Identification of Their Epitope Reveals the Structural Basis for the Mechanism of Action of the Immunosuppressive Antibodies Basiliximab and Daclizumab

Priority Report

Mascha Binder,1 Friederike-Nora Vo¨gtle,1 Stefan Michelfelder,1 Fabian Mu¨ller,1 Gerald Illerhaus,1 Sangeeth Sundararajan,1 Roland Mertelsmann,1 and Martin Trepel1,2

1Department of Hematology and Oncology and 2Institute for Molecular Medicine and Cell Research, University of Freiburg Medical Center, Freiburg, Germany

h Abstract high-affinity IL-2R (a gc; ref. 6), in which the IL-2 interaction with a h g Interleukin-2 (IL-2) and its receptor (IL-2R) play a major role the chain initiates the sequential recruitment of the and c a in cellular immunity. The monoclonal antibodies basiliximab subunits(7).Tcellsexpressthe chain (CD25) only on activation. and daclizumab directed against the IL-2R subunit CD25 are Therefore, blocking of this receptor efficiently and specifically widely used to prevent graft or host rejection after allogeneic interferes with the cellular immune response, whereas other tissue transplantation. Although these antibodies have been components of the immune system remain unaffected. An anti- used for this purpose for many years, their common epitope CD25 antibody [initially referred to as ‘‘anti-Tac’’ (8)] has been within the CD25 protein is unknown. We screened a random modified to facilitate its application in patients for activated T- phage display library to isolate peptides specifically binding to cell suppression. At present, two such anti-Tac derivatives are basiliximab. A striking amino acid sequence motif was used clinically: basiliximab, a chimeric antibody containing human constant regions and murine variable regions, and enriched. This motif is homologous to the peptide ERIYHFV comprising amino acid positions 116 to 122 within the daclizumab, a humanized antibody whose only remaining murine extracellular domain of CD25, suggesting that this is the parts are the CDR regions. These therapeutic antibodies are used basiliximab epitope. Basiliximab and daclizumab binding of for IL-2 signaling suppression to prevent (5) or treat (1) acute selected phage was specific, as no binding was observed to allograft rejections, acute graft-versus-host disease (2, 3), auto- isotype antibody controls. Phage binding could be inhibited by immune disorders (9), and malignancies, such as T-cell leukemias the cognate peptide. In cells expressing mutant CD25, binding (4), with high response rates and very few side effects. of basiliximab was abolished when two or more amino acids of The mechanism of action of these antibodies is mediated the suspected epitope were changed. In contrast, basiliximab primarily by inhibition of IL-2 binding to CD25 (10). Thus, binding remained unaffected in cells expressing CD25 versions downstream events mediating IL-2/IL-2R signaling are impeded. with mutations outside this epitope. We therefore conclude In addition, these antibodies may also act through antibody- that the (116)ERIYHFV(122) string within CD25 is the epitope directed cellular cytolysis and complement-mediated cytotoxicity in recognized by basiliximab and daclizumab. This epitope Tcells(11). The epitope within the CD25 molecule recognized by anti-Tac overlaps with the interaction site of CD25 and IL-2, thus revealing the structural basis for the inhibition of IL-2R and its derivatives basiliximab and daclizumab has been unknown binding by this class of immunosuppressive antibodies. thus far. Phage display library screenings are a valuable tool for the [Cancer Res 2007;67(8):3518–23] exploration of epitopes recognized by antibodies (12–14). Phage displaying a peptide that mimics the epitope of a given antibody can be selectively enriched through panning on antibody-coated Introduction matrices (15, 16). In this study, we used such phage peptide library Interleukin-2 (IL-2) and its receptor (IL-2R) play a pivotal role as screenings to analyze the unknown epitope of the monoclonal signaling molecules in cellular immune responses. Therefore, this antibody basiliximab. A striking peptide sequence motif was ligand-receptor interaction has been considered a very good target recovered, which is homologous to a peptide string within CD25. in the clinical treatment of acute allograft rejection, graft-versus- We substantiate the hypothesis that this is the epitope recognized host disease, T-cell–mediated autoimmune disease, and certain by basiliximab and show that this epitope overlaps with the hematologic malignancies (1–5). previously characterized interaction site of CD25 and IL-2, Binding of IL-2 to the IL-2R expressed on T cells elicits cellular revealing the structural basis for the inhibition of IL-2 binding to changes primarily related to proliferation and differentiation. The its receptor by this group of immunosuppressive antibodies. IL-2R is composed of the a, h,andgc subunits. On activation, they assemble to form the intermediate-affinity IL-2R (hg )orthe c Materials and Methods Tissue culture. 293T cells were used with kind permission of David Baltimore (California Institute of Technology, Pasadena, CA) and main- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). tained in DMEM containing 10% fetal bovine serum and 1% penicillin and Requests for reprints: Martin Trepel, Department of Hematology and Oncology, streptomycin. University of Freiburg Medical Center, Hugstetter Strasse 55, D-79106 Freiburg, Phage display library screening. The fUSE5 vector and K91Kan Germany. Phone: 49-761-2707357; Fax: 49-761-2707357; E-mail: [email protected] bacteria were from George Smith (University of Missouri, Columbia, MO). freiburg.de. I2007 American Association for Cancer Research. fUSE5-based phage peptide libraries displaying seven random residues doi:10.1158/0008-5472.CAN-06-3919 flanked by two cysteines (CX7C) were made as described (12). The

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degenerate oligonucleotide was TGT(NNB)7TGT. Second strand was evaluated for daclizumab, as both therapeutic antibodies derive synthesized using sequenase (Amersham) and the primer 5¶-TTCGG- from the same parental murine antibody (8). Like basiliximab, the ¶ CCCCAGCGGCCCC-3 . The fUSE5 plasmid was digested with SfiI, and CWYHYIWEC phage bound specifically to daclizumab but not to the insert was digested with BglI. Plasmid and insert were ligated and control antibodies (Fig. 2C). This suggests that the selected transformed into MC1061. Phage was purified from the bacterial culture peptides structurally mimic the common epitope recognized by using polyethylene glycol-NaCl precipitation. The library was screened on basiliximab (Novartis, Basel, Switzerland) basically as described previously daclizumab and basiliximab. (13). Briefly, polystyrene 96-well plates were coated with 0.5 mg/mL CWYHYIWEC phage binding to basiliximab can be competi- basiliximab (Roche, Basel, Switzerland) in PBS. The wells were blocked tively blocked by the cognate peptide. To confirm that the with 3% bovine serum albumin and incubated with 1010 transducing units peptides displayed on the enriched phage bind the antibody of the library for 1 h. In the third and fourth round of selection, the independently of other phage components, competition experi- library was precleared on rituximab. Bound phage was eluted by adding a ments were done using recombinant peptides. Therefore, the K91Kan culture and amplified by overnight growth. Randomly selected peptide CWYHYIWEC displayed on the phage clone that had clones from the fourth panning round were sequenced (GATC, Konstanz, shown the strongest antibody binding was produced as a Germany). glutathione S-transferase (GST) fusion protein and tested for its Single clone binding assays. Individual phage clones were tested for ability to inhibit phage binding to basiliximab. GST-CWYHYIWEC, binding to basiliximab, daclizumab (Roche), or control proteins as indicated following the protocol of the initial screening. The number of bound phage but not GST alone, inhibited phage binding to basiliximab in a was determined by growing the recovered phage-bacteria mixture on Luria- dose-dependent manner (Fig. 2D; Supplementary Fig. S2). This Bertani agar containing 40 Ag/mL tetracycline. indicates that the CWYHYIWEC phage mimics the basiliximab Glutathione S-transferase fusion proteins. The oligonucleotide epitope by means of the displayed library peptide and that the encoding the phage-derived peptide was synthesized, digested with BamHI conformation of this peptide is independent of the phage capsid and EcoRI, and ligated into pGEX-2TK (Amersham). Proteins were protein context. expressed in BL21 and purified following the manufacturer’s instructions. The selected phage displayed peptides are homologous to Mutation of CD25. The pReceiverM02_CD25 plasmid encoding human F E L CD25. The selected consensus motif /Y- /H-W- /V shows sequence CD25 was obtained from RZPD (Heidelberg, Germany). CD25 variants with homology to the peptide string Y-H-F-V comprising amino acid point mutations were generated using the QuikChange Site-Directed positions 119 to 122 within the extracellular domain of CD25 Mutagenesis kit (Stratagene) as described (17). Five mutants were established as indicated (Fig. 3B) using suitable primers (Supplementary (SwissProt database). In fact, the amino acids flanking the YHFV Table S1). peptide string within CD25 contain additional homologies to at Transfection and fluorescence-activated cell sorting analysis. 293T least some of the selected phage peptides (Fig. 2A). Taking these cells were transfected with wild-type or mutant CD25 plasmids as additional residues into account, we hypothesized that the epitope indicated using PolyFect (Qiagen). After 24 h, cells were stained using recognized by basiliximab consists of the strand ERIYHFV (amino basiliximab at 10 Ag/mL or a polyclonal goat anti-human IL-2Ra antibody acid positions 116–122) within CD25. In congruence with this, the (R&D Systems) at 7 Ag/mL for 30 min. Secondary antibodies were FITC- labeled rabbit anti-human (Jackson) at 2.3 Ag/mL or rabbit anti-goat antibodies (Dianova) at 13 Ag/mL. Stained cells were analyzed using FACSCalibur (BD Biosciences).

Results and Discussion Phage library biopanning on basiliximab yields specific peptides. To identify the epitope of the anti-Tac monoclonal antibody and its derivatives, we screened a random CX7C phage display peptide library (C = cysteine, X = any amino acid) on immobilized basiliximab. In the fourth selection round, phage bound 525 stronger to basiliximab than to the rituximab control, indicating that basiliximab-specific clones had been enriched (Fig. 1). Sequencing of the inserts of 18 clones recovered from the basiliximab wells of this selection round revealed their striking similarity. The consensus motif contains two aromatic amino acids separated by a single E or H and flanked either upstream or downstream by the branched side chain amino acids L or V. This F E L consensus motif can be summarized as /Y- /H-W- /V (Fig. 2A). The amino acid N between the aromatic amino acids occurred numerically as often as H but was still not considered to be part of the motif as it only occurred in one single clone. The selected phage clones specifically bind basiliximab and daclizumab. All of the selected clones, but not insertless fd-tet Figure 1. Enrichment of phage selected on basiliximab over four selection control phage, bound specifically and strongly to basiliximab but rounds. A CX7C phage display peptide library was selected on immobilized basiliximab. Bound phage was recovered by K91 bacterial infection, amplified not to the rituximabcontrol (Fig. 2 B). Specificity of the phage clone overnight, purified, and subjected to the next selection round. Negative CWYHYIWEC was confirmed using an independent control preselection of the library was done in selection round three and four. The antibody (infliximab; Supplementary Fig. S1). To verify that the selected phage was tested for binding to basiliximab and the control antibody rituximab after each selection round. Bacteria transduced by recovered phage selected peptides bind basiliximab at the paratope region of the were grown on Luria-Bertani plates containing tetracycline to determine the antibody, binding of the CWYHYIWEC phage clone was also number of transducing units (TU) by colony counting. www.aacrjournals.org 3519 Cancer Res 2007; 67: (8). April 15, 2007

Figure 2. Characterization of phage clones binding to basiliximab. A, sequences of peptides from a random CX7C phage display library selected over four rounds on immobilized basiliximab. A total of 18 randomly picked clones was sequenced. Sequences are aligned for the consensus motif using a single-letter code. Letters are colored according to the biochemical property of the amino acids when they have sequence homology to the CD25 protein. Red, aromatic amino acid; magenta, positively charged amino acid; blue, branched side chain; orange, negatively charged amino acid; green, heterocyclic amino acid. Phage clone CWYHYIWEC is depicted in reverse orientation (C!N) to highlight its homology to the natural CD25 sequence. Numbers in brackets, amino acid position. B, phage displaying the selected peptides binds specifically to basiliximab but not to the control isotype antibody rituximab. Transducing units (5 107) of phage displaying no peptide insert (fd-tet) or the peptides enriched by selection on basiliximab were incubated on immobilized basiliximab or the rituximab control, respectively. Bound phage was recovered by K91 bacterial infection. Transduced bacteria were grown on Luria-Bertani plates containing tetracycline to determine the number of transducing units by colony counting. Columns, mean of triplicate platings; bars, SE. C, the CWYHYIWEC phage also binds to daclizumab. Phage displaying CWYHYIWEC or no peptide insert (fd-tet) was incubated on immobilized daclizumab and quantified after recovery as described in (A). Columns, mean of triplicates; bars, SE. D, the fusion protein GST-CWYHYIWEC blocks binding of CWYHYIWEC phage to basiliximab. Transducing units (5 107) of CWYHYIWEC phage were incubated on immobilized basiliximab in the presence or absence of various concentrations of GST-CWYHYIWEC or GST alone, respectively. Bound phage was recovered and quantified as described in (A). Data are relative values compared with binding in the absence of GST. Points, mean of triplicate platings; bars, SE.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Basiliximab Epitope strongest basiliximab-binding phage CWYHYIWEC showed a have been enriched, as it possessed closest sterical resemblance to remarkably high degree of sequence similarity to CD25, especially the original peptide string within CD25. Of note, 50% of all enriched when read from COOH terminus to NH2 terminus (Fig. 2A). There peptide sequences could be read in both forward and reverse are possible explanations for the reversed order of the amino acids orientation without losing their homology to the 116 to 122 in the CWYHYIWEC clone compared with the original sequence of segment of CD25 (Fig. 2A), supporting this assumption. This the presumed epitope. Importantly, the peptide sequence theoret- suggests that the orientation of the peptide backbone does not ically having optimal binding characteristics may not have been fundamentally affect the binding characteristics of the peptides present in the phage library in the forward orientation at all. Only enriched in our screenings on basiliximab. f1% to 1% of all conceivable amino acid combinations are Interestingly, the selected peptides contained a considerable represented in a CX7C state of the art-made phage library. If the number of aromatic amino acids, and in defined positions, the amino N-to-C-direction of the sequence is not crucial for the binding acids tyrosine (Y), tryptophane (W), and phenylalanine (F) seemed to characteristics of the peptide, the CWYHYIWEC sequence may be interchangeable. Such predominance of aromatic amino acids in

Figure 3. The peptide string comprising amino acid positions 116 to 122 of CD25 is the epitope recognized by basiliximab. A, phage displaying the presumed epitope sequence (phage ‘‘CD25’’) binds specifically to basiliximab but not to the control antibody rituximab. Transducing units (5 107) of phage displaying the ERIYHFV peptide of CD25 (amino acid positions 116–122, flanked by two cysteines in accordance with the constraint of the peptide library) were incubated on immobilized basiliximab or the control antibody rituximab, respectively. Phage without peptide insert (fd-tet) served as controls. Bound phage was recovered by K91 bacterial infection. Transduced bacteria were grown on Luria-Bertani plates containing tetracycline to determine the number of transducing units by colony counting. Columns, mean of triplicate platings; bars, SE. B, CD25 wild-type and mutant sequences established by site-directed mutagenesis in the plasmid pReceiverM02_CD25. Numbers in brackets, amino acid position. Amino acids are given in single-letter code. Bold and underlined letters, mutated amino acids. C, the region surrounding amino acid position 120 of CD25 is critical for basiliximab binding. CD25-negative 293T cells were transfected as described in Materials and Methods with the pReceiverM02_CD25 plasmid in wild-type sequence or mutations thereof (B). Binding of basiliximab or a control polyclonal antibody was assessed by FACS analysis 24 h after transfection. Basiliximab bound to wild-type CD25, CD25_M1, CD25_M4, and CD25_M5, whereas no binding was detected to CD25_M2 and CD25_M3. www.aacrjournals.org 3521 Cancer Res 2007; 67: (8). April 15, 2007

Figure 4. Model of the IL-2R binding to IL-2. Magenta, IL2; yellow, contact site of IL-2Ra and IL-2; red, basiliximab epitope. E116 and V122 mark the first and the last amino acid of the epitope. Orange, overlap of IL-2 binding and basiliximab binding amino acids within the IL-2R. The model was established using the software Cn3D 4.1 based on the published crystal structure (6).

the selected peptide motifs has been reported in previous epitope recognized by basiliximab, we tested basiliximab binding to mapping studies using phage display (13, 18, 19). In fact, aromatic cells expressing CD25 with mutations in the suspected epitope amino acids and hydroxylated amino acids, such as serine, are region or an unrelated downstream region, respectively. We particularly suited for molecular recognition patterns in protein- transfected 293T cells, which are CD25 negative, with the plasmid protein interactions (20, 21). For antibody-antigen interactions, pReceiverM02_CD25 encoding CD25 or the mutants thereof tyrosine seems to be especially important (22). Although this (Fig. 3B). CD25 expression and integration into the membrane phenomenon has been described for paratopes of antibodies, it is was verified by staining with a polyclonal CD25 antibody. Twenty- remarkable that a similar pattern seems to apply for epitopes as well, four hours after transfection, >70% of the transfected cells were at least when they are optimized by random library biopanning. positive for CD25 as assessed by fluorescence-activated cell sorting Peptides with additional aromatic residues not present in the natural (FACS) analysis (Fig. 3C, white columns). Binding of basiliximab to epitope may have been enriched in the panning because these residues cells expressing wild-type CD25 was approximately the same as to enhance affinity. The replacement of functionally important amino cells expressing CD25 with control mutations surrounding amino acids by slightly different equivalents may thus improve binding of a acid position 210 (mutants M4 and M5). Likewise, exchange of the peptide, as it may lead to a better structural mimicry of the epitope. A amino acid in position 119 (Y!D; mutant M1) had no negative similar finding has recently been described for paratopes of synthetic effect on basiliximab binding. In contrast, exchange of two or more antibodies by Fellouse et al. (22). This suggests that for epitope amino acids in this region (mutants M2 and M3) totally abolished mapping approaches using phage display functional classes of amino basiliximab binding to the CD25-transfected cells (Fig. 3C, black acids rather than single specific residues have to be considered. columns). This confirms that the peptide string surrounding amino Phage displaying the natural CD25 sequence specifically acid position 120 within the extracellular domain of CD25 is critical bind basiliximab. Although all peptides of the enriched phage for the binding of basiliximab. clones showed a certain degree of sequence similarity to the CD25 The binding sites of IL-2 and basiliximab in CD25 overlap. segment comprising amino acids 116 to 122, none of the selected As IL-2 has been previously crystalized in complex with the IL-2R peptides had sequence identity to this presumed epitope region. To (7), it is possible to compare the binding site of IL-2 to IL-2Ra further explore if the presumed CD25 epitope binds to basiliximab, (CD25) with the localization of the basiliximab epitope identified in we designed phage expressing the natural CD25 sequence, flanked our study. Figure 4 illustrates the structure of IL-2 in complex with by cysteines like in the library-derived peptides. This CERIYHFVC the three receptor subunits. The IL-2/IL-2Ra interface consists of phage (termed ‘‘CD25 phage’’), but not fd-tet control phage, 20 IL-2 residues and 21 IL-2Ra residues (Fig. 4, yellow) on different specifically bound to basiliximab (Fig. 3A), although binding was loops of the molecule. The basiliximab epitope characterized here weaker compared with the phage selected from the random library. comprises the seven amino acids (116) E-R-I-Y-H-F-V (122) (Fig. 4, This may be due to improved structural epitope mimicry by amino red and orange), which include three of the IL-2 contact residues acid exchange as discussed above. (118) I-Y-H (120) (Fig. 4, orange; ref. 7). We therefore conclude that Mutation of the presumed epitope within the extracellular this overlap in contact sites is the structural basis for the domain of CD25 abrogates binding of basiliximab. To prove basiliximab-mediated inhibition of IL-2 binding to IL-2Ra.Asa that the ERIYHFV peptide string within CD25 is the epitope consequence of this inhibition, the assembly of the IL-2R and thus

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Basiliximab Epitope downstream signaling events leading to proliferation and differen- Acknowledgments tiation are prevented in vivo. Received 10/22/2006; revised 12/11/2006; accepted 2/20/2007. Taken together, we identified the epitope targeted by the Grant support: Deutsche Jose´ Carreras Leuka¨mie-Stiftung grant R03/08 (M. Trepel). immunosuppressive antibody basiliximab within the extracellular The costs of publication of this article were defrayed in part by the payment of page domain of CD25 and showed that the structural basis for inhibition charges. This article must therefore be hereby marked advertisement in accordance a with 18 U.S.C. Section 1734 solely to indicate this fact. of IL-2 binding to IL-2R is given by a topic overlap of binding sites We thank Florian Otto for helpful discussions, Kerstin Schmidt for technical support, of IL-2 and basiliximab. and George Smith for generously supplying the fUSE5 plasmid and K91Kan bacteria.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Identification of Their Epitope Reveals the Structural Basis for the Mechanism of Action of the Immunosuppressive Antibodies Basiliximab and Daclizumab

Mascha Binder, Friederike-Nora Vögtle, Stefan Michelfelder, et al.

Cancer Res 2007;67:3518-3523.

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