Interleukin-2 mutants with enhanced {alpha}-receptor subunit binding affinity

Balaji M. Rao1, Andrew T. Girvin1, Thomas Ciardelli2, Douglas A. Lauffenburger1 and K.Dane Wittrup1,3

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Stimulation of T-cells by IL-2 has been exploited for treatment of metastatic renal carcinoma and melanoma. However, a narrow therapeutic window delimited by negligible stimulation of T-cells at low picomolar concentrations and undesirable stimulation of NK cells at nanomolar concentrations hampers IL-2-based therapies. We hypothesized that increasing the affinity of IL-2 for IL-2R{alpha} may create a class of IL-2 mutants with increased biological potency as compared with wild-type IL-2. Towards this end, we have screened libraries of mutated IL-2 displayed on the surface of yeast and isolated mutants with a 15–30-fold improved affinity for the IL-2R{alpha} subunit. These mutants do not exhibit appreciably altered bioactivity at 0.5–5 pM in steady-state bioassays, concentrations well below the IL-2R{alpha} equilibrium binding constant for both the mutant and wild-type IL-2. A mutant was serendipitously identified that exhibited somewhat improved potency, perhaps via altered endocytic trafficking mechanisms described previously.

Keywords: {alpha}-receptor subunit binding affinity/interleukin-2 mutants/T-cell stimulation


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Interleukin-2 (IL-2) (Theze et al., 1996Go) is a 133 amino acid cytokine that induces proliferation of antigen-activated T-cells and stimulation of NK cells. The proliferation of T-cells stimulated by IL-2 has been exploited for the treatment of metastatic renal carcinoma and melanoma (Fyfe et al., 1995Go; Atkins et al., 1999Go). However, a narrow therapeutic window has hampered IL-2 therapies: undesirable inflammatory responses are activated at IL-2 concentrations above 100 pM through stimulation of NK cells (Smith, 1993Go; Jacobson et al., 1996Go), while stimulation of T cells is not achieved below 1 pM. Given the very rapid systemic clearance of IL-2 [an initial clearance phase with a half-life of 12.9 min followed by a slower phase with a half-life of 85 min (Konrad et al., 1990Go)], it is difficult to maintain therapeutic concentrations of IL-2 (1–100 pM) for a sustained period.

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{alpha}), p75 (IL-2Rß) and p64 (IL-2R{gamma}), which span the cell membrane (Nelson and Willerford, 1998Go). NK cells in general express only the IL-2Rß and IL-2R{gamma} subunits (Voss et al., 1992Go), so enhanced affinity for IL-2R{alpha} 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., 1994Go). 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{gamma} (Shanafelt et al., 2000Go).

Display technologies such as phage display (Parmley and Smith, 1988Go) and yeast surface display (Boder and Wittrup, 1997Go), 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., 1991Go), interleukin-6 (Toniatti et al., 1996Go) and ciliary neutrotrophic growth factor (Saggio et al., 1995Go), using phage display. IL-2 has been functionally displayed on phage (Buchli et al., 1997Go), 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{alpha}. 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, 1995Go). Increasing the affinity of IL-2 for IL-2R{alpha} 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-2–IL-2R complex is internalized upon ligand binding and the different components undergo differential sorting (Hemar et al., 1995Go). IL-2R{alpha} is recycled to the cell surface, whereas IL-2 associated with the IL-2–IL-2Rß{gamma} complex is routed to the lysosome and degraded. Increasing the affinity of IL-2 for IL-2R{alpha} 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., 2000Go). Further, IL-2–IL-2R{alpha} on one cell can augment IL-2 signaling on another cell (Eicher and Waldmann, 1998Go). IL-15, which exhibits picomolar binding affinity for its private IL-2R{alpha} subunit, also performs such juxtacrine signaling (Dubois et al., 2002Go). Hence it is conceivable that increasing the affinity of IL-2 for IL-2R{alpha} 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 15–30-fold increase in IL-2R{alpha} binding affinity does not contribute to improved IL-2 potency.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Yeast surface display of IL-2

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 pCT–IL-2, by induction in medium containing galactose (Boder and Wittrup, 1997Go). 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 Aga2p–IL-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 Aga2p–IL-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, 2000Go), to detect the presence of IL-2 fusions on the yeast cell surface.

A soluble ectodomain of IL-2R{alpha} (Wu et al., 1999Go), expressed in insect cell culture, was purified and biotinylated. Yeast cells were labeled with biotinylated soluble IL-2R{alpha} as described (Boder and Wittrup, 2000Go), Labeling with soluble IL-2R{alpha} 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., 1996Go; Zaccolo and Gherardi, 1999Go). 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., 1999Go).

Detailed protocols for screening yeast polypeptide libraries have been described (Boder and Wittrup, 2000Go). Yeast cells from the IL-2 library were labeled with biotinylated soluble IL-2R{alpha} at a concentration of 0.2–0.8 nM and saturating concentration of mAb 12CA5 against the HA epitope tag, at 37°C, for 30 min–1 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 FLAG–BAP 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 3000–7000 IL-2R{alpha}ß{gamma} and 200 000–300 000 IL-2R{alpha} (Hori et al., 1987Go; Arima et al., 1992Go). 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 streptavidin–phycoerythrin 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{gamma} (Teshigawara et al., 1987Go). 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 streptavidin–phycoerythrin 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, 1999Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Functional expression of IL-2 on the surface of yeast

Although IL-2 has been displayed on bacteriophage previously (Buchli et al., 1997Go), 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, 1997Go). Expression of the Aga2p–IL-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 Aga2p–IL-2 fusion (Figure 1A). IL-2 displayed on the surface of yeast binds specifically to the soluble ectodomain of IL-2R{alpha} (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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Fig. 1. IL-2 is functionally displayed on the surface of yeast. (A) Labeling of IL-2 displaying yeast with saturating concentration of anti-c-myc antibody (9e10). (B) Labeling of IL-2 displaying yeast with 52 nM soluble IL-2R. (C) Labeling of D1.3 (an irrelevant negative control scFv) displaying yeast with saturating concentration of anti-c-myc antibody (9e10). (D) Labeling of D1.3 displaying yeast with 52 nM soluble IL-2R{alpha}.

 
Screening of IL-2 library for clones with improved binding to IL-2R{alpha}

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{alpha}. 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{alpha}, normalized to the number of IL-2 fusions on the yeast surface by labeling with mAb 12CA5 (Figure 2).



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Fig. 2. Ensemble of clones with better binding for IL-2R{alpha} compared with wild-type C125S. Labeling with saturating concentration of anti-HA antibody (12CA5) and 0.4 nM IL-2R{alpha} (2x106 cells, 100 µl volume) at 37°C. (A) Wild-type (C125S). (B) Population isolated after four rounds of sorting.

 
Twenty mutants were sequenced (Table I) and seven distinct sequences were obtained from the 20 clones sequenced. The most frequently occurring mutations (V69A and Q74P) cluster in a region predicted to be at the IL-2/IL-2R{alpha} interface, by a homology model of IL-2 binding to its receptor subunits (Figure 3). Further, the mutant M6 has a mutation I128T, which is close to the predicted IL-2/IL-2ß and IL-2/IL-2R{gamma} interface (Bamborough et al., 1994Go; Berman et al., 2000Go)


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Table I. Mutations in IL-2 clones with greater affinity for IL-2Ra compared with C125S: seven distinct sequences were obtained out of 20 clones sequenced
 


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Fig. 3. Locations of mutations on a model of IL-2–receptor complex. IL-2 is shown in red, IL-2R{alpha} subunit in blue, IL-2Rß subunit in white, IL-2R{gamma} subunit in gray. The residues where mutations were encountered, in improved IL-2 mutants, are marked Q74 (orange), V69 (brown) and I128 (green).

 
Binding of IL-2 mutants to KIT-225 cells expressing a large excess of IL-2R{alpha}

The IL-2 mutants isolated by yeast surface display were tested in soluble form for tighter binding to IL-2R{alpha} 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{alpha}. C1 represents the mutation predicted to be close to the IL-2/IL-2ß and IL-2/IL-2R{gamma} 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{alpha} over IL-2Rß and IL-2R{gamma}, the binding data obtained correspond to IL-2R{alpha} binding. Thus, M6 and M1 have a higher binding affinity for IL-2R{alpha} 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 0.01–400 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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Fig. 4. Binding of solubly expressed IL-2 mutants to KIT-225 cells expressing a large excess of IL-2R{alpha}, at 37°C. Data shown are representative data from at least two experiments, for each mutant or wild-type IL-2, The binding curves look similar at 4°C (data not shown).

 


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Fig. 5. Binding of solubly expressed IL-2 mutants to YT-2C2 cells expressing IL-2Rß and IL-2R{gamma}. Different symbols denote different data sets.

 

where Fobs = observed fluorescence, L0 = initial ligand concentration and C = proportionality constant.

The Kd for M6 and M1 can be estimated to be 1–2 nM (1.1 ± 0.08 nM for M6 and 1.7 ± 0.4 nM for M1). This represents roughly a 15–30-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., 2002Go). 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{alpha}-bound IL-2 (Liparoto et al., 2002Go). 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{gamma} 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{gamma}

The binding of M1, M6 and C1 to YT-2C2 cells expressing IL-2Rß and IL-2R{gamma} 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., 2002Go). 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 M1’s mutation sites, predicted to be on the opposite side from IL-2’s contacts with IL-2Rß.


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Table II. Binding affinities of IL-2 mutants for IL-2Rß on YT-2C2 cells
 
Proliferation of IL-2 dependent KIT-225 cells in response to IL-2 mutants

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{alpha} on biological potency. At low concentrations (0.5 pM) and long times, C1 and M6 caused ~50–60% 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{alpha} as M6, did not. The observed increase in affinity of IL-2 for IL-2R{alpha} 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{alpha} alone is not responsible for the increased potency of M6.



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Fig. 6. Proliferation of IL-2-dependent KIT-225 cells in response to wild-type (C125S) IL-2 and IL-2 mutants. (a) Number of viable KIT-225 cells in culture with time, at different concentrations of wild-type (C125S) IL-2. Error bars indicate the standard deviation for three separate cultures. (b) Ratio of number of viable cells in culture with the mutants to number of cells in culture with wild-type (C125S) IL-2. The concentrations are indicated on the plots. Error bars indicate the standard deviation for three separate cultures.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We hypothesized that increasing the affinity of IL-2 for its {alpha}-receptor subunit would create an IL-2 mutant with improved biological potency, for reasons described earlier (see Introduction). To this end, IL-2 mutants with improved affinity for IL-2R{alpha} were selected from a yeast-displayed randomly mutated library. The mutants obtained were tested for proliferation of a T-cell line (KIT-225). The concentrations at which the KIT-225 proliferation assays were carried out lie in the picomolar range (0.5–5 pM); however, the equilibrium dissociation constants for the IL-2 mutants selected lie in the nanomolar range. One of the predicted mechanisms for IL-2 mutants with increased IL-2R{alpha} binding affinity to have increased biological potency is an increased concentration of IL-2 localized at the cell surface, by binding to IL-2R{alpha}. Under the steady-state conditions of the bioactivity assay, the increase in occupancy of IL-2R{alpha} would not be significantly different for the mutants compared with wild-type IL-2. The T-cell response to the IL-2 mutants is therefore not detectably different from wild-type IL-2. A greater increase in occupancy of IL-R{alpha} would conceivably lead to an increase in potency, through increase in the local concentration of IL-2, at the cell surface. We hypothesize that a greater difference in T-cell response, in these assays, may be observed for mutants with IL-2R{alpha} affinity in the picomolar range. However, on the basis of our results, we can conclude that a 15–30-fold increase in affinity for IL-2R{alpha} does not result in a corresponding increase in biological potency in proliferation assays at picomolar concentrations, as described. The quantitative relationship between such steady-state, low-concentration assays and the pharmacological situation in vivo is not clear, however, given the rapid renal clearance of parenterally administered IL-2.

The mutations responsible for the higher affinity for IL-2R{alpha} (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{gamma} owing to the extremely weak affinity of interaction between IL-2 and IL-2R{gamma} (Liparoto et al., 2002Go).

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{alpha} and IL-2Rß, compared with wild-type, whereas M1, with increased affinity for IL-2R{alpha}, has slightly decreased biological potency. Also, as explained earlier, there would not be a significant increase in IL-2R{alpha} 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{alpha}. 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., 2000Go). 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{alpha}, sufficient to drive greater receptor occupancy in the 0.1–10 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.


    Acknowledgement
 
Chiron Corporation provided financial support for this work.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received June 13, 2003; revised July 30, 2003; accepted September 26, 2003





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