Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37°C

Christilyn P. Graff1, Kerry Chester2, Richard Begent2 and K.Dane Wittrup1,3

1Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA and 2Royal Free Hospital, University College London, Gower Street, London WC1E 6BT, UK

3 To whom correspondence should be addressed. E-mail: wittrup{at}mit.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37°C. Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry. Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.

Keywords: affinity/carcinoembryonic antigen/directed evolution/scFv/tumor targeting


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carcinoembryonic antigen (CEA) has long been identified as a tumor-associated antigen (Gold and Freedman, 1965Go). Originally classified as a protein expressed only in fetal tissue, CEA has now been identified as present in several normal adult tissues. These tissues are primarily epithelial in origin, including cells of the colon, stomach, tongue, esophagus, cervix, sweat glands and prostate (Nap et al., 1988Go, 1992Go; Prall et al., 1996Go). Not surprisingly, tumors of epithelial origin, as well as their metastases, contain CEA as a tumor-associated antigen. Although the presence of CEA itself does not indicate transformation to a cancerous cell, the localization of CEA in the cell is indicative. In normal epithelial tissue, CEA is restricted to the apical surface of the cell (Hammarstrom, 1999Go) and so secreted CEA does not gain access to circulation. As an example of normal tissue expression, a healthy adult excretes 50–70 mg of CEA in feces per day (Matsuoka et al., 1991Go) but has <10 µg/l in the blood. In contrast to normal tissue, cancerous cells will express CEA over the entire surface (Hammarstrom, 1999Go). Furthermore, in cancer the malignant glandular structure permits leakage of secreted CEA into the blood. It is this loss of polarity that allows the use of serum CEA levels as a diagnostic for recurrence of the disease in colon cancer (Graham et al., 1998Go).

Carcinoembryonic antigen is a 180 000 kDa protein with ~50% carbohydrate content. It has seven domains, with a single N-terminal Ig variable domain and six domains homologous to the Ig constant domain (Williams and Barclay, 1988Go). Several distinct epitopes have been identified in CEA (Hammarstrom et al., 1989Go). Multiple monoclonal antibodies have been raised against CEA for research purposes and as diagnostic tools (Nap et al., 1992Go). More recently, single chain antibody fragments have been isolated from phage display libraries to be used in radioimmunodetection and radioimmunotherapy (Chester et al., 1994Go; Osbourn et al., 1999Go). Of particular interest is the MFE-23 scFv (Chester et al., 1994Go). This antibody fragment has been shown to target colon cancer effectively for radioimmunodetection in vivo (Begent et al., 1996Go). MFE-23 has also been used in radioimmunoguided surgery of colorectal cancer (Mayer et al., 2000Go). Its rapid clearance from the bloodstream (due to its low molecular weight) is advantageous over full-sized monoclonal antibodies by reducing the time from injection to surgery. MFE-23 has also been produced as a fusion protein to carboxypeptidase G2 (CPG2) and TNF{alpha}. Both fusions have shown promise for therapy (Chester et al., 2000aGo,bGo; Cooke et al., 2002Go).

One avenue of improvement that may increase the efficacy of MFE-23 is to increase the retention time of the antibody in the tumor relative to normal tissue (Graff and Wittrup, 2003Go). This can be accomplished through directed evolution. A previous affinity maturation trial with another anti-CEA scFv (CEA6) produced several affinity-improved mutants, the best of which had a 7-fold reduction in the off-rate over CEA6 (Osbourn et al., 1999Go). When these scFvs were tested for their tumor targeting efficiency in mice, the highest affinity antibody showed no difference in tumor uptake relative to the parental scFv at 24 h, but persisted at the tumor site for somewhat longer than the original scFv (Jackson et al., 1998Go). Although these results are encouraging, the modest improvement in the off-rate in that study raises the question of whether a larger improvement in off-rate would result in further improvements in tumor targeting.

In this study, MFE-23 was engineered to increase the retention time of the antibody in the tumor relative to normal tissue. In order to reduce the likelihood of immunogenicity, the antibody was first resurfaced to present a more ‘human’ Fv framework surface (Pedersen et al., 1994Go; Roguska et al., 1994Go). The crystal structure of MFE-23 shows structural similarity to another crystallized human antibody. This resemblance allowed for the determination of 28 residues in MFE-23 that were >30% solvent-accessible and chemically different from or similar to the human antibody (TR1.9) (Boehm et al., 2000Go). For humanization of MFE-23, the residues suggested in Boehm et al. were changed to the corresponding residues in the human antibody. Equilibrium titration verified that altering these 28 residues resulted in no detectable change in CEA binding affinity. This resurfaced scFv is termed hMFE.

Increased affinity of the hMFE scFv may increase the retention time of the antibody in a tumor, as shown previously (Osbourn et al., 1999Go) and as predicted by mathematical modeling (Graff and Wittrup, 2003Go). A critical parameter determining tumor retention is the dissociation rate constant (koff). After two rounds of mutagenesis and screening of yeast surface-displayed libraries, several variants of hMFE were isolated which showed a 10-, 100- and 1000-fold improvement in the off-rate over the original scFv, hMFE. The greatest improvement corresponds to a half-life for binding to CEA of ~4–7 days at 37°C (versus 10 min for hMFE).

A final parameter addressed in this study was the stability of the scFv. In order to function in a therapeutic setting, the scFv must be resistant to thermal denaturation at 37°C (Willuda et al., 1999Go). Furthermore, stability has been shown to correlate closely with expression efficiency (Shusta et al., 1999Go) and the affinity-matured hMFE mutants were found to express poorly. For these reasons, all affinity-improved mutants were subjected to stability maturation. All stability-improved mutants retained their affinity gains and soluble expression levels were greatly increased. Yeast cells displaying the stabilized, highest affinity mutant retained ~80% binding activity after incubation at 37°C for 9 days.

It is important to note that the off-rate achieved in this study is amongst the slowest known for antibody–protein antigen interactions. However, a particular significance of this panel of very stable antibody fragments is that they span three orders of magnitude in off-rate improvement. This series of scFvs will therefore permit the definitive determination of the issue of the impact of affinity on efficacy in anti-CEA tumor therapy.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cloning of MFE-23 and hMFE surface display plasmids

The MFE-23 scFv was PCR amplified from the MFE-23his vector (Casey et al., 1995Go). The resulting 750 bp fragment was digested with NheI and BamHI, gel purified and ligated into the yeast surface display vector pCTCON. The pCTCON vector is a variant of pCT302 (Boder and Wittrup, 1997Go) that contains a unique BamHI site 5' to the c-myc coding sequence. Twenty-eight residues of the MFE-23 scFv were identified for resurfacing (Boehm et al., 2000Go). The resurfaced humanized version (hMFE) was designed using yeast optimum codons with an NheI site immediately 5' to the gene and a BamHI site immediately 3' to the gene. The gene was synthesized by Synthetic Genetics (San Diego, CA). The hMFE scFv was excised from the Synthetic Genetics vector with NheI and BamHI and cloned into pCTCON. The two display vectors, pCTMFE23 and pCThMFE, were transformed into yeast strain EBY100 (Boder and Wittrup, 1997Go) by the lithium acetate method of Gietz and co-workers (http://tto.trends.com). To test expression of the Aga2p–MFE-23 and Aga2p–hMFE fusions, cells were grown at 30°C for 24 h in SD-CAA (2% dextrose, 0.67% yeast nitrogen base, 1% casamino acids) to OD600 = 5.0–7.0. Cells were then transferred to SG-CAA (2% galactose, 0.67% yeast nitrogen base, 1% casamino acids) to a starting OD600 = 1.0 and grown for 16–20 h at 20 or 37°C.

To perform an equilibrium titration, cells displaying the MFE-23 and hMFE scFvs were labeled at multiple concentrations with biotinylated CEA and incubated at 37°C for 1–24 h. In order to limit depletion of antigen, the labeling volume was increased with decreasing CEA concentration. Cells were washed with cold PBS–BSA and labeled with a 1:100 dilution of 9E10 on ice. Cells were washed and labeled with secondary reagents goat-anti-mouse–FITC (1:50) (Sigma) and streptavidin–PE (1:100) (Pharmingen) on ice. Cells were washed and resuspended for analysis on a Coulter EPICS XL. The mean fluorescence of the labeled population was plotted against the CEA concentration and data were fitted to calculate the dissociation constant.

Soluble production and detection of MFE-23 and hMFE

The pCTMFE-23 and pCThMFE vectors were digested with NheI and XhoI. The digests were isolated on a 1% agarose gel and the 750 bp scFvs were excised from the gel and purified with the Qiagen gel purification kit. The scFv was ligated into the pRS4420 vector (Boder et al., 2000Go), which was also digested with NheI and XhoI and gel purified. The new vectors, pRSMFE-23 and pRShMFE, were digested with BamHI and XhoI to remove the c-myc epitope tag. The digests were isolated on a 1% agarose gel and the ~6700 bp backbone was purified with a Qiagen gel purification kit. The oligos were annealed and ligated into the backbone. The resulting vectors, MFE23–His and hMFE-his, contain the scFv followed by a C-terminal His6 tag. The two secretion vectors, MFE23–His and hMFE-his, along with the pRS314 vector were transformed into yeast strain YVH10 (Shusta et al., 1998Go) by the lithium acetate method of Gietz and co-workers (http://tto.trends.com). To test soluble expression of the scFvs, cells were grown at 30°C for 48 h in SD-SCAA [2% dextrose, 0.67% yeast nitrogen base, 1% synthetic casamino acids (Ura-, Trp-, Leu-)] to OD600 = 5.0–7.0. Cells were pelleted and resuspended in SG-SCAA [2% galactose, 0.67% yeast nitrogen base, 1% casamino acids (Ura-, Trp-, Leu-) supplemented with 1 mg/ml of BSA] and grown for 36–48 h at 20 or 37°C.

For detection of the scFv, samples were run on a 12% polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was blocked in a milk solution (1.5 mg of non-fat dry milk in 30 ml of TBST [8 g/l NaCl, 3.6 g/l Tris base, 1 ml/l Tween-20 solution (pH 7.6)] at room temperature for 1 h. The membrane was washed 1 x 15 min and 2 x 5 min in 30 ml of TBST. The membrane was incubated with the tetra-His antibody (Qiagen) (1:800 dilution) for 1 h at room temperature and was washed 1 x 15 min and 2 x 5 min in 30 ml of TBST. The membrane was incubated with a goat anti-mouse–HRP antibody (1:2000) and streptavidin–HRP (1:1500) for 20 min at room temperature. The membrane was washed 1 x 15 min and 4 x 5 min in 30 ml of TBST. The membrane was developed with ECL reagents. The chemiluminescent signal was detected with a Bio-Rad Fluor-S Imager for 2–30 min. Samples were quantitated with Quantity one software.

Construction and screening of hMFE library

A random scFv library of the hMFE scFv was created by adapting the nucleotide analogue method of Zaccolo and Gherardi (1999)Go. The expression cassette of hMFE was amplified by PCR with the T3 and T7 promoter standard primers. Five mutagenic PCR conditions were used: 250 mM dPTP and 8-oxo-dGTP/five cycles; 25 mM dPTP and 8-oxo-dGTP/10 cycles; 2.5 mM dPTP and 8-oxo-dGTP/10 cycles; 25 mM dPTP and 8-oxo-dGTP/20 cycles; 2.5 mM dPTP and 8-oxo-dGTP/20 cycles. Other PCR components were 1 ng of pCThMFE, 250 mM each dNTP, 0.5 mM each primer, 3 units Taq polymerase (Gibco), 1 x Gibco PCR buffer supplemented with 2 mM MgCl2. The reaction was cycled as follows: 94°C 1 min, 50°C 1 min, 72°C 3.5 min. PCR products from 10 and 20 cycles were isolated on a 1% agarose gel. Four 2200 bp fragments were excised from the gel and purified with a gel purification kit (Qiagen). PCR products from five cycles were diluted 1:10 for further amplification. The PCR fragments were then amplified in the absence of the nucleotide analogues for 25 cycles using Taq polymerase. The PCR conditions were identical with those listed above, with the exception of extension time at 72°C, which was changed to 3 min and 10 s. PCR products were purified using a PCR purification kit (Qiagen). Purified PCR fragments were digested with NheI and BamHI, gel purified and ligated overnight at 16°C into pCThMFE. Ligation reactions were transformed into 10 aliquots of DH5a-FT cells (Life Technologies). Transformants were pooled and aliquots were plated to determine the library size. The diversity was calculated to be 105. The library was amplified in LB/Amp50/Carb50 medium at 37°C and plasmid DNA was purified with a Qiagen Maxi-prep kit. Ten clones were selected for sequencing from the library and the mutagenesis rate was calculated to be 0.2–5%. Library DNA was transformed into yeast strain EBY100 (Boder and Wittrup, 1997Go) by the lithium acetate method of Gietz and co-workers (http://tto.trends.com). Transformants were pooled in SD-CAA (2% dextrose, 0.67% yeast nitrogen base, 1% casamino acids) and aliquots were plated to determine library diversity. The library was passaged twice in SD-CAA to reduce the concentration of untransformed EBY100.

The library was grown to OD600 = 10.0 in SD-CAA. Cells were transferred to SG-CAA to OD600 = 1.0 and grown for 18 h at 37°C. The hMFE library was screened by the equilibrium method as described in Boder and Wittrup (1998)Go. The optimal biotinylated CEA concentration was calculated from the mathematical model. Concentrations were selected to screen for 3- and 10-fold improvements. Yeast cells displaying mutated hMFE scFv were incubated in 0.35 or 0.2 nM biotinylated CEA at 37°C. CEA purified from primary human tumor samples (Calbiochem) was biotinylated by the succinimidyl ester method on primary amines (Molecular Probes.) Cells were washed with cold PBS–BSA (8 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na2HPO4, 0.24 g/l KH2PO4, 1 mg/ml BSA) and labeled with a 1:100 dilution of the monoclonal antibody 9E10 on ice (Covance). Cells were washed with cold PBS–BSA and labeled with secondary reagents streptavidin–phycoerythrin (1:100) (Pharmingen) and goat anti-mouse–FITC (1:50) (Sigma) on ice. Cells were washed with PBS–BSA and resuspended at a concentration of 107 cells/ml and sorted on a Becton Dickinson FACStar flow cytometer (Flow Cytometry Center, MIT Cancer Research Center) with a sort rate of ~4000 cells/s. Cells were collected with gate settings designed to collect the cells displaying the highest PE fluorescent signal (CEA binding) per FITC fluorescent signal (scFvs on surface). Four rounds of sorting and regrowth were performed to isolate a highly enriched (100%) population of improved mutants. Mutants were analyzed from both the third- and fourth-round sorts.

Analysis of clones from equilibrium sort

Four individual clones were chosen at random from each of the third- and fourth-round sorts for analysis by equilibrium titration. For each clone, 10 samples of 106 cells were incubated with various concentrations of biotinylated CEA at 37°C for 1–24 h. Cells were washed with PBS–BSA and labeled with a 1:100 dilution of the monoclonal antibody 9E10 on ice. Cells were washed and labeled with secondary reagents streptavidin–PE (1:100) and goat-anti-mouse–FITC (1:50). Cells were washed again and resuspended at a concentration of ~106 cells/ml for analysis on a Coulter EPICS XL flow cytometer using Expo v.2 software. All clones with improved dissociation constants were selected for sequence analysis. Plasmids were rescued from the yeast cells using a Zymoprep (Zymogen) kit following the manufacturer's protocol and transformed into XL1-blue competent cells (Stratagene). Escherichia coli cultures were grown overnight in LB–AMP100 and plasmid DNA was purified using a Qiagen Miniprep kit. Sequencing of the scFv ORF was done using the dideoxy terminator method on a Perkin-Elmer Applied Biosystems Model 377 DNA sequencer at the MIT Cancer Research Center Polymer Laboratory.

Construction and screening of hLib2 library

A second random scFv library based on clones isolated from the first library was constructed by adapting the sexual PCR method of Stemmer (1994)Go. The expression cassette of improved round one mutants and enriched pooled library was amplified with the T3 and T7 promoter standard primers. PCR products (~10 mg) were digested to <200 bp with DNase I. Samples were equilibrated for 5 min at 15°C, then digested with 1 unit of DNase I (Boehringer) for 90 s at 15°C. DNase I was inactivated by heating at 90°C for 10 min. Samples were purified with the Qiagen Qiax II kit. Five DNase digestion mixtures were used (Table I), combining equal amounts of each component. Digested products were recombined following the method of Stemmer, replacing Taq polymerase with Pfu polymerase and cycling for 45 cycles. Six recombination mixtures were used, one for each of the five digestion mixtures and one combining all five digestion mixtures. PCR conditions were as follows: 94°C 3 min (1 cycle); 94°C 1 min, 55°C 1 min, 72°C 1 min + 5 s/cycle (45 cycles); 72°C 7 min (1 cycle). Final amplification was performed with primers nested ~50 and ~100 bp from the ends of the scFv ORF and in the presence of 2.25 mM MnCl2 and 0.375 mM MgCl2 to introduce further mutations. PCR products were purified by gel electrophoresis, digested with NheI and BamHI and ligated into the pCThMFE backbone. A portion of the library (25%) was created by the nucleotide analogue method as described previously. Ligation reactions were transformed into XL10-Gold competent cells (Stratagene), pooled and aliquots were plated to determine library diversity. The library size was calculated to be 2 x 105. Library DNA was purified and transformed into EBY100 as above.


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Table I. Mutants from equilibrium library screen

 
The second random hMFE library was selected with a kinetic screen as described by Boder and Wittrup (1998)Go. Yeast cells displaying mutant scFv were incubated in 2 nM biotinylated CEA at 37°C. Cells were washed with cold PBS–BSA and then resuspended in a 100-fold excess of CEA (~250 nM) and returned to 37°C. The optimal competition time was calculated from Boder and Wittrup (1998)Go; however, this time was not used in order to reduce the length of the experiment. First round competition was 1 day, second round was 1 day, third round was 1.2 days and fourth round was 2 days. Cells were washed with PBS–BSA and labeled with a 1:100 dilution of 9E10 (Covance) on ice. Cells were washed in PBS–BSA and labeled with secondary reagents as described above. Cells were washed with PBS–BSA and resuspended to a concentration of 107 cells and sorted on a Becton Dickinson FACStar flow cytometer. Four rounds of sorting and regrowth were performed to isolate a highly enriched (100%) population of improved mutants.

Analysis of clones isolated from kinetic sort

Ten individual clones were chosen at random from the fourth sort for off-rate analysis. For each clone, 107 cells were incubated in 2 nM biotinylated CEA at 37°C for 3 h. Cells were washed with PBS–BSA and transferred to microcentrifuge tubes (106 cells/tube) and incubated in a 100-fold excess of CEA (Calbiochem) at 37°C for various amounts of time. Cells were washed and incubated in a 1:50 dilution of 9E10 (Covance) on ice, then washed and incubated in secondary reagents streptavidin–PE (1:100) and goat anti-mouse–FITC (1:50) on ice. Cells were washed and resuspended at a concentration of 106 cells/ml for analysis on a Coulter Epics XL flow cytometer. All clones with improved off-rates were selected for sequence analysis. Plasmid rescue and sequencing were performed as described above.

Soluble competition assay

In order to measure the off-rate of sm3E in soluble form, purified sm3E scFv samples were incubated in 4.8 nM biotinylated CEA at 37°C for 3 h in binding buffer (PBS, 0.05% Triton X-100, 1 mM PMSF, 0.5 mM iodoacetamide, 2.5 mM EDTA, 0.02% NaN3, 0.1% BSA). At designated time points, a 10-fold excess of CEA (Calbiochem) was added to the scFv–biotinylated CEA mixture. Each time point was sampled at least in triplicate and the entire experiment was repeated three independent times with the scFv sm3E. To measure an infinite competition time, 4.8 nM biotinylated CEA and a 10-fold excess of CEA were pre-mixed and sm3E was added to this solution. A 96-well plate (Immulon 2 HB, Dynex) was coated with an anti-His4 antibody (Qiagen) at a concentration of 10 µg/ml overnight at 4°C. Sample wells were blocked with blocking solution (PBS, 0.02% NaN3, 3% BSA) for 2 h at room temperature. The plate was washed four times with TBST. The sm3E–biotinylated CEA–CEA solutions were added to the wells and incubated for 1 h at 37°C. The plate was washed five times with TBST and a 1:1000 dilution of streptavidin–europium (Wallac) in assay buffer was added to the wells and incubated at 4°C for 30 min. The plate was washed seven times with TBST. Enhancement solution was added to each of the wells and incubated at room temperature for 20 min. Fluorescence was measured with a Victor 1420 Multilabel Counter.

Stability screen of hMFE library

The first random hMFE library was also screened for improved stability of the scFv. Cells were induced at 37°C. Yeast cells were incubated in 10 nM biotinylated CEA at 37°C. Cells were transferred to microcentrifuge tubes, washed with ice cold PBS–BSA and incubated in a 1:100 dilution of 9E10 (Covance) on ice. Cells were labeled with secondary reagents as described previously. Cells were sorted at a concentration of 107 cells/ml on a Becton Dickinson FACStar flow cytometer. Cells were sorted with gate settings designed to collect the highest FITC signal for the CEA binding population. Three rounds of sorting and regrowth were performed to isolate an enriched population of better displayed mutants. Sixteen mutants were analyzed from the third sort. For each clone, cells were labeled with 10 nM bio-CEA and 9E10. Six clones with higher display levels were selected for sequence analysis. Plasmids were rescued and sequenced as described previously. The single common stabilizing mutation, VL W47L, was added back to m10B and m3E with site-directed mutagenesis.

Soluble production of mutants

The pCTm10B, pCTm3E, pCTm10B47L and pCTm3E47L vectors were digested with NheI and BamHI. The digests were isolated on a 1% agarose gel and the 750 bp scFvs were excised from the gel and purified with the Qiagen gel purification kit. The four scFvs were ligated into the MFE23-His vector, which was also digested with NheI and BamHI and gel purified. Insertion of the product was confirmed by restriction digest. The secretion plasmids were transformed into the yeast strain YVH10 as previously described. Soluble production was performed as previously described, with protein induction at 37°C for 48 h. Samples were detected by an anti-His4 western blot.

Construction of destabilized scFv library

The two cysteines in the light chain of m3E and m3E47L were changed to valine and alanine, respectively. A QuikChange kit (Stratagene) was used to perform site-directed mutagenesis, following the manufacturer's protocol. Two random scFv libraries were created based on m3E-VLcysout and m3E-VLcys47L using the nucleotide analogue method. The expression cassette of each scFv was amplified by PCR with the T3 and T7 promoter standard primers. Three mutation PCR conditions were used: 250 mM analogues/5 cycles, 25 mM analogues/12 cycles, 2.5 mM analogues/12 cycles. Amplification, purification and ligation were performed as described previously. Ligation reactions were transformed into Ultramax DH5a-FT cells (Invitrogen). Both libraries were 105 in size. Growth of transformants and plasmid purification were performed as described previously.

Screening of destabilized scFv libraries

For the m3E-VLcysout library, cells were incubated in 5 nM biotinylated CEA and 1:50 9E10 at 37°C. Cells were washed with cold PBS–BSA and resuspended in a 1:50 dilution of goat anti-mouse–FITC and 1:100 streptavidin–PE and incubated on ice. Cells were washed with cold PBS–BSA and resuspended to a concentration of 107 cells/ml for sorting. For the m3E-VLcys47L library, cells were incubated in 1 nM biotinylated CEA at 37°C. Cells were washed with PBS–BSA, resuspended in a 1:50 dilution of 9E10 and incubated on ice. Cells were washed with cold PBS–BSA and resuspended in a 1:50 dilution of goat anti-mouse–FITC and 1:100 streptavidin–PE and incubated on ice. Cells were washed with cold PBS–BSA and resuspended to a concentration of 107 cells/ml for sorting on a Beckton Dickinson FACStar flow cytometer. Sort windows were set to collect the cells with the highest CEA binding per number of scFvs on the surface for both libraries. Three rounds of sorting and regrowth were performed to isolate a highly enriched population for both sorts. Twenty clones were analyzed from each of the second and third sorts of the m3E-VLcys47L library. For each clone, cells were labeled with 1 nM biotinylated CEA at 37°C and 9E10 on ice. Ten clones with higher display levels and CEA binding were selected for sequence analysis. Twenty clones were analyzed from the third sort of the m3E-VLcysout library. For each clone, cells were labeled with 5 nM biotinylated CEA at 37°C and 9E10 on ice. Five clones with higher display levels and CEA binding were selected for sequence analysis. Plasmids were rescued and sequenced as described previously.

Construction of more stable high-affinity mutant

Four mutations were selected from the destabilized library sorts. Multi-QuikChange and QuikChange kits (Stratagene) were used to add these mutations back to hMFE47L, m9B47L, m10B47L and m3E47L with site-directed mutagenesis following the manufacturer's protocol. Insertion of the mutations was confirmed by sequence analysis.

Soluble production of high-stability, high-affinity mutant

The restabilized hMFE, m9B, m10B and m3E scFv display vectors were digested with NheI and BamHI. The digests were isolated on a 1% agarose gel and the 750 bp scFv was excised from the gel and purified with a Qiagen gel purification kit. The scFvs were ligated into the MFE23–His vector, which was also digested with NheI and BamHI and gel purified. Insertion of the scFv was confirmed by restriction digest and sequence analysis. The secretion plasmid was transformed into the yeast strain YVH10 as previously described. Soluble production was performed as previously described, with protein induction at 37°C for 48 h. Samples were detected by an anti-His4 western blot.

For large-scale production of each of the mutants, a 5 ml culture was grown for 36–48 h at 30°C in SD-SCAA [2% dextrose, 0.67% yeast nitrogen base, 1% synthetic casamino acids (Ura-, Trp-, Leu-)]. The 5 ml culture was then used to inoculate a 1 l culture, which was grown for 36–48 h at 30°C in SD-SCAA. Cells were pelleted and transferred to a 1 l culture of SG-SCAA [2% galactose, 0.67% yeast nitrogen base, 1% casamino acids (Ura-, Trp-, Leu-) supplemented with 0.42 mg/ml of BSA] and induced at 30°C for 48 h. The supernatant containing the scFv was concentrated with an Amicon stirred ultrafiltration cell (molecular weight cut-off 10 kDa) and exchanged 200–1000-fold with column buffer (300 mM NaCl, 50 mM NaH2PO4, pH 7.9). The scFv was purified from the yeast supernatant using the histidine tag by IMAC (immobilized metal-affinity chromatography). Supernatant was incubated with nickel resin (Qiagen) for 2 h at room temperature and then loaded on to a column. Resin was washed with 5–7 column volumes with wash buffer (300 mM NaCl, 50 mM NaH2PO4, 20 mM imidazole, pH 7.9). The scFv was eluted from the column with elution buffer (300 mM NaCl, 50 mM NaH2PO4, 250 mM imidazole, pH 7.9). The protein eluted predominantly in the second and third elution fractions. All fractions containing scFv were combined and samples were exchanged with PBS to reduce the imidazole concentration to <1 mM.

Soluble binding assays

In order to assess the stability of the scFvs, a 96-well plate (Immulon 2 HB, Dynex) was coated with an anti-His4 antibody (Qiagen) at a concentration of 10 µg/ml overnight at 4°C. Sample wells were blocked with blocking solution (PBS, 0.02% NaN3, 3% BSA) for 2 h at room temperature. The plate was washed four times with TBST (200 mM NaCl, 50 mM Tris, 0.1% Tween-20, pH 8.0). The scFv sample was incubated at 37, 70 and 84°C for 1 h in binding buffer (PBS, 0.05% Triton X-100, 1 mM PMSF, 0.5 mM iodoacetamide, 2.5 mM EDTA, 0.02% NaN3, 0.1% BSA). Samples were then incubated with 25 nM biotinylated CEA for 1 h at 37°C. The scFv–biotinylated CEA solution was added to the wells and incubated for 1 h at 37°C. The plate was washed five times with TBST. A 1:1000 dilution of streptavidin–europium (Wallac) in assay buffer was added to the wells and incubated at 4°C for 30 min. The plate was washed seven times with TBST. Enhancement solution was added to each of the wells and incubated at room temperature for 20 min. Fluorescence was measured with a Victor 1420 Multilabel Counter.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resurfacing humanization of MFE-23 scFv

Two general alternative approaches have been described for humanizing a murine Fv. ‘Loop grafting’ involves substitution of murine CDRs into a homologous human framework, followed by fine tuning of several key framework residues (Baca et al., 1997Go; Rader et al., 1998Go). ‘Resurfacing’ involves changing only the solvent-exposed residues in a murine framework to their human homologs. A possible pitfall of resurfacing, which has not yet been experimentally reported, is potential retention of T-cell epitopes involving buried murine framework residues. However, a benefit of resurfacing is that it generally has a less negative impact on antigen-binding affinity. Resurfacing was recently applied to the antibody cantuzumab and, in a clinical trial with 37 patients, no instances of HAHA were observed (Tolcher et al., 2003Go). The open reading frames of the MFE-23 scFv and humanized MFE-23 (hMFE) scFv were subcloned into the yeast display vector, pCTCON, a variation of pCT302 (Boder and Wittrup, 1997Go) containing a BamHI site. This construct contains an HA tag N-terminal and a c-myc tag C-terminal of the scFv, to allow detection of scFv expression independent of CEA binding. Twenty-eight changes were made to the MFE-23 scFv in order to produce hMFE (Boehm et al., 2000Go). Display of the full-length fusion of each scFv was confirmed on the yeast surface by labeling with an anti-c-myc antibody. Both scFvs also bound biotinylated CEA. Single-chain antibody fragments are often only produced at 20°C in the yeast display system owing to instability of the protein (Boder and Wittrup, 1997Go). Antibody production was also tested at 37°C to measure the relative stability of MFE-23 and hMFE. Higher levels of each scFv were detected on the surface (as measured by fluorescence) for expression at 37°C versus 20°C. An equilibrium titration was performed to measure the dissociation constant (KD) of MFE-23 and hMFE at 37°C. Both scFvs bound CEA with a similar KD (Figure 1). MFE-23 and hMFE were also produced solubly at 37°C. Resurfacing did not alter the CEA binding affinity and was found to increase soluble expression levels slightly.



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Fig. 1. CEA binding affinity of MFE-23 scFv and resurfaced hMFE. Equilibrium binding constants KD for CEA at 37°C. Titrations were performed by labeling yeast cell surface-displayed scFvs with biotinylated CEA at various concentrations and detecting binding with streptavidin–phycoerythrin and flow cytometry. hMFE expressed at 20°C (KD = 8.3 ± 0.5 nM) (circles); MFE-23 expressed at 20°C (KD = 8.3 ± 1.3 nM) (squares); hMFE expressed at 37°C (KD = 8.5 ± 0.6 nM) (diamonds); MFE-23 expressed at 37°C (KD = 7.5 ± 0.8 nM) (triangles).

 
hMFE affinity maturation by equilibrium selection

In order to affinity mature the hMFE scFv, random mutagenesis was performed on the entire scFv by error-prone PCR with an error rate from 0.2 to 5%. The error rate was controlled over this range by using the mutagenic nucleotide analogues 8-oxo-dGTP and dPTP (Zaccolo and Gherardi, 1999Go) and by varying the number of PCR cycles (see Materials and methods). The resulting library contained 105 clones. The library was transformed into the yeast surface display strain EBY100, over-sampling the library size by 10-fold. An equilibrium sort was performed to isolate scFvs with an improved dissociation constant, with optimal selection parameters calculated as previously described (Boder and Wittrup, 1998Go).

The KD of hMFE was measured as 8.5 nM on the yeast surface (Figure 1). For a 10-fold improvement in the KD, the optimal CEA labeling concentration was calculated to be 0.2 nM. A highly enriched population of improved scFvs was isolated after four flow cytometry sorts. The progression of the enrichment is shown in Figure 2. Four clones were analyzed by equilibrium titration from each of the third and fourth sorts (Figure 2D and E) and all had an improved dissociation constant. Seven unique clones were isolated (Table I). Six of the seven clones contained the VLS50L mutation in the CDR 2 loop of the light chain. One clone, m10B, contained only the S50L mutation. Since m10B maintains high affinity for CEA when the other mutations are not present, only the S50L change is necessary for the improvement in affinity. The highest affinity clone (m10B) has a dissociation constant of 0.08 nM and a dissociation rate constant of 6.0 x 10–6 s–1 (Figure 3). This corresponds to a 100-fold improvement in the affinity and a 200-fold improvement in the dissociation rate over the wild-type scFv hMFE. The only clone that did not contain the S50L mutation was m9B. It has a dissociation constant of 0.8 nM (Figure 3A) and a dissociation rate of 7.5 x 10–5 s–1 (Figure 3B), which represent a 10- and 16-fold improvement over hMFE.



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Fig. 2. Progressive enrichment of improved-affinity hMFE mutants, with hMFE library labeled with 0.2 nM biotinylated CEA at equilibrium. CEA binding is on the y-axis and surface expression level on the x-axis. (A) hMFE sample; (B) library mutated by error-prone PCR, with sort window drawn on figure; (C) sample of amplified first sort population, with sort window drawn for second sort; (D) sample of amplified second sort population, with sort window drawn for third sort; (E) sample of amplified third sort population, with sort window drawn for fourth sort.

 


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Fig. 3. Antigen-binding analysis of clones isolated from hMFE library screens. (A) Equilibrium titration. hMFE (KD = 8.5 ± 0.5 nM) (circles); m9B (KD = 0.8 ± 0.1 nM) (squares); m10B (KD = 0.08 ± 0.01 nM) (diamonds). (B) Dissociation rates were measured by first labeling yeast displaying the scFv of interest with biotinylated CEA. Then, the labeled cells were washed and incubated with an excess of non-biotinylated CEA. Dissociation of labeled CEA from the cells was followed at the single-cell level by flow cytometry and streptavidin–PE labeling, as described in Materials and methods. hMFE [koff = (1.2 ± 0.1) x 10–3 s–1] (circles); m9B [koff = (7.5 ± 0.4) x 10–5 s–1] (squares); m10B [koff = (6.0 ± 0.4) x 10–6 s–1] (diamonds). (C) Dissociation rate analysis of clones isolated from hMFE library and hLib2 library sorts. m9B (squares); m10B (diamonds); m3E [koff = (1.2 ± 0.4) x 10–6 s–1] (triangles). (D) Confirmation of scFv dissociation kinetics by soluble competition ELISA, as described in Materials and methods. Complexes were formed between soluble biotinylated CEA and sm3E scFv, then competition with a 10-fold excess of non-biotinylated CEA was performed for the designated times. ScFv was captured on microplates via a C-terminal His6 tag and captured biotinylated CEA detected with streptavidin–europium. In three independent experiments, the dissociation rate constant koff was curve fitted to 1.7 x 10–6, 2.9 x 10–6 and 1.6 x 10–6 s–1.

 
Further affinity maturation by kinetic selection

To improve the affinity further, a second library was constructed by random mutagenesis and DNA shuffling. For random mutagenesis (~25% of the final library), the conditions were the same as those used to create the first library. Several different populations were combined for the shuffled portion of the library. These included the third and fourth sorted populations from the equilibrium sort, the first hMFE library, hMFE, m9B and m10B. The resulting library contained 2 x 105 clones. A kinetic sort was used to isolate clones with slower dissociation, with optimal selection conditions calculated as previously described (Boder and Wittrup, 1998Go). The optimal competition time was determined based on the best mutant from the first sort (m10B), the signal-to-noise ratio and the desired improvement in the off-rate. For a 3-fold improvement in koff, the optimal competition time with non-fluorescent CEA was calculated to be 7 days. In order to reduce the time necessary to isolate improved clones, competition times shorter than the optimal time were selected. Mutants with slower off-rates were isolated by competition with non-fluorescent CEA for 1–3 days (first sort, 1 day; second sort, 1 day; third sort, 1.2 days; fourth sort, 2 days). A highly enriched population was isolated after four sorts. Ten clones were analyzed by competition and all had slower dissociation kinetics. Eight unique clones were isolated (Table II). Seven of the eight clones contained the VLS50L and VLP36L mutations. The most improved mutant (m3E) has a measured dissociation rate constant of 1.2 x 10–6 s–1 (Figure 3C). As it happens, m3E is simply the combination of m9B and m10B from the previous cycle of mutagenesis and screening. The very slow dissociation kinetics for m3E–CEA should correspond essentially to irreversible targeting in vivo (see Discussion), indicating that further affinity maturation is not warranted. To confirm the slow dissociation kinetics of the scFv–CEA complex, sm3E (a stabilized mutant of m3E) was expressed solubly and the kinetics of dissociation from soluble CEA were determined by competition immunoassay (Figure 3D). The dissociation rate constant measured in this assay was (2.1 ± 0.7) x 10–6 s–1, within error of the value measured on the yeast surface in the flow cytometric assay. scFv presented multivalently on the yeast cell wall has been observed previously to produce avidity effects with multivalent antigens. When such avidity effects occur, they are manifest in the two-dimensional flow cytometric dot plots such as Figure 2 as non-linear, non-diagonal groupings: bound antigen ligand levels would increase greater than proportionally with increasing surface expression levels. This effect is absent for the data in Figure 2 and all such data for sm3E. Furthermore, soluble scFv, even if dimerized, would exhibit avidity effects quantitatively distinct from those for cell-displayed multivalency. The quantitative significant agreement between soluble and surface-displayed dissociation rate measurements is not consistent with the presence of any avidity effect.


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Table II. Mutants from kinetic library screen

 
Expression and stability maturation

Unfortunately, increased affinity correlates with decreased expression for this particular series of affinity mutants (data not shown). As a practical matter, it was necessary to improve expression yields significantly. In order to accomplish this, yeast display expression maturation was performed. A correlation has been shown previously to exist between surface display levels and thermal stability of the protein, as well as soluble protein production (Shusta et al., 2000Go). The initial hMFE library was sorted for increased display levels of the scFv in the non-affinity matured context. Cells were labeled at a CEA concentration of 5 nM, chosen in order to isolate mutants that were improved in display levels only and not in affinity. After three sorts, 16 clones were analyzed for increased display levels. Six exhibited a 2.5-fold improvement in display, four of which were unique clones (data not shown). All four clones contained the VLW47L mutation. This mutation was added to the m10B and m3E clones, increasing display levels 2.5- and 1.5-fold, respectively (data not shown). Soluble expression levels were also increased (data not shown); however, further improvements were necessary.

Destabilization by disulfide removal, followed by restabilization

Previous work has shown that Fv cysteines can be replaced with a valine and alanine and then mutated to isolate clones that improve the stability of the scFv in the absence of the disulfide bond, with subsequent reintroduction of the disulfide bond to stabilize the scFv further (Proba et al., 1998Go). The two cysteines were pairwise removed from the heavy chain or the light chain of m3E and replaced with valine and alanine. Curiously, removal of the VH cysteines does not significantly alter expression of m3E. By contrast, removal of the VL cysteines destabilizes m3E significantly (Figure 4). It can be seen that introduction of W47L restabilizes the disulfide deleted VL, but not back to wild-type levels. With this in mind, two libraries were constructed based on m3E and m3E47L. The new constructs, m3E-cysout and m3E-cys47L, were used as the template for two new libraries. Random mutagenesis was performed on the entire scFv with an estimated error rate from 0.2 to 3%. Both libraries contained 105 clones. The libraries were labeled at a CEA concentration of 2.5 nM (m3E-cysout library) and 0.5 nM (m3E-cys47L). A higher CEA concentration was used with the m3E-cysout library because too little CEA binding was detected at the lower concentration. Stabilized clones were isolated by enriching for those cells with the highest display levels and CEA binding. Twenty clones were analyzed from the third sort of the m3E-cysout library and 19 were improved in display levels over m3E-cysout. Five were sequenced, four of which were unique (data not shown). Forty clones were analyzed for increased display levels and CEA binding from each of the second and third m3E-cys47L library sorts. All 40 were improved over m3E-cys47L. Ten of the most improved clones were sequenced, nine of which were unique (data not shown). Several mutants reverted the F36L affinity mutation back to phenylalanine. This result is consistent with the library being a comprehensive sample of the point mutations since the F36L reversion is a known (albeit trivial and undesirable) stabilizing mutation.



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Fig. 4. Destabilization of m3E by removal of the two VL cysteines by site-directed mutagenesis. All samples were labeled at 10 nM CEA. (A) m3E; (B) m3E-cysout; (C) m3E-cys47L.

 
Combination of mutations to construct a stable, well-expressed, high-affinity scFv

The stabilizing mutations VLN20T, VLS31P, VLW47L and VLM78V were introduced to hMFE, m9B, m10B and m3E. The mutation VLA13V was also added to m3E. These stabilized mutants are denoted by addition of an ‘s’ prefix to their names. Location of the mutations is shown in Figure 5. Introduction of these mutations increases the yeast surface display levels 4-, 5- and 6-fold for m9B, m10B and m3E, respectively. The soluble expression level was also increased for the series (Figure 6). The shMFE scFv is secreted in yeast shake flask culture at ~20 mg/l, sm9B at ~17 mg/l, sm10B at ~12.5 mg/l and sm3E at ~8 mg/l. This level of production for sm3E is a 100-fold improvement over the m3E scFv. The CEA dissociation kinetics of the stabilized scFvs were relatively unchanged. The scFv sm3E has a dissociation constant equal to 30 pM and a dissociation rate constant equal to 1.2 x 10–6 s–1 at 37°C and 3.0 x 10–7 s–1 at 25°C. Estimation of the association rate constants obtained from the ratio koff/KD indicates that kon for sm3E is several fold slower than for hMFE (data not shown).



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Fig. 5. Locations of mutations increasing affinity, expression and stability in sm3E. The residues in parentheses correspond to resurfacing humanization sites.

 


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Fig. 6. Secretion of mutant scFvs in shake-flask yeast culture. Supernatants were examined by western blot with antibodies against the C-terminal His6 tag present on all constructs. Samples: hMFE (1), m9B (2), m10B (3), m3E (4), shMFE (5), sm9B (6), sm10B (7) and sm3E (8). A description of mutations is given in the text.

 
Stability of the final mutant was assessed and compared with the stability of the wild-type hMFE scFv. After incubation at 37°C for 9 days, yeast displaying sm3E retained ~80% binding to CEA. Similar values were obtained for hMFE. The soluble version of the sm3E mutant exhibited comparable stability to the wild-type hMFE scFv at elevated temperatures after restabilization as well. Since the high-affinity mutant without the stabilizing mutations (m3E) is poorly expressed, a direct comparison between soluble m3E and sm3E cannot be made. However, yeast-displayed sm3E does exhibit similar thermal stability properties to hMFE and the soluble expression level is approximately four times higher than hMFE for the well-expressed, high-affinity mutant.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, the MFE-23 scFv antibody fragment was engineered to improve tumor targeting through the use of directed evolution and yeast surface display. To accomplish this goal, three properties were addressed: potential human immunogenicity, affinity and stability. A series of scFvs were engineered that span three orders of magnitude in off-rate and are extremely stable at 37°C. These designed molecules are amenable to testing in animal biodistribution studies to determine the importance of affinity in tumor targeting.

Since framework amino acids can often contribute to affinity gains (Low et al., 1996Go; Saviranta et al., 1998Go; Boder et al., 2000Go; Daugherty et al., 2000Go), the entire scFv was subjected to mutagenesis. The nucleotide analogues dPTP and 8-oxo-dGTP were used since higher error rates can be achieved than with conventional error-prone PCR and higher rates of mutagenesis more effectively search sequence space (Zaccolo and Gherardi, 1999Go; Daugherty et al., 2000Go). DNA shuffling was also performed in subsequent steps because random recombination of mutations is more efficient than manual selection and recombination of the changes. As it happens, the most improved clone (m3E) is simply the combination of VLF36L and VLS50L from the previous cycle of mutagenesis and screening. Both libraries constructed in this study were ~105 in size, which would be considered small by comparison with most phage display libraries. However, the dramatic affinity improvements obtained indicate that library size was not a limiting factor in this case. The mutations of interest are both in the light chain. The change that led to the greatest improvement in affinity, VLS50L, is in the CDR 2 loop of the light chain. From inspection of the crystal structure, the side chain of this residue protrudes into the cavity where contact with the antigen may occur. The other mutation that increases the affinity is VLF36L. It is not in a CDR loop and occurs at the interface of the light and heavy chain, directly below this cavity. Its contribution to CEA binding is not as readily apparent as that at position 50. The crystal structure of MFE-23, as well as a homology model of the CEA–MFE-23 interaction, has led to speculation as to the location and importance of certain contact residues (Boehm and Perkins, 2000Go). Most of the CEA contacts in the theoretical model of the complex involve residues located in the CDR loops of the heavy chain. There is a band of six acidic residues in MFE-23 (Asp-H31, Asp-H52, Glu-H53, Asp-H56, Glu-H58 and Glu-L1) that appears to interact with a band of six basic residues in CEA (Boehm et al., 2000Go). Previous mutational studies have also highlighted the importance of several residues in the H3 loop (Read et al., 1995Go). The VHY100bP mutation eradicated binding, a VHE53K change decreased binding and the VHT98A mutation led to a modest improvement in binding. While all six CDR loops were shown to interact with CEA in the homology model, it is possible that the loops of the heavy chain were already optimized for this interaction. While several mutations were isolated which were located in the CDR loops of the heavy chain, they seem to be neutral in effect.

The hMFE scFv was already fairly stable at 37°C prior to affinity maturation. Single-chain antibody fragments are often only produced at 20°C in the yeast display system owing to instability of the protein (Boder and Wittrup, 1997Go). However, higher levels of the hMFE scFv were detected on the surface when produced at 37°C than at 20°C. Unfortunately, increased affinity correlated with decreased expression for the isolated mutant series. This necessitated screening for higher display of the scFv, which has been shown to correlate with the soluble expression level and thermal stability of the protein (Shusta et al., 2000Go). The goal of this screen was to engineer the affinity mutants in such a manner as to return soluble expression capacity and thermal stability to at least the level of the hMFE scFv. A total of five stabilizing mutations were selected as significant from the three stability sorts. Four to five of these changes were added to each of the affinity mutants. In the cases where less than five of the changes were incorporated in the stabilized mutants, inclusion of the additional mutations did not confer greater stability. All of the stabilized mutants contained VLN20T, VLS31P, VLW47L and VLM78V. Position 20 was resurfaced in the humanization portion of the project to asparagine. The change to threonine was a reversion back to the original MFE-23 residue. It is not unusual in the case of humanization to change residues selectively back to their original amino acid to restore function. The key is to find a balance between the desired goal, humanization in this case, and any potential drawbacks such as a decrease in soluble production levels and/or stability. The change to leucine at position 47 was isolated in the first sort and proved to increase significantly soluble production levels and stability for all clones. This mutation was also isolated from the destabilized, cysteine removed library. Position 78 in the light chain is a buried residue in a pocket away from the CDR loops. The change to valine may have created better packing of the side chains within this pocket. In the course of humanization, residues 77, 79 and 80 were all changed to the corresponding consensus amino acids in human frameworks. It is also possible that these three changes dictated the change at position 78. The VLS31P mutation is located in CDR 1 of the light chain. While inclusion of this change slightly increased soluble expression levels, it was incorporated to offset small affinity losses as a result of the other changes. The VLA13V mutation was only included in the stabilized version of m3E. This residue is not solvent exposed, with its side chain facing into the same pocket as position 78. The change from alanine to the larger side chain of valine may also fill a gap within this pocket. It is interesting that for the highest affinity mutant, the stabilized version (sm3E) is only 6-fold better displayed, but its soluble expression level increased 100-fold from ~0.08 to 8 mg/l. This difference highlights a caveat to this approach. While the general progression holds that as surface display levels increase so do soluble expression levels, the magnitude of this improvement is not always proportional. Yeast cells displaying sm3E retained ~80% binding to CEA after incubation at 37°C for 9 days. Through the stabilization process, we were able to return the highest affinity mutant to similar thermal stability as the hMFE scFv. Addition of the five stabilizing mutations to the high-affinity mutant also increased soluble production levels 4-fold over the wild-type hMFE scFv.

The highest affinity mutant isolated in this study has the slowest reported dissociation rate constant engineered for an antibody against a protein antigen. Previous affinity maturation studies against protein antigens using phage display produced several scFvs or Fabs with affinities in the picomolar range (Yang et al., 1995Go; Schier et al., 1996Go; Pini et al., 1998Go). Yang and co-workers engineered a 13 pM Fab (koff = 1.2 x 10–6 s–1) against the human envelope glycoprotein gp120 of HIV-1. This was accomplished by CDR walking mutagenesis (Yang et al., 1995Go). Schier et al. (1996)Go produced a 15 pM scFv (koff = 8.0 x 10–6 s–1) against the tumor antigen erbB2 by mutagenesis of the VL and VH CDR3 loops. Pini et al. (1998)Go designed a 54 pM scFv (koff = 6.0 x 10–6 s–1) against the ED-B domain of fibronectin by mutagenesis of CDR residues. At 25°C, the sm3E scFv engineered in this study exhibits a koff = 3.0 x 10–7 s–1 for a dissociation half-time of 27 days. Of greater practical interest, however, is the dissociation rate under physiological conditions, at 37°C: koff = (1.85 ± 0.73) x 10–6 s–1 (averaged from ELISA and cell surface assays) and a dissociation half-time of several days.

Beyond the implications in the field of directed evolution, we have engineered an scFv that may eventually prove valuable in a therapeutic setting. These single-chain fragments span three orders of magnitude in off-rate against the tumor-associated CEA, the greatest with a half-life of ~7 days. Success of antibody targeting may depend on the metabolic turnover of targeted antigen (Graff and Wittrup, 2003Go). Several antigens routinely used as targets cover a broad range of values for internalization or shedding of the antigen. ErbB2, a protein targeted in several forms of cancer, is internalized with a half-life of ~17 min (Worthylake et al., 1999Go). CD20, a B-cell surface antigen used in the treatment of non-Hodgkin's lymphoma, is shed with a half-life of 1 day (Press et al., 1994Go). CEA, the target in this study, is shed with a half-life of ~7 days (Shi et al., 1983Go; Bidart et al., 1999Go; Stein et al., 1999Go). By affinity maturing an antibody with a half-life equal to the turnover half-life of the antigen, we have engineered an antibody with effectively irreversible binding to CEA. Because CEA is a stable target with a long half-life, differences in tumor retention for the series of scFvs is predicted to be dominated by the off-rate of the antibody and not the half-life of CEA. With this in mind, the molecules designed in this study can be used to determine the impact of affinity on efficacy in tumor targeting.


    Acknowledgments
 
This work was supported by a Koch cancer seed grant and by NSF BPEC.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received December 17, 2003; revised April 2, 2004; accepted April 8, 2004.

Edited by Dario Neri