DAB389 interleukin-2 receptor binding domain mutations. Cytotoxic probes for studies of ligand-receptor interactions.

Site-directed mutagenesis was used to generate point mutations in the diphtheria toxin-related fusion protein, DAB389 interleukin-2 (IL-2). Thr-439, in the IL-2 receptor binding domain of the fusion toxin, was changed to a Pro residue. The resultant fusion toxin, DAB389 IL-2(T439P), was 300-fold less cytotoxic than wild type DAB389 IL-2, partially as the result of a 100-fold decrease in binding affinity for the high affinity form of the IL-2 receptor. However, DAB389 IL-2(T439P) stimulated DNA synthesis to a greater extent than expected. Studies of intoxication kinetics indicated that the increased stimulation might result from an increased contact time between the mutated IL-2 receptor binding domain and the receptor, perhaps due to a decreased internalization rate. Another mutant, DAB389 IL-2(Q514D), in which a Gln residue at position 514 was changed to an Asp, was 2000-fold less cytotoxic than wild type DAB389 IL-2. This mutant had a 50-fold decrease in binding affinity, did not stimulate DNA synthesis and also had a reduced rate of intoxication. Gln-514 appears to play a role in receptor binding and activation, whereas Thr-439 appears to be involved with receptor binding and signaling internalization of the fusion toxin-receptor complex.

Site-directed mutagenesis was used to generate point mutations in the diphtheria toxin-related fusion protein, DAB 389 interleukin-2 (IL-2). Thr-439, in the IL-2 receptor binding domain of the fusion toxin, was changed to a Pro residue. The resultant fusion toxin, DAB 389 IL-2(T439P), was 300-fold less cytotoxic than wild type DAB 389 IL-2, partially as the result of a 100-fold decrease in binding affinity for the high affinity form of the IL-2 receptor. However, DAB 389 IL-2(T439P) stimulated DNA synthesis to a greater extent than expected. Studies of intoxication kinetics indicated that the increased stimulation might result from an increased contact time between the mutated IL-2 receptor binding domain and the receptor, perhaps due to a decreased internalization rate. Another mutant, DAB 389 IL-2(Q514D), in which a Gln residue at position 514 was changed to an Asp, was 2000-fold less cytotoxic than wild type DAB 389 IL-2. This mutant had a 50-fold decrease in binding affinity, did not stimulate DNA synthesis and also had a reduced rate of intoxication. Gln-514 appears to play a role in receptor binding and activation, whereas Thr-439 appears to be involved with receptor binding and signaling internalization of the fusion toxin-receptor complex.
Interleukin-2 (IL-2) 1 is a 133-amino acid lymphokine protein secreted by activated T-cells. Binding of IL-2 to its high affinity receptor stimulates processes that result in gene activation, DNA synthesis, internalization of the IL-2⅐IL-2 receptor complex, and proliferation of IL-2-dependent T cells (1)(2)(3)(4)(5)(6). The high affinity form of the IL-2 receptor is composed of at least three subunits, ␣, ␤, and ␥. The ␣ subunit forms a heteromeric complex with the ␤ subunit that functions to bind IL-2 to the surface of activated T cells (7). The ␤ and ␥ subunits associate in a ligand-dependent fashion and appear to be involved with intracellular signaling (8). Mutational studies have been performed on IL-2 in an attempt to define the structure/function relationships between IL-2 and the receptor subunits (9 -13). Buchli and Ciardelli (14) identified a Gln residue at position 126 of IL-2 that was involved with binding to the ␤/␥ portion of the high affinity receptor. IL-2 in which Gln-126 was mutated to an Asp residue resulted in an analog with greatly reduced biological activity. Another analog was created in which a Thr residue at position 51 was changed to a Pro residue (15). This analog, despite a decreased binding affinity, increased DNA synthesis of stimulated human peripheral blood lymphocytes to a much greater extent than expected. The authors postulated that the Thr-51 3 Pro mutation slowed the internalization rate of the ligand-receptor complex, thereby allowing a greater time interval for signaling activation.
The interleukin-2 diphtheria toxin-related fusion protein, DAB 389 IL-2, is composed of amino acid residues 2-133 of IL-2, genetically fused to the first 386 residues of diphtheria toxin (DT) (16,17). The fusion toxin is targeted to cells that express the high affinity form of the IL-2 receptor and is internalized by receptor mediated endocytosis. The fusion toxin is processed and the catalytic domain of DT is translocated across the endocytic membrane, into the cell cytosol, where it ADP-ribosylates elongation factor 2, leading to an irreversible inhibition of protein synthesis and subsequent cell death (18 -20).
In the present study, we introduced the Q126D and T51P mutations described above, as well as an E106K mutation, into the IL-2 receptor binding domain of DAB 389 IL-2. We studied the effects of these mutations on cytotoxicity, binding affinity, and kinetics of cytotoxicity. We also created analogous mutations, in which the catalytic domain of DT was mutated to a nontoxic form, so we could study the effects of the IL-2 receptor binding domain on stimulation of DNA synthesis. Our results indicate that the Gln residue is involved with binding affinity and activation of DNA synthesis, and that activation may affect cytotoxicity. The Thr residue appears to affect receptor binding and signaling internalization of the fusion toxin-receptor complex. The Glu residue that was mutated does not appear to play a critical role in the IL-2 binding domain of the fusion toxin.

EXPERIMENTAL PROCEDURES
Plasmid, Bacterial Strains, and Fusion Toxin Products-A schematic representation of the gene encoding DAB 389 IL-2, the restriction digest sites used, and the single amino acid residue changes are shown in Fig.  1. The plasmid encoding DAB 389 IL-2(T439P) was created by a 306nucleotide MluI to HindIII exchange from the plasmid encoding the T51P mutation in IL-2 (15). This exchange encompassed the region of IL-2 containing the Thr-51 3 Pro mutation. The plasmids encoding DAB 389 IL-2(Q514D) and DAB 389 IL-2(E494K) were created by PCR mutagenesis of the wild type, DAB 389 IL-2 gene, and cassette exchange encompassing the mutated site (21). In the case of the plasmid encoding DAB 389 IL-2(Q514D), a 229-nucleotide XbaI to SalI exchange was performed, and for DAB 389 IL-2(E494K), a 241-nucleotide XbaI to HindIII cassette exchange was performed. The E149S mutation in the DT catalytic domain was introduced into each of the above mutants by an NcoI to SphI exchange from the gene encoding DA(E149S)B 389 IL-2. In all cases the constructs were sequenced by the dideoxy chain termina-* This work was supported in part by Public Health Service Grant CA-60934 from the NCI, National Institutes of Health (to J. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ Supported by National Institutes of Health Grant R01 GM 52858-01, American Cancer Society Grant FRA 385, and the Norris Cotton Cancer Center. 1 The abbreviations used are: IL-2, interleukin-2; DT, diphtheria toxin; PCR, polymerase chain reaction. tion method (22) using a Sequenase reagent kit (U. S. Biochemical Corp.) The mutant fusion toxins, and the corresponding mutations in IL-2, are listed in Table I.
Escherichia coli JM101 was the host strain for plasmid propagation, and the HMS174 or HMS174 DE3 strain (Novagen, Madison, WI) was used as host for the expression of DAB 389 IL-2 and all the mutants.
Oligonucleotide Synthesis-Oligonucleotides were synthesized on an Applied Biosystems model 391 PCR Mate DNA synthesizer. The oligonucleotides were removed from the columns and deprotected as recommended by Applied Biosystems. The oligonucleotides were vacuumdried, resuspended in TE buffer, and the concentration determined using absorbance A 260 readings (23).
Polymerase Chain Reaction (PCR)-PCR was performed using a PCR reagent kit (Perkin Elmer Corp.). The conditions for PCR were 1 min at 95°C for strand separation, 1 min at 37°C for primer hybridization, and 1 min at 72°C for the polymerase activity. The cycle was repeated 25 times.
The plasmids encoding DAB 389 IL-2, DA(E149S)B 389 IL-2, DAB 389 IL-2(E494K), and DA(E149S)B 389 IL-2(E494K) were transformed into HMS174 for expression. The bacteria were propagated to an A 600 ϭ 0.8 in LB/ampicillin/maltose, and protein expression was induced by the addition of the coliphage derivative, CE6. The expressed proteins formed inclusion bodies under these conditions, and these inclusion bodies were isolated, denatured, and refolded as described previously (24). After refolding overnight at 4°C, the proteins were concentrated using a Filtron concentrator and an M r 10,000 cut-off filter (Filtron, Northborough, MA). The proteins were then purified further by ion exchange chromatography on DEAE-Sepharose (Pharmacia Biotech Inc.). The fusion toxins were applied to the column and washed extensively with 10 mM phosphate buffer, pH 7.2. The proteins were eluted using a linear 0 -0.8 M KCl gradient.
All protein concentrations were determined using Pierce protein assay reagent. The proteins were analyzed for purity by electrophoresis on a 12% SDS-polyacrylamide gel, stained with Coomassie Blue (Fig. 2).
Cytotoxicity Assays-HUT 102/6TG cells were maintained in complete RPMI 1640 medium (Life Technologies, Inc.) (supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT): 2 mM glutamine, 50 IU/ml penicillin, and 50 g/ml streptomycin). For the cytotoxicity assays, 5 ϫ 10 4 cells in 100 l of complete medium were seeded into each well of a 96-well, V-bottomed microtiter dish (Linbro). The fusion toxins were added so the final volume was 200 l/well, and the fusion toxin concentrations ranged from 10 Ϫ7 M down to 10 Ϫ12 M. The plates were incubated for 18 h at 37°C in a 5% CO 2 atmosphere and then centrifuged at 170 ϫ g for 5 min. The medium was aspirated and replaced with 200 l of leucine-free minimal essential medium (Life Technologies, Inc.) containing 1.0 Ci/ml [ 14 C]leucine (280 mCi/mmol, DuPont NEN), 2 mM L-glutamine, 50 g/ml streptomycin, and 50 IU/ml penicillin. The cells were pulsed for 90 min and then centrifuged at 170 ϫ g for 5 min. The medium was removed, and the cells were lysed by addition of 60 l/well 0.4 M KOH and incubated at room temperature for 10 min. The proteins were precipitated out by addition of 140 l/well 10% trichloroacetic acid and incubating for another 10 min at room temperature. The insoluble proteins were collected on glass fiber filters using a PhD cell harvester (Cambridge Technology, Inc., Watertown, MA). Radioactivity was determined according to standard methods. All assays were performed in quadruplicate, and medium alone served as a control.
Binding Assays-The displacement of 125 I-labeled recombinant IL-2 from the high affinity form of the IL-2 receptor was measured following the method of Wang and Smith (25). HUT102/6TG cells were harvested and washed thoroughly in complete RPMI 1640 medium. The cells (1 ϫ 10 6 /point) were incubated with 200 pM 125 I-labeled recombinant IL-2 (0.7 Ci/pmol, DuPont NEN) in the presence or absence of increasing concentrations of unlabeled recombinant IL-2, DAB 389 IL-2, or the related mutant fusion toxins. The cells were overlaid on a mixture of 80% 550 fluid (Accumetric Inc., Elizabethtown, KY) and 20% paraffin oil (density ϭ 1.03 g/ml) and incubated at 37°C for 20 min. The reactions were centrifuged in a microcentrifuge for 2 min. The cell pellet, representing bound ligand, and the aqueous phase, representing unbound ligand, were counted in a Beckman Gamma 5500 counter. The apparent dissociation constants (K i values) were determined based on the concentration of unlabeled ligand required to displace 50% 125 Ilabeled recombinant IL-2 binding. (The K i for DAB 389 IL-2(T439P) is an estimate determined from Fig. 4, as an IC 50 was not reached, even at the highest concentration of toxin tested).
Activation Assay-CTLL-2 cells were maintained in complete RPMI, as above, that was further supplemented with 5 M 2-mercaptoethanol and 1 nM recombinant IL-2. For the assays, the CTLL-2 cells were washed twice in the above medium, without IL-2, and seeded in the medium, again without IL-2, at 1.5 ϫ 10 4 cells/well in a 96-well, Vbottomed microtiter plate. The catalytic domain mutants of the fusion toxins, or recombinant IL-2, were added to final concentrations ranging from 10 Ϫ7 M down to 10 Ϫ12 M. The cells were incubated at 37°C for 24 h and then 1.0 Ci of [ 3 H]thymidine (6.7 Ci/mmol, DuPont NEN) was added to each well. The cells were pulsed for 6 h at 37°C, harvested on glass fiber filters, and radioactivity was determined using standard methods. Each assay point was performed in triplicate or quadruplicate, and the assays were performed several times for each fusion toxin (Fig. 5). The results for activation reported in Table II  Kinetic Assays-HUT 102/6TG cells were seeded in 100 l of complete medium, at a concentration of 5 ϫ 10 4 cells/well, as described above for the cytotoxicity assays. The cytotoxic forms of the fusion toxins were added to a final concentration of 10 Ϫ9 M, and the cells were incubated for 1, 3, 5, 9, 12, 15, or 18 h before pulsing and harvesting, as described above. RESULTS A schematic representation of the gene encoding DAB 389 IL-2 is shown in Fig. 1. The mutations in the IL-2 receptor binding domain of the gene, where nucleotides were changed to encode different amino acid residues, are indicated. The Glu 3 Ser mutation in the catalytic domain of DT is also indicated. The mutations in IL-2, corresponding to the mutations in the IL-2 receptor binding domain of the fusion toxins, are shown in Table I DA(E149S)B 389 IL-2(T439P), DAB 389 IL-2(Q514D), and DA-(E149S)B 389 IL-2(Q514D) were expressed and purified as described under "Experimental Procedures," Following purification, the proteins were separated by electrophoresis on 12% SDS-polyacrylamide gels and stained with Coomassie Blue (Fig. 2). The full-length forms of the proteins all migrated at approximately 57.7 kDa, in agreement with their calculated molecular weights. Western blot analysis was also performed and indicated the proteins were all immunoreactive with DT antibody (data not shown).
DAB 389 IL-2 possessed an IC 50 of 2.2 ϫ 10 Ϫ12 M in the cytotoxicity assay (Fig. 3, Table II). The corresponding catalytic domain mutation, DA(E149S)B 389 IL-2, in which a Glu residue was changed to a Ser residue, was not cytotoxic (Table II). The apparent binding affinities (K i values) were 3.6 ϫ 10 Ϫ10 M and 5.7 ϫ 10 Ϫ10 M, respectively (Fig. 4, Table II). DA(E149S)B 389 IL-2 was tested for stimulation of DNA synthesis by a CTLL-2 cell [ 3 H]thymidine incorporation assay, and a representative assay is shown in Fig. 5. The stimulation by 10 Ϫ8 M DA(E149S)B 389 IL-2 was 83% that of the stimulation induced by 10 Ϫ8 M IL-2 (Table II). The stimulation by 10 Ϫ8 M DAB 389 IL-2 was only 4% that of 10 Ϫ8 M IL-2. DAB 389 IL-2(E494K), in which a Glu residue in the IL-2 binding domain was changed to a Lys residue (Fig. 1), possessed an IC 50 of 1.6 ϫ 10 Ϫ11 M, approximately 7-fold less than the wild type cytoxicity (Fig. 3, Table II). The K i for the high affinity form of the IL-2 receptor was 2.9 ϫ 10 Ϫ9 M, approximately 8-fold less than the binding affinity of the wild type (Fig. 4, Table II). DAB 389 IL-2(E494K) stimulated DNA synthesis to 3% of the level of 10 Ϫ8 M rIL-2 (Table II). The corresponding fusion toxin with the E149S catalytic domain mutation, DA(E149S)B 389 IL-2(E494K), was not cytotoxic, possessed a K i of 6.6 ϫ 10 Ϫ9 M, and stimulated CTLL-2 [ 3 H]thymidine incorporation by 63% compared to the same concentration, 10 Ϫ8 M, of rIL-2 (Fig. 4, Table II).
DAB 389 IL-2(T439P) (Fig. 1) possessed an IC 50 of 6.5 ϫ 10 Ϫ10 M (Fig. 3, Table II) and a K i of 3.5 ϫ 10 Ϫ8 M (Fig. 4, Table II). This represents approximately a 300-fold decrease in cytotoxicity and approximately 100-fold less binding affinity when compared to wild type fusion toxin. 10 Ϫ8 M DAB 389 IL-2(T439P) stimulated DNA synthesis in CTLL-2 cells to 5% of the level of 10 Ϫ8 M rIL-2. The E149S form of DAB 389 IL-2(T439P) was not cytotoxic and possessed a K i of 1.8 ϫ 10 Ϫ8 M (Table II) Table II). The cytotoxicity was therefore, approximately 2000-fold less than that of wild type DAB 389 IL-2. The K i was approximately 50fold less than that of wild type. DAB 389 IL-2(Q514D) stimulated CTLL-2 cell DNA synthesis by 2% compared to rIL-2 (Table II). DA(E149S)B 389 IL-2(Q514D) was not cytotoxic, the K i was 2.0 ϫ 10 Ϫ9 M and, in contrast to the other noncytotoxic forms of the fusion toxins, at a concentration of 10 Ϫ8 M, stimulated CTLL-2 [ 3 H]thymidine incorporation to only 8% of the level of 10 Ϫ8 M rIL-2 stimulation (Fig. 5, Table II).
The above results were of interest as the T439P IL-2 binding domain mutant stimulated DNA synthesis by CTLL-2 cells to a greater extent than one would expect, given the K i results. Also, the Q514D IL-2 binding domain mutant was less cytotoxic than expected given the K i results. For this reason, kinetic assays of cytotoxicity were performed. DAB 389 Table II.  IL-2(T439P) and DAB 389 IL-2(Q514D) exhibited reduced rates of intoxication (Fig. 6).

DISCUSSION
Studies of the relationships between IL-2 and its receptors are important to identify the various functions of IL-2, as well as the structural elements involved with these functions. This information is required for the design of analogs with expanded therapeutic applications. In the present work we use the IL-2directed fusion toxin, DAB 389 IL-2, to further study some amino acid residues previously identified as involved in the processing of IL-2 and its receptors. The purpose of this work was to expand on the knowledge already obtained for these residues, to ascertain how these residues affect the function of the fusion toxin, and to demonstrate the potential of DAB 389 IL-2 as a useful agent for the study of ligand-receptor interactions.
Buchli and Ciardelli (14) created an IL-2 analog in which a Gln residue at position 126 of IL-2 was changed to an Asp residue. Their results indicated that the Asp-126 mutant stimulated [ 3 ]thymidine incorporation in human T-lymphocytes and CTLL-2 cells to a lesser degree than wild type IL-2. The binding affinity of the D126 mutant was greatly decreased, and the loss was due to a disruption of the ␤/␥ receptor subunit interaction. The authors postulated that cross-linking of the ␤/␥ receptor subunits is the likely signaling event for activity of IL-2, and that Gln-126 is involved with binding and cross-linking the subunits, either as a contact position or allosterically.
We constructed and studied DAB 389 IL-2(Q514D), which contains the analogous mutation in the IL-2 receptor binding domain of DAB 389 IL-2. For comparison, we also studied DAB 389 IL-2(E494K), a form of DAB 389 IL-2 containing a mutation in the IL-2 binding domain that we already knew exerted minimal effects on cytotoxicity. DAB 389 IL-2(Q514D) was 2000fold less cytotoxic than wild type DAB 389 IL-2 and possessed a decreased binding affinity, and the corresponding protein with the catalytic domain mutation did not stimulate [ 3 H]thymidine incorporation. The cytotoxicity kinetic assays indicate that DAB 389 IL-2(Q514D) inhibited protein synthesis at a slower rate than wild type DAB 389 IL-2. The rate of inhibition reflects the rate of binding and toxin entry into the cell cytosol. These results indicate that Gln-514, in the receptor binding domain of DAB 389 IL-2, is involved with binding affinity, stimulation of DNA synthesis and rate of toxin internalization. These results are all consistent with the findings for the Gln-126 residue of IL-2. DAB 389 IL-2(E494K) was 7-fold less cytotoxic than DAB 389 IL-2, probably as a direct result of the 8-fold decrease in binding affinity. The rate of protein synthesis inhibition was the same as for DAB 389 IL-2. DA(E149S)B 389 IL-2(E494K) stimulated [ 3 H]thymidine incorporation, although not as much as wild type, DA(E149S)B 389 IL-2. The effects imposed by the Glu-494 mutation to a Lys probably result from structural changes in the IL-2 binding domain of the fusion toxin, leading to the decrease in binding affinity.
Chang et al. (15) discovered that mutating Thr-51 of IL-2 to a Pro residue resulted in an IL-2 analog with a decreased binding affinity, but the corresponding loss in stimulation of DNA synthesis was much lower than expected. They postulated that either the Thr-51 3 Pro mutation resulted in a conformational change that partially mimicked a change required for IL-2 to facilitate the ␤/␥ subunit cross-linking necessary for signaling activation, or the Thr-51 3 Pro mutation slowed the internalization rate for the ligand-bound receptor complex, thereby allowing a greater time for the signaling interval. The corresponding residue in the IL-2 receptor binding domain of DAB 389 IL-2 was mutated to create DAB 389  Table II. IL-2(T439P). DAB 389 IL-2(T439P) was 300-fold less cytotoxic than wild type and possessed a decreased binding affinity, but still stimulated DNA synthesis as well as the control mutation, DAB 389 IL-2(E494K). SDS-polyacrylamide gel electrophoresis of DAB 389 IL-2(T439P) (Fig. 2, lane 3) shows this protein was subject to breakdown after freezing and thawing, probably due to conformational effects the Thr-439 3 Pro mutation had on the protein. (DAB 389 IL-2(T439P) was originally isolated as full-length protein, as shown by gel electrophoretic analysis performed during the purification procedures; data not shown.) DAB 389 IL-2(T439P) and DAB 389 IL-2(Q514D) both possessed poor binding affinities, but DAB 389 IL-2(T439P) was 10-fold more cytotoxic. The fact that DAB 389 IL-2(T439P) stimulated DNA synthesis and DAB 389 IL-2(Q514D) did not lead us to speculate that the difference in stimulatory effect may account for the difference in cytotoxicity, i.e. stimulation of DNA synthesis leads to enhancement of cytotoxicity. The cytotoxicity kinetics assay for DAB 389 IL-2(T439P) showed that this mutant fusion toxin, like DAB 389 IL-2(Q514D), possessed a decreased rate of cytotoxicity compared to the wild type and DAB 389 IL-2(E494K). This leads to the conclusion that the Thr-439 3 Pro mutation in the IL-2 receptor binding domain of DAB 389 IL-2, and the corresponding Thr51 to Pro mutation in IL-2, exerted greater than expected stimulatory effects at least partially due to a decreased rate of internalization.
The results from these studies confirm and expand on the previous findings for studies performed with IL-2. The Asp residue at position 126 of IL-2 is involved with binding of IL-2 to its receptors. The rate of internalization of the IL-2-receptor complex is decreased and, possibly due to a decrease in ␤/␥ cross-linking, signaling stimulation of DNA synthesis is decreased. Additionally, mutation of this residue in the IL-2 binding domain of the fusion toxin DAB 389 IL-2 decreases cytotoxicity to a greater degree than expected, indicating that the loss in stimulation of DNA synthesis, affects cytotoxicity. It appears that the stimulatory effect of the IL-2 binding domain on DNA synthesis enhances cytotoxicity. The Thr residue at position 51 of IL-2 is involved with binding, and this effect may be conformational. The rate of ligand-receptor internalization is decreased when this residue is changed to a Pro, and an increase in DNA stimulation occurs. This effect results in a greater than expected cytotoxicity when the corresponding residue in the IL-2 binding domain of DAB 389 IL-2 is mutated.