional effects on binding affinities of displayed scFvs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
FN18 antibody

The monoclonal antibody FN18 (IgG1) was generated by immunizing mice with rhesus peripheral blood mononuclear cells (Nooij et al., 1986aGo). FN18 reactivity was found to be restricted to rhesus T cells and thymocytes and encompassed both CD4+ and CD8+ T cells (Nooij et al., 1986bGo). The antibody precipitated two proteins from rhesus T cells of 27 and 22 kDa, suggesting properties similar to the anti-human CD3 antibodies UCHT1, OKT3 and Leu-4 (Nooij et al., 1986bGo). The clone secreting FN18 was kindly supplied by Margreet Jonker, Biomedical Primate Research Center, Rijswijk and was produced and purified by the National Cell Culture Center, Minneapolis, MN.

Plasmids, bacterial and yeast strains, antibodies

pYD1, Saccharomyces cerevisiae EBY100 and anti-V5 monoclonal antibody were purchased from Invitrogen. PET17b and Escherichia coli BL21 (DE3) pLysS were purchased from Novagen. UCHT1 monoclonal antibody (IgG1) was made available by Peter Beverley, Imperial Cancer Research Fund, London, and was produced and purified by the National Cell Culture Center. Goat anti-mouse antibody conjugated with PE/FITC was purchased from CALTAG. Streptavidin Alexa Fluor-488 conjugate and streptavidin–FITC were from Molecular Probes. Goat anti-mouse IgG-HRP was purchased from Santa Cruz Biotechnology and used to detect the expression of FN18 scFv or UCHT1 scFv in E.coli BL21 (DE3) pLysS.

Preparation of biotin-labeled CD3{varepsilon}{gamma} ecto heterodimer ligands

Human or monkey CD3{varepsilon}{gamma} ecto heterodimers were prepared as described (Wang and Neville, 2004Go). The amino groups were derivatized with iminothiolane (Sigma) and the resulting SH groups were reacted with 3-(N-maleimidylpropionyl)biocytin (Molecular Probes) as follows. To 1 mg of human or monkey {varepsilon}{gamma} ecto heterodimer (based on OD280 and A0.1% 1.36) in 275 µl of 20 mM Tris–HCl, pH 8.0 with 125 mM NaCl and 5% glycerol, 25 µl of 25 mM of freshly made up iminothiolane were added (in the same buffer), mixed well and overlaid with argon and incubated at room temperature for 1 h. The reaction mixture was purified over Sephadex G25 Fine (Amersham Biosciences) in 0.1 M sodium bicarbonate, pH 8.3, 1 mM EDTA, 5% glycerol and the major heterodimer fraction was identified by detection of the free–SH and/or SDS gels. SH derivatization was determined (Vanaman and Stark, 1970) by reacting 20 µl with Ellman's reagent (Sigma) and was generally two SH groups per mole of heterodimer. A 1 mg amount of 3-(N-maleimidylpropionyl)biocytin was dissolved in 100 µl of DMSO and 20 µl of this were added to 280 µl of the purified thiolated {varepsilon}{gamma} heterodimer and incubated for 1 h at room temperature. The reaction mixture was fractionated on a new Sephadex G25 Fine column and washed with PBS, pH 8.0, containing 5% glycerol. Substitution of borate for bicarbonate in the pH 8.3 buffer increases the labeling consistency. Large-scale labeling is accomplished by a proportional increase in all reagents.

Rebuilding yeast display vector pYD1

The rebuilding plan is shown schematically in Figure 1A. pYD1 is the parental vector with the surface expression cassette located C-terminal to the Aga2p yeast membrane-associated protein and pYD5 is the modified vector in the reverse configuration. Figure 1B shows the sequence of the Aga2 signal peptide and how an NheI site was added by a silent mutation just proximal to the last three residues of the signal peptide before the cleavage site. Figure 1C shows how an EcoRI site was added downstream from the NheI site in pYD1 leading to pYD5. This permits cloning in scFv inserts having the sequence ASVLA-(scFv)-EF into pYD5, where AS and EF are NheI and EcoRI sites. We used a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and followed the manufacturer's instructions: sense primer, (5' TCA ATA TTT TCT GTT ATT GCT AGC GTT TTA GCA CAG GAA CTG ACA ACT ATA TGC 3'); antisense primer, (5' GCA TAT AGT TGT CAG TTC CTG TGC TAA AAC GCT AGC AAT AAC AGA AAA TAT TGA 3'). We defined this mutated construct as pYD2. The two fragments, NheI–VLAEcoRI–V5–(G4S)2BamHI, sense primer carrying NheI and EcoRI, (5' CTA GCT AGC GTT TTA GCA GAA TTC GGT AAG CCT ATC CCT AAC CCT 3'), antisense primer carrying BamHI, (5' CGC GGA TCC ACC ACC ACC AGA ACC ACC ACC ACC CGT AGA ATC GAG ACC GAG GAG 3'), BglII–(G4S)–Aga2–stop–PmeI–HindIII, sense primer carrying BglII, (5' GGA AGA TCT GGT GGT GGT GGT TCT CAG GAA CTG ACA ACT ATA TGC 3'), antisense primer carrying PmeI and HindIII, (5' CCC AAG CTT GTTTAAAC TCA AAA AAC ATA CTG TGT GTT TAT GGG 3') were PCR amplified with high fidelity cloned pfu DNA polymerase. They were digested with NheI–BamHI and BglII–HindIII, respectively. The two fragments were co-cloned into pET17b between NheI and HindIII sites. The construct was confirmed by sequencing and in-frame checking by expression in E.coli BL21 (DE3) pLysS and western blot analysis with goat anti-mouse IgG-HRP. The insert was then subcloned into pYD2 between the NheI and PmeI sites. We defined the final construct as pYD5.



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Fig. 1. (A) Schematic of the commercially available yeast display vector pYD1 (Invitrogen) in which the displayed protein is tethered at its N-terminus to Aga2p mating protein through the Xpress epitope and a (G4S)3 linker (left) and the rebuilt vector pYD5 in the reverse orientation (right). (B) The amino acid and DNA sequences of the aga2 signal peptide illustrating the creation of a silent NheI site three residues before the signal peptidase cleavage site. (C) An outline of the rebuilding plan for pYD5 utilizing the newly created NheI site. (D) The DNA sequence between NheI site and PmeI site in pYD5.

 
Adding peptide spacers between aga2 signal peptide and the display protein cassette

In order to optimize cleavage between the aga2 signal peptide and the display cassette, various dipeptide and tetrapeptide spacers were added by incorporating the corresponding DNA sequence into the sense PCR primer to amplify the FN18 scFv or UCHT1 scFv. EA (FN18), (5' CTA GCT AGC GTT TTA GCA GAG GCT GAC ATT GTT ATG TCT CAA TCT 3'), AG (FN18), (5' CTA GCT AGC GTT TTA GCA GCT GGT GAC ATT GTT ATG TCT CAA TCT 3'), LE (FN18), (5' CTA GCT AGC GTT TTA GCA TTG GAG GAC ATT GTT ATG TCT CAA TCT 3'), EF (FN18), (5' CTA GCT AGC GTT TTA GCA GAG TTC GAC ATT GTT ATG TCT CAA TCT 3'), EAEA (FN18), (5' CTA GCT AGC GTT TTA GCA GAG GCT GAG GCT GAC ATT GTT ATG TCT CAA TCT 3'), EA (UCHT1), (5' CTA GCT AGC GTT TTA GCA GAG GCT GAC ATC CAG ATG ACC CAG ACC 3'), AG (UCHT1), (5' CTA GCT AGC GTT TTA GCA GCT GGT GAC ATC CAG ATG ACC CAG ACC 3').

FN18 scFv and UCHT1 scFv

A codon-optimized UCHT1 scFv DNA sequence for yeast Pichia pastoris was used (Woo et al., 2002Go). A codon-optimized FN18 scFv DNA sequence for yeast P.pastoris was used (Z.Wang and D.M.Neville,Jr, unpublished data). To clone FN18 scFv into pYD1 the sense PCR primer carrying the EcoRI site, (5' CCG GAA TTC GAC ATT GTT ATG TCT CAA TCT 3') and antisense primer carrying the XhoI site, (5' CCG CTC GAG AGA GGA GAC GGT GAC AGA GGT 3') were used. To clone UCHT1 scFv into pYD1 the sense PCR primer carrying the EcoRI site, (5' CCG GAA TTC GAC ATC CAG ATG ACC CAG ACC 3') and antisense primer carrying the XhoI site, (5' CCG CTC GAG AGA GGA GAC AGT GAC AGT AGT 3') were used. To clone UCHT1 scFv into pYD5 the sense PCR primer carrying the NheI site (listed previously) and adding the dipeptide spacer section and antisense primer carrying the EcoRI site, (5' CCG GAA TTC AGA GGA GAC AGT GAC AGT AGT 3') were used. To clone FN18 scFv into pYD5 the sense PCR primer carrying NheI site (listed previously) and the antisense primer carrying the EcoRI site, (5' CCG GAA TTC AGA GGA GAC GGT GAC AGA GGT 3') were used.

Yeast display of anti-CD3 scFv antibodies in pYD1 and pYD5 vectors

Transformation of the plasmid DNA into EBY100 was conducted with electroporate transformation (Bio-Rad Gene Pulser). A single colony or 100 µl of frozen stock of EBY100 was inoculated into 50 ml of YPD medium and cultured overnight to OD600nm = 1.3–1.5. The cells were harvested by centrifugation at 1500 g for 5 min and washed with 50 ml of ice-cold H2O by centrifugation for 5 min at 1500 g. They were washed again with 25 ml of ice-cold H2O and then washed with 10 ml of ice-cold 1 M sorbitol. The cell pellets were resuspended with 100 µl of 1 M sorbitol. Then 80 µl of cells and 5–10 µg of the plasmid DNA were mixed and transferred into a 0.2 cm cuvette. They were incubated for 5 min on ice and electroporated (voltage 1500 V, capacitance 25 µF, resistance 200 ohm). Then 1 ml of 1 M sorbitol was immediately added and incubated at 30°C for 2 h without shaking. The cells were spread on an HSM–Trp-Ura (Invitrogen) plate containing 0.67% YNB (Biogene, Irvine, CA), with ammonium sulfate, without amino acids, without dextrose, 2% raffinose, (Sigma) 1.5% agar (Difco). The colonies grew up in 3–4 days.

For the yeast display we basically followed the modified Invitrogen protocol. Briefly, a single yeast colony was inoculated into 5 ml HSM medium (as above but without agar) and grown overnight at 30°C with shaking (250 r.p.m.). The absorbance of the cell culture was read at 600 nm. The OD600 should be between 2 and 5. The cell culture was centrifuged at 1500 g for 5 min at room temperature. The cell pellet was resuspended and induced in HSM medium substituting galactose for raffinose at 20°C for 24–30 h with shaking (250 r.p.m.). The optimal induction time was between 24 and 30 h. Yeast samples were stored on ice prior to FACS analysis. We substituted raffinose for dextrose to eliminate the inhibition of induction by residual dextrose. HSM plates without tryptophan are required for selection of pYD1 or pYD5 transformants. For plating we add 1.5% agar (Difco) to the growth medium. We added 20 µg/ml of ampicillin to the liquid medium or plating medium to suppress bacterial contamination.

FACS analysis of yeast displayed scFvs and KD determination

A total of 1 x 107 cells from a freshly growing induced culture were washed twice in cold PBS–0.1% BSA pH 7.4 (PBS–BSA), spun down at 5000 g at 4°C (Eppendorf 5417R) and resuspended in 100 µl of PBS–BSA. A 10 µl volume was removed and mixed with 90 µl of PBS–BSA and set aside in a clean Eppendorf tube as negative control. To the remaining 90 µl of cell suspended cells, 2 µl of anti-V5 antibody were added and incubated on ice for 30 min, then 10 µl were removed and mixed with 90 µl of PBS–BSA and set aside in a clean tube for the V5 control. Stock biotinylated CD3 ecto-{varepsilon}{gamma} was diluted to yield different desired concentrations, then 10 µl of cells + anti-V5 were added for each {varepsilon}{gamma} concentration to be tested and incubated at room temperature for 30 min, then cooled on ice for 15 min. Cells were washed with cold buffer twice and spun at 5000 g for 2 min at 4°C, the liquid aspirated and 100 µl of secondary antibody mixture were added for 30 min on ice (3 µl of goat anti-mouse PE–100 µl of PBS–BSA and 1.5 µl of Strep-488–100 µl PBS–BSA or streptavidin–FITC). Cells were washed with cold PBS–BSA pelleted by centrifugation and the liquid was aspirated. Just prior to FACS sample injection (Beckman Coulter Cytomics FC-500), 0.5 ml of PBS–BSA were added to the stained centrifuged cells. A homogeneous population of yeast cells was identified on FACS by plotting log side scattering versus log forward scattering and a gate was drawn around this population and 104 events were counted and analyzed using RXP software. A gate line was drawn parallel to the {varepsilon}{gamma} fluorescence axis that just excluded the unstained negative control population. This gate was completed as a rectangle that included the V5 only stained population that represented the zero concentration of {varepsilon}{gamma} and the associated blank {varepsilon}{gamma} mean fluorescence intensity (MFI). The log channel {varepsilon}{gamma} fluorescence value was plotted versus the log V5 channel fluorescence value and the MFI values were tabulated at each {varepsilon}{gamma} concentration and plotted on a linear scale versus {varepsilon}{gamma} concentration. This plot was fitted using non-linear least-squares by KaleidaGraph software (Synergy Software, Reading, PA) using the hyperbolic equation y = (m1 + m2)M0/(m3 + M0), where y = MFI at the given ligand concentration, m1 = MFI of zero ligand control, m2 = MFI at saturation minus m1 and m3 = KD, the equilibrium dissociation constant (Yeast Display scFv Antibody Library User's Manual, Pacific Northwest National Laboratory, Richland, WA; http://www.sysbio.org/dataresources/index.stm) (Feldhaus et al., 2003Go; Colby et al., 2004Go).

Mutagenesis of scFv libraries and selection of higher affinity mutants

Mutagenesis was performed using the nucleotide analogue method (Zaccolo and Gherardi, 1999Go) as detailed by Graff et al. (2004)Go. For this preliminary study only one round of mutagenesis was performed on FN18 scFv. Higher affinity clones were selected on an Epics Elite ESPcell sorter (Beckman-Coulter) using an equilibrium screen monitoring V5 and monkey CD3{varepsilon}{gamma} staining (Colby et al., 2004Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Rebuilding yeast display vector and adding a peptide spacer between the aga2 signal peptide and the displayed protein

Figure 1A shows a schematic of the rebuilt display vector pYD5. The N-terminus of the displayed protein is free, in contrast to the original vector pYD1. The single restriction sites NheI and EcoRI are available for cloning the desired displayed protein into the vector. The rebuilt vector without an scFv insert displayed the V5 epitope strongly (MFI ~30 compared with ~3.5 for pYD1). However, after cloning the FN18 scFv insert (NheI–VLA–FN18 scFv–EcoRI) into pYD5, we could not detect the staining either with anti-V5 mAb or monkey CD3{varepsilon}{gamma} ecto heterodimer ligand. We noted that the vector without an scFv insert placed the EF EcoRI dipeptide at the terminus of the aga2 signal peptide. On the basis of previous studies we hypothesized that the +1 and +2 amino acid sequence of the N-terminus of the aga2 display fusion protein is critical for the processing of the aga2 signal peptidase (Degryse et al., 1992Go; Parekh et al., 1995Go). We therefore tried adding four different peptide spacers between the aga2 signal peptide and the N-terminus of FN18 scFv. EF, EA, EAEA and AG were all successful in inducing V5 display in the presence of the FN18 scFv (MFI ~3.5). AG but not EA was successful for the scFv of UCHT1. Although EA can be cut away by STE13, it is sometimes insufficient (Brake, 1990Go). AG seemed relatively innocuous on the basis of bulk and neutrality and was chosen as our default peptide spacer. Consequently, the new cloning insert became (NheI–VLAAG–FN18 scFv–EcoRI).

FACS and KD comparisons of UCHT1 scFv and FN18 scFv in pYD5 and pYD1

FACS analysis of anti-human and anti-monkey anti-CD3 scFvs displayed on the pYD1 and pYD5 vectors are shown in Figure 2. V5 epitope staining is displayed on the y-axis and CD3{varepsilon}{gamma} on the x-axis. The concentrations of CD3{varepsilon}{gamma} used in this study were 1500 and 500 nM for human and monkey CD3{varepsilon}{gamma}, respectively. The pYD1 vector exhibited strong staining of the V5 epitope but no staining of monkey CD3{varepsilon}{gamma} above the blank value (vertical gate) was detected (top left panel). The lack of pYD1 FN18 scFv staining above the zero concentration CD3{varepsilon}{gamma} blank was replicated in three separate experiments with three different CD3{varepsilon}{gamma} preparations at 1500 nM. In contrast, weak staining with monkey CD3{varepsilon}{gamma} was observed with the rebuilt pYD5 vector (Figure 2, top right panel). This finding was replicated in three additional experiments with three different CD3{varepsilon}{gamma} preparations at 1500 nM, the CD3{varepsilon}{gamma} staining being 1.7-fold higher than the zero concentration CD3{varepsilon}{gamma} blank (data not shown). The higher affinity anti-human anti-CD3 scFv stained well in both vectors (Figure 2, bottom panels), but pYD5 CD3{varepsilon}{gamma} staining was more intense in pYD5 than pYD1.



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Fig. 2. FACS analysis of anti-human and anti-monkey anti-CD3 scFvs displayed on the pYD1 and pYD5 vectors. V5 epitope staining is displayed on the y-axis and CD3{varepsilon}{gamma} on the x-axis. The concentrations of CD3{varepsilon}{gamma} used in this study were 1500 and 500 nM for human and monkey CD3{varepsilon}{gamma}, respectively. The pYD1 vector exhibits strong staining of the V5 epitope but no staining of CD3{varepsilon}{gamma}. However, weak staining with CD3{varepsilon}{gamma} is observed with the rebuilt pYD5 vector. The higher affinity anti-human anti-CD3 scFv stained well in both vectors, but is more intense in pYD5.

 
When CD3{varepsilon}{gamma} staining above background occurs, the distribution of detectable double staining events can be approximated around a diagonal line. This reflects a linear relationship between the V5 epitope staining (classical 1:1 staining) and CD3{varepsilon}{gamma} staining as individual yeast cells display varying amounts of scFv and the associated V5 epitope. This linear relationship is consistent with a model in which the dye-associated streptavidin binds to only one scFv associated CD3{varepsilon}{gamma} molecule and excludes binding models with a higher order of dye-associated potentially multivalent streptavidin binding (Graff et al., 2004Go).

Varying the CD3{varepsilon}{gamma} over a wide range and plotting the MFI of CD3{varepsilon}{gamma} staining versus CD3{varepsilon}{gamma} concentration as shown in Figures 3 and 4 further illuminates the differences between the pYD5 and pYD1 vectors. Figure 3 shows the data for the scFv of UCHT1 in pYD1 (circles) and pYD5 (squares) and the accompanying least-squares fits and the fitted parameters. The upper inset table is for pYD1 revealing a fitted KD of 3.9 nM whereas the lower inset table is for pYD5 revealing a fitted KD of 1.5 nM. These differences were replicated and the average KD ratio increase of pYD1/pYD5 was found to be 3.1 ± 0.4 SD, n = 3. These binding data are fitted fairly well (R > 0.994) by the hyperbolic binding equation that is first order in each component. The goodness of the fit excludes binding models that invoke higher order concentration terms.



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Fig. 3. MFI for human CD3{varepsilon}{gamma} (x-axis) is plotted over a wide range of CD3{varepsilon}{gamma} concentrations for the pYD1 vector (circles) and pYD5 (squares) displaying the scFv of UCHT1. The accompanying least-squares fits and the fitted parameters are shown. The upper inset table is for pYD1 revealing a fitted KD of 3.9 nM and the lower inset table is for pYD5 revealing a fitted KD of 1.5 nM.

 


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Fig. 4. The axes are similar to Fig. 3 except that the displayed protein is scFv of FN18 with the corresponding CD3{varepsilon}{gamma} ligand. pYD5 data points are circles along with the fitted curve (long dashed lines). The lower inset table gives the fitted parameters with a fitted KD of ~7.5 µM. Triangles represent the data points of FN18 scFv in pYD1. None of the MFI values exceeds the blank value of V5 staining without CD3{varepsilon}{gamma} staining. The squares represent the data points of the selected scFv FN18 mutant L1. The fitted KD of the L1 mutant is ~1.3 µM (upper inset table).

 
The plot for MFI versus CD3{varepsilon}{gamma} concentration for FN18 scFv in pYD5 is shown in Figure 4 (circles) along with the fitted curve (long dashed lines). The lower inset table gives the fitted parameters with a fitted KD of ~7.5 µM. The associated error is fairly large and probably reflects the difficulties inherent in performing accurate binding studies that employ wash steps in the face of rapid off rates. Seven independent KD studies were performed on pYD5 FN18 wild-type and yielded a mean KD value of 5.0 µM, a median of 5.6 µM and a standard error of 1.8 µM. Four independent studies were performed with the pYD1 vector displaying wild-type FN18 scFv. None of these data sets could be fitted by the equation. In two cases none of the points were above the blank value as illustrated by the triangles in Figure 4. In a third case the signal was only 1.3 times the blank value and deemed unreliable. A fourth case failed to show any saturation and could only be fitted by a linear function (data not shown). The squares in Figure 4 represent the data points of the selected scFv FN18 mutant L1. The fitted KD of the L1 mutant is ~1.3 µM (upper inset table). The associated error is much smaller than that of the lower affinity wild-type scFv.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The affinity of the anti-human anti-CD3 scFv antibody UCHT1 is attenuated by N-terminal tethering to foreign proteins (Hexam et al., 2001Go; Thompson et al., 2001Go). In the process of performing affinity maturation on the anti-monkey anti-CD3 scFv FN18 we suspected that the same attenuation was taking place in the pYD1 display vector because the affinity of the displayed scFv for its CD3{varepsilon}{gamma} ligand was too low to measure. Attempts to increase the avidity of this reaction by adding the biotinylated ligand with fluorescent-labeled streptavidin in a one step binding reaction were not successful. We therefore rebuilt pYD1 changing the tethering of the displayed protein from N-terminus to C-terminus. The new vector, pYD5, displayed the FN18 scFv and permitted measurement of the affinity to its CD3{varepsilon}{gamma} ligand that was in the micromolar range. This in turn allowed us to select mutants of FN18 scFv with higher affinity. The pYD5 vector is likely to be useful in protein display when N-terminal tethering is suspected of attenuating the ligand binding reaction, particularly when the reaction is of low affinity. Displaying a higher affinity scFv in both vectors permitted a direct comparison of the equilibrium affinities in each vector. The affinity in the rebuilt pYD5 vector was found to be increased by ~3-fold. In addition the maximum binding capacity was increased by ~20%. The construction of a free N-terminal displayed protein necessitated removing any epitope N-terminal to the displayed protein. This reduces some of the diagnostic advantage present in pYD1 that is useful when display is compromised. However the distal V5 epitope of pYD5 provides ample information as to the state of display.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Jim Nagle and the NINDS Sequencing Facility for performing DNA sequence analysis on mutated scFvs.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received January 10, 2005; revised May 11, 2005; accepted May 13, 2005.

Edited by Jane Osbourn





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