1Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, MIT 66-552, Cambridge, MA 02139 and 2Dartmouth Medical School, Hanover, NH 03755, USA
3 To whom correspondence should be addressed. e-mail: wittrup{at}mit.edu
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Abstract |
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Keywords: -receptor subunit binding affinity/interleukin-2 mutants/T-cell stimulation
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Introduction |
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The biological activity of IL-2 in activated T cells is mediated through a multi-subunit IL-2 receptor complex (IL-2R) consisting of three cell-surface subunits: p55 (IL-2R), p75 (IL-2Rß) and p64 (IL-2R
), which span the cell membrane (Nelson and Willerford, 1998
). NK cells in general express only the IL-2Rß and IL-2R
subunits (Voss et al., 1992
), so enhanced affinity for IL-2R
might be expected to increase the specificity of IL-2 for activated T cells relative to NK cells. Manipulation of the binding affinities to these receptor subunits might be used to alter the biological response to IL-2 and potentially create an improved therapeutic. Screening of over 2600 IL-2 variants created by combinatorial cassette mutagenesis has led to the isolation of an IL-2 variant (L18M, L19S) with increased potency (Berndt et al., 1994
). Site-directed mutagenesis was also utilized to isolate IL-2 variants causing reduced stimulation of NK cells via reduced binding to IL-2Rß and IL-2R
(Shanafelt et al., 2000
).
Display technologies such as phage display (Parmley and Smith, 1988) and yeast surface display (Boder and Wittrup, 1997
), are powerful tools that can be used for screening large libraries of protein variants for altered binding properties. Variants with enhanced receptor binding affinities have been isolated for human growth hormone (Lowman et al., 1991
), interleukin-6 (Toniatti et al., 1996
) and ciliary neutrotrophic growth factor (Saggio et al., 1995
), using phage display. IL-2 has been functionally displayed on phage (Buchli et al., 1997
), but improved mutants have not previously been engineered by phage display.
Here we present IL-2 engineering by directed evolution with yeast surface display, to generate mutants with increased affinity for IL-2R. This is the first reported affinity maturation of IL-2 for a receptor subunit. T-cell response to IL-2 depends on the number of IL-2R occupied by IL-2 via (1) the concentration of IL-2, (2) the number of IL-2R molecules on the cell surface and (3) the number of IL-2R occupied by IL-2, i.e. the affinity of binding interaction between IL-2 and IL-2R (Smith, 1995
). Increasing the affinity of IL-2 for IL-2R
at the cell surface will increase receptor occupancy within a limited range of IL-2 concentration, and also raise the number of IL-2 molecules localized at the cell surface. The IL-2IL-2R complex is internalized upon ligand binding and the different components undergo differential sorting (Hemar et al., 1995
). IL-2R
is recycled to the cell surface, whereas IL-2 associated with the IL-2IL-2Rß
complex is routed to the lysosome and degraded. Increasing the affinity of IL-2 for IL-2R
may shift trafficking of internalized IL-2 towards recycling, causing decreased degradation of IL-2, and hence favorably affect T-cell response (Fallon et al., 2000
). Further, IL-2IL-2R
on one cell can augment IL-2 signaling on another cell (Eicher and Waldmann, 1998
). IL-15, which exhibits picomolar binding affinity for its private IL-2R
subunit, also performs such juxtacrine signaling (Dubois et al., 2002
). Hence it is conceivable that increasing the affinity of IL-2 for IL-2R
may create a class of IL-2 mutants with increased biological potency as compared with wild-type IL-2. However, in the steady-state bioassays reported here, a 1530-fold increase in IL-2R
binding affinity does not contribute to improved IL-2 potency.
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Materials and methods |
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The IL-2 gene was subcloned into the pCT302 backbone at NheI and BamHI restriction sites. A serine was introduced at position 125 by site-directed mutagenesis to obtain what will be termed wild-type C125S IL-2 (equivalent to ProleukinTM). This vector is termed pCTIL-2.
IL-2 was expressed as an Aga2p protein fusion in Saccharomyces cerevisiae EBY100 transformed with vector pCTIL-2, by induction in medium containing galactose (Boder and Wittrup, 1997). A hemagglutinin (HA) epitope tag is expressed N-terminal to IL-2, whereas a c-myc epitope tag is attached to the C-terminus of Aga2pIL-2 fusion. The HA epitope tag can be detected by immunofluorescent staining using a mouse monoclonal antibody (mAb) 12CA5 (Roche Molecular Biochemicals) along with a goat anti-mouse antibody conjugated with fluorescein isothiocyanate (FITC). The c-myc epitope tag can be detected using a mouse mAb 9e10 (Covance) and a goat anti-mouse antibody conjugated with R-phycoerythrin (PE). Detection of the c-myc epitope tag at the C-terminus of the Aga2pIL-2 fusion is indicative of display of the full-length IL-2 fusion on the yeast cell surface. Yeast cells were labeled with mAb 9e10 as described (Boder and Wittrup, 2000
), to detect the presence of IL-2 fusions on the yeast cell surface.
A soluble ectodomain of IL-2R (Wu et al., 1999
), expressed in insect cell culture, was purified and biotinylated. Yeast cells were labeled with biotinylated soluble IL-2R
as described (Boder and Wittrup, 2000
), Labeling with soluble IL-2R
is indicative of the IL-2 fusion on the yeast surface being functional. Yeast displaying an irrelevant single-chain antibody (scFv), D1.3, was used as a negative control.
Construction and screening of IL-2 library
The wild-type IL-2 coding sequence was subjected to random mutagenesis by error-prone polymerase chain reaction (PCR). The error rate was controlled by varying cycles of PCR amplification in the presence of nucleotide analogs 8-oxodGTP and dPTP (Zaccolo et al., 1996; Zaccolo and Gherardi, 1999
). The PCR product obtained was further amplified by PCR without the nucleotide analogs. The final PCR product was transformed into yeast along with linearized pCT-IL-2. Homologous recombination in vivo in yeast between the 5' and 3' flanking 50 base pairs of the PCR product with the gapped plasmid resulted in a library of approximately 5x106 IL-2 variants (Raymond et al., 1999
).
Detailed protocols for screening yeast polypeptide libraries have been described (Boder and Wittrup, 2000). Yeast cells from the IL-2 library were labeled with biotinylated soluble IL-2R
at a concentration of 0.20.8 nM and saturating concentration of mAb 12CA5 against the HA epitope tag, at 37°C, for 30 min1 h. Labeling with an antibody against one of the epitope tags is necessary to normalize for the number of IL-2 fusions on the yeast surface. The cells were washed, labeled with streptavidin conjugated with R-phycoerythrin (PE) (Pharmingen) and a goat anti-mouse antibody conjugated with FITC. The cells were then sorted on a Cytomation Moflo (first two sorts) or a Beckton Dickinson FACStar flow cytometer to isolate clones with improved binding to soluble IL-2R, relative to wild-type IL-2. Four rounds of sorting by flow cytometry were carried out, with regrowth and reinduction of surface expression between each sort. After the fourth sort, DNA from 20 individual clones was extracted using a Zymoprep kit (Zymo Research). The DNA was amplified by transforming into XL-1 Blue cells (Stratagene). Sequences of the IL-2 mutants were determined by DNA sequencing.
IL-2 mutants isolated by flow cytometry were subcloned into secretion vectors and secreted in yeast shake-flask cultures, with an N-terminal FLAG epitope tag and a C-terminal c-myc epitope tag. The mutants were purified by FLAG immunoaffinity chromatography (Sigma). Quantification of IL-2 concentration was performed using quantitative western blotting, with a FLAGBAP protein standard (Sigma) and mutant M6 as standards. The stock protein concentrations obtained were 11.7 ± 1.2 µM for wild-type C125S (six measurements) IL-2, 20.7 ± 1.4 µM for M6 (four measurements), 25.3 ± 6.1 µM for M1 (four measurements) and 3.3 ± 0.6 µM for C1 (eight measurements).
KIT-225 cell proliferation assay
KIT-225 is a human IL-2 dependent T-cell line, expressing roughly 30007000 IL-2Rß
and 200 000300 000 IL-2R
(Hori et al., 1987
; Arima et al., 1992
). KIT-225 cells were cultured in RPMI 1640 supplemented with 20 pM IL-2, 10% FBS, 200 mM L-glutamine, 50 units/ml penicillin and 50 µg/ml gentamycin.
KIT-225 cells were cultured in medium without IL-2 for 6 days. The cell culture medium was changed after 3 days. On the sixth day, the cells were transferred into medium containing wild-type IL-2 or IL-2 mutants at different concentrations at 105 cells/ml. Cell culture aliquots were taken at different times and the viable cell density was determined using the Cell-titer GloTM (Promega) assay.
Binding of IL-2 mutants to KIT-225 and YT2C2 cells
KIT-225 cells were incubated (106 cells in 100 µl) with soluble IL-2 or mutants at 37°C for 30 min, at pH 7.4. The cells were washed with ice-cold PBS, pH 7.4, containing 0.1% BSA and labeled with a biotinylated antibody against the FLAG epitope followed by streptavidinphycoerythrin on ice. The cells were washed again and the mean single-cell fluorescence was determined using an EPICS-XL flow cytometer.
YT-2C2 is a human NK cell line expressing 20 000 IL-2Rß
(Teshigawara et al., 1987
). YT-2C2 cells were cultured in the same medium as KIT-225 cells, without IL-2. YT-2C2 cells were incubated (106 cells in 100 µl) with the IL-2 mutants on ice for 30 min, at pH 7.4. The cells were washed with ice-cold PBS (pH 7.4, 0.1% BSA) and labeled with a biotinylated antibody against the FLAG epitope followed by streptavidinphycoerythrin on ice. The cells were washed again and the mean single-cell fluorescence was determined using an EPICS-XL flow cytometer. The equilibrium dissociation constants were determined using a global fit. The 66% confidence intervals were calculated as described. (Lakowicz, 1999
).
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Results |
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Although IL-2 has been displayed on bacteriophage previously (Buchli et al., 1997), directed evolution using phage display, to obtain IL-2 mutants with improved binding for the IL-2R subunits, has not been reported. IL-2 was expressed on the surface of yeast cells, on the assumption that expression in a eukaryotic system would produce a higher fraction of correctly folded protein. IL-2 was expressed as a fusion to the Aga2p agglutinin subunit, on the surface of yeast (Boder and Wittrup, 1997
). Expression of the Aga2pIL-2 fusion on the surface of yeast was measured by immunofluorescent labeling of the c-myc epitope tag attached to the C-terminus of the Aga2pIL-2 fusion (Figure 1A). IL-2 displayed on the surface of yeast binds specifically to the soluble ectodomain of IL-2R
(Figure 1B), whereas negative control yeast displaying an irrelevant scFv, D1.3, does not (Figure 1D). The presence of the c-myc tag indicates that the full-length IL-2 fusion is displayed on the yeast cell surface. Figure 1C shows immunofluorescent labeling of the c-myc tag on negative control yeast, displaying D1.3, indicating the presence of D1.3 fusions on the yeast cell surface.
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A yeast-displayed library of IL-2 mutants with a diversity of 5x106 clones was constructed by error-prone PCR. This library was screened through four rounds of sorting by flow cytometry, with regrowth and reinduction of surface expression between each sort, to isolate clones with improved binding to soluble IL-2R. The ensemble of clones after four rounds of sorting shows improved binding relative to wild-type IL-2 at 0.4 nM soluble IL-2R
, normalized to the number of IL-2 fusions on the yeast surface by labeling with mAb 12CA5 (Figure 2).
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The IL-2 mutants isolated by yeast surface display were tested in soluble form for tighter binding to IL-2R in its physiologically relevant context on the surface of KIT-225 cells. Three different mutants were tested: M6 (V69A, Q74P, I128T), M1 (V69A, Q74P) and C1 (I128T). We chose to test mutant M6 (and mutants derived from M6) based on the observation that M6, and not the other six mutants, exhibited slightly improved biological potency in preliminary KIT-225 cell proliferation assays (data not shown), described subsequently. M1 represents the two most frequently occurring mutations. We hypothesized, on the basis of the homology model of IL-2 binding to its receptor subunits, that the subset of mutations in M6 represented by M1 would be sufficient for increased binding affinity for IL-2R
. C1 represents the mutation predicted to be close to the IL-2/IL-2ß and IL-2/IL-2R
interface.
Figure 4 shows representative data for binding of M6, M1, C1 and wild-type (C125S) IL-2 to KIT-225 cells, at 37°C. M6 and M1 have similar binding to KIT-225 cells, whereas C1 exhibits similar binding to wild-type (C125S) IL-2. Since the KIT-225 cells express a large excess of IL-2R over IL-2Rß and IL-2R
, the binding data obtained correspond to IL-2R
binding. Thus, M6 and M1 have a higher binding affinity for IL-2R
on the surface of KIT-225 cells, as compared with C1 and wild-type (C125S) IL-2. The fluorescence data, in Figure 5, for concentrations of 0.01400 nM, were used to obtain a gross estimate of the Kd for M6 and M1. An equation describing a simple one-step binding equilibrium was used to fit the data.
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where Fobs = observed fluorescence, L0 = initial ligand concentration and C = proportionality constant.
The Kd for M6 and M1 can be estimated to be 12 nM (1.1 ± 0.08 nM for M6 and 1.7 ± 0.4 nM for M1). This represents roughly a 1530-fold minimum improvement in binding affinity, relative to a wild-type Kd value of 28 nM for C125A IL-2, a mutant with alanine at position 125 (Liparoto et al., 2002). The errors represent variations due to the errors in estimating concentrations using quantitative western blotting. This calculation underestimates the binding affinity compared with the actual value (overestimates the Kd) owing to the following systematic errors. (1) The cell density used in the binding assay represents severe ligand (IL-2)-depleting conditions. For Equation 1 to hold true, the initial ligand concentration must be approximately equal to the free ligand concentration in solution at equilibrium. This assumption breaks down at concentrations less than
10 nM for the experimental setup used and the free ligand concentration is less than the initial ligand concentration. This leads to an overestimate of Kd (i.e. an underestimate of binding affinity). (2) Internalization of ligand-bound receptors occurs at 37°C. The internalization rate of ligand-bound receptors can be assumed to be proportional to the fraction of ligand-bound receptors, leading to an overestimate of Kd (underestimate of binding affinity).
The equilibrium dissociation constant (Kd) for C1 and C125S cannot be estimated from these data, owing to the rapid dissociation of IL-2R-bound IL-2 (Liparoto et al., 2002
). The receptor-bound IL-2 dissociates during the several wash steps involved in the experiment. This leads to a very low fluorescence signal, even at high concentrations for C1 and C125S. M6 and M1 were also assayed for binding at these high concentrations for consistency. The increase in fluorescence signal beyond 400 nM concentrations of M6 and M1 may be due to binding to IL-2Rß and IL-2R
on KIT225 cells and non-specific binding at micromolar concentrations of M6 and M1. In summary, the data in Figure 4 provide only a crude estimate of Kd, but definitively demonstrate that M1 and M6 exhibit substantial, qualitative improvements in binding affinity on the T-cell surface, relative to C125S and C1.
Binding of IL-2 mutants to YT-2C2 cells expressing IL-2Rß and IL-2R
The binding of M1, M6 and C1 to YT-2C2 cells expressing IL-2Rß and IL-2R was determined (Figure 5). A global fit was used to estimate the equilibrium dissociation constants (Kd). These values are given in Table II. The Kd values are consistent with reported affinities for the binding of IL-2 to IL-2Rß (Liparoto et al., 2002
). M1 was found to have a significantly lower binding affinity for IL-2Rß than wild-type, M6 and C1. This is interesting in the light of M1s mutation sites, predicted to be on the opposite side from IL-2s contacts with IL-2Rß.
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The proliferation of a T-cell line (KIT-225) in response to the IL-2 mutants was studied to evaluate the effect of increase in affinity of IL-2 for IL-2R on biological potency. At low concentrations (0.5 pM) and long times, C1 and M6 caused
5060% greater proliferation of IL-2 dependent KIT-225 cells in cell culture, compared with wild-type (C125S) IL-2 and M1. The proliferation of KIT-225 cells in culture with the different mutants, at different initial concentrations, is shown in Figure 6. It was surprising that both M6 and C1 had slightly improved biological potency whereas M1, with comparable affinity to IL-2R
as M6, did not. The observed increase in affinity of IL-2 for IL-2R
did not have an appreciable effect on biological potency for mutant M1 in this steady-state assay, suggesting that such an increase in affinity for IL-2R
alone is not responsible for the increased potency of M6.
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Discussion |
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The mutations responsible for the higher affinity for IL-2R (V69A, Q74P) cause a decrease in affinity for IL-2Rß. One of the reasons for the decreased biological activity of M1 relative to M6 may be this decrease in affinity for IL-2Rß. We could not analyze the effect of the selected mutations on the binding affinity for IL-2R
owing to the extremely weak affinity of interaction between IL-2 and IL-2R
(Liparoto et al., 2002
).
M6 and C1 exhibit slightly improved biological potency relative to wild-type in the T-cell proliferation assays described. C1 has no appreciable change in affinity for IL-2R and IL-2Rß, compared with wild-type, whereas M1, with increased affinity for IL-2R
, has slightly decreased biological potency. Also, as explained earlier, there would not be a significant increase in IL-2R
receptor occupancy under the particular conditions of the T-cell proliferation assays. These observations suggest that the increased potency of M6 in the T-cell proliferation assays is through a mechanism unrelated to the increase in affinity for IL-2R
. Previous studies have investigated the increased biological potency of 2D1, a mutant of IL-2. 2D1 internalized by receptor-mediated endocytosis is recycled to a greater extent than wild-type IL-2, leading to decreased depletion of 2D1 in cell culture and hence improved biological potency (Fallon et al., 2000
). Increased recycling of internalized M6 and C1 could be a potential mechanism for increased biological potency of M6 and C1, relative to wild-type IL-2.
Our results establish the proof of concept of a strategy to isolate IL-2 mutants with tailored binding characteristics and characterize T-cell response to these mutants. The YT-2C2 cell-binding assay provides a convenient preliminary test to check and ensure that the mutants selected do not have their affinities for IL-2Rß greatly weakened. The IL-2 mutants did not show increased potency in T-cell proliferation assays at low picomolar concentrations. Conversely, none of the seven isolated mutants showed loss of biological potency compared with wild-type IL-2 in preliminary assays (data not shown). This work lays the foundation for the generation and characterization of IL-2 mutants with further improved affinities for IL-2R, sufficient to drive greater receptor occupancy in the 0.110 pM concentration range. In addition, bioassays designed to mimic better the transient nature of IL-2 exposure in vivo may highlight the altered properties of these mutants.
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Acknowledgement |
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Received June 13, 2003; revised July 30, 2003; accepted September 26, 2003