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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: CD3/FN18/pYD5/scFv/yeast display
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The monoclonal antibody FN18 (IgG1) was generated by immunizing mice with rhesus peripheral blood mononuclear cells (Nooij et al., 1986a). FN18 reactivity was found to be restricted to rhesus T cells and thymocytes and encompassed both CD4+ and CD8+ T cells (Nooij et al., 1986b
). 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., 1986b
). 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 streptavidinFITC 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 ecto heterodimer ligands
Human or monkey CD3 ecto heterodimers were prepared as described (Wang and Neville, 2004
). 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
ecto heterodimer (based on OD280 and A0.1% 1.36) in 275 µl of 20 mM TrisHCl, 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 freeSH 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
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, NheIVLAEcoRIV5(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)Aga2stopPmeIHindIII, 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 NheIBamHI and BglIIHindIII, 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.
|
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., 2002). 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 = 1.31.5. 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 510 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 HSMTrp-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 34 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 2430 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 PBS0.1% BSA pH 7.4 (PBSBSA), spun down at 5000 g at 4°C (Eppendorf 5417R) and resuspended in 100 µl of PBSBSA. A 10 µl volume was removed and mixed with 90 µl of PBSBSA 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 PBSBSA and set aside in a clean tube for the V5 control. Stock biotinylated CD3 ecto- was diluted to yield different desired concentrations, then 10 µl of cells + anti-V5 were added for each
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 PE100 µl of PBSBSA and 1.5 µl of Strep-488100 µl PBSBSA or streptavidinFITC). Cells were washed with cold PBSBSA pelleted by centrifugation and the liquid was aspirated. Just prior to FACS sample injection (Beckman Coulter Cytomics FC-500), 0.5 ml of PBSBSA 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
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
and the associated blank
mean fluorescence intensity (MFI). The log channel
fluorescence value was plotted versus the log V5 channel fluorescence value and the MFI values were tabulated at each
concentration and plotted on a linear scale versus
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., 2003
; Colby et al., 2004
).
Mutagenesis of scFv libraries and selection of higher affinity mutants
Mutagenesis was performed using the nucleotide analogue method (Zaccolo and Gherardi, 1999) as detailed by Graff et al. (2004)
. 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
staining (Colby et al., 2004
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (NheIVLAFN18 scFvEcoRI) into pYD5, we could not detect the staining either with anti-V5 mAb or monkey CD3
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., 1992
; Parekh et al., 1995
). 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, 1990
). 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 (NheIVLAAGFN18 scFvEcoRI).
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 on the x-axis. The concentrations of CD3
used in this study were 1500 and 500 nM for human and monkey CD3
, respectively. The pYD1 vector exhibited strong staining of the V5 epitope but no staining of monkey CD3
above the blank value (vertical gate) was detected (top left panel). The lack of pYD1 FN18 scFv staining above the zero concentration CD3
blank was replicated in three separate experiments with three different CD3
preparations at 1500 nM. In contrast, weak staining with monkey CD3
was observed with the rebuilt pYD5 vector (Figure 2, top right panel). This finding was replicated in three additional experiments with three different CD3
preparations at 1500 nM, the CD3
staining being 1.7-fold higher than the zero concentration CD3
blank (data not shown). The higher affinity anti-human anti-CD3 scFv stained well in both vectors (Figure 2, bottom panels), but pYD5 CD3
staining was more intense in pYD5 than pYD1.
|
Varying the CD3 over a wide range and plotting the MFI of CD3
staining versus CD3
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.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boder,E.T. and Wittrup,K.D. (2000) Methods Enzymol., 328, 430434.[ISI][Medline]
Brake,A.J. (1990) Methods Enzymol., 185, 408421.[Medline]
Colby,D.W., Kellogg,B.A., Graff,C.P., Yeung,Y.A., Swers,J.S. and Wittrup,K.D. (2004) Methods Enzymol., 388, 348358.[CrossRef][ISI][Medline]
Contreras,J.L., Jenkins,S., Eckhoff,D.E., Hubbard,W.J., Lobashevsky,A., Bilbao,G., Thomas,F.T., Neville,D.M.,Jr and Thomas,J.M. (2003) Am. J. Transplant., 3, 128138.[CrossRef][ISI][Medline]
Degryse,E., Dietrich,M., Nguyen,M., Achstetter,T., Charlier,M., Charpigny,G., Gaye,P. and Martal,J. (1992) Gene, 118, 4753.[CrossRef][ISI][Medline]
Fechner,J.H.,Jr, Vargo,D.J., Geissler,E.K., Graeb,C., Wang,J., Hanaway,M.J., Watkins,D.J., Piekarczyk,M., Neville,D.M.,Jr and Knechtle,S.J. (1997) Transplantation, 63, 13391345.[CrossRef][ISI][Medline]
Feldhaus,M.J. et al. (2003) Nat. Biotechnol., 21, 163170.[CrossRef][ISI][Medline]
Graff,C.P., Chester,K., Begent,R. and Wittrup,K.D. (2004) Protein Eng. Des. Sel., 17, 293304.
Hexham,J.M. et al. (2001) Mol. Immunol., 38, 397408.[CrossRef][ISI][Medline]
Hubbard,J.W., Moore,J.K., Contreras,J., Smyth,C.A., Chen,Z.W., Lobashevsky,A.L., Nagata,K., Neville,D.M. and Thomas,J.M. (2001) Hum. Immunol., 62, 479487.[CrossRef][ISI][Medline]
Ma,S., Hu,H., Thompson,J., Stavrou,S., Scharff,J. and Neville,D.M.,Jr (1997) Bioconj. Chem., 8, 695701.[CrossRef][ISI][Medline]
Neville,D.M.,Jr, Scharff,J., Rigaut,K., Hu,H., Shiloach,J., Slingerland,W. and Jonker,M. (1996) J. Immunother., 19, 8592.[ISI]
Nooij,F.J., Borst,J.G., Van Meurs,G.J., Jonker,M. and Balner,H. (1986a) Eur. J. Immunol., 8, 975979.
Nooij,F.J., Jonker,M. and Balner,H. (1986b) Eur. J. Immunol., 8, 981984.
Parekh,R., Forrester,K. and Wittrup,D. (1995) Protein Expr. Purif., 4, 537545.
Thomas,J.M., Eckhoff,D.E., Contreras,J.L., Lobashevsky,A.L., Hubbard,W.J., Moore,J.K., Cook,W.J., Thomas,F.T. and Neville,D.M.,Jr (2000) Transplantation, 69, 24972503.[CrossRef][ISI][Medline]
Thomas,J., Contreras,J., Smyth,C., Lobashevsky,A., Jenkins,S., Hubbard,W., Eckhoff,D., Stavrou,S., Neville,D. and Thomas,F. (2001) Diabetes, 50, 12271236.
Thompson,J. et al. (2001) Protein Eng., 14, 10351041.[CrossRef][ISI][Medline]
Vanaman,T.C. and Stark,G.R. (1970) J. Biol. Chem., 245, 35653573.
Wang,Z. and Neville,D.M.,Jr (2004) Mol. Immunol., 40, 11791188.[CrossRef][ISI][Medline]
Woo,J.H., Liu,Y.Y., Mathias,A., Stavrou,S., Wang,Z., Thompson,J. and Neville,D.M.,Jr (2002) Protein Expr. Purif., 25, 270282.[CrossRef][ISI][Medline]
Zaccolo,M. and Gherardi,E. (1999) J. Mol. Biol., 285, 775783.[CrossRef][ISI][Medline]
Received January 10, 2005; revised May 11, 2005; accepted May 13, 2005.
Edited by Jane Osbourn
|