Basiliximab Signaling by Therapeutic Antibody Structural Basis for The

Structural Basis for the Blockage of IL-2 Signaling by Therapeutic Antibody Basiliximab

This information is current as Jiamu Du, Hui Yang, Dapeng Zhang, Jianchuan Wang, of September 27, 2021. Huaizu Guo, Baozhen Peng, Yajun Guo and Jianping Ding J Immunol 2010; 184:1361-1368; Prepublished online 23 December 2009; doi: 10.4049/jimmunol.0903178

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Structural Basis for the Blockage of IL-2 Signaling by Therapeutic Antibody Basiliximab

Jiamu Du,* Hui Yang,*,† Dapeng Zhang,‡ Jianchuan Wang,*,† Huaizu Guo,‡ Baozhen Peng,* Yajun Guo,‡ and Jianping Ding*

IL-2 signaling plays a central role in the initiation and activation of immune responses. Correspondingly, blockage of this pathway leads to inhibition of the immune system and would provide some therapeutic benefits. Basiliximab (Simulect), a therapeutic mAb drug with specificity against IL-2Ra of T cells, was approved by U.S. Food and Drug Administration in 1998. It has been proven to be effective in the suppression of the IL-2 pathway and hence has been widely used to prevent allograft rejection in organ transplantation, especially in kidney transplants. In this study, we report the crystal structure of the basiliximab Fab in complex with the ectodomain of IL-2Ra at 2.9 A˚ resolution. In the complex structure, the Fab interacts with IL-2Ra with extensive hydrophobic and hydrophilic interactions, accounting for a high binding affinity of 0.14 nM. The Ag binding site of basiliximab Downloaded from consists of all six CDR loops that form a large binding interface with a central shallow hydrophobic groove surrounded by four hydrophilic patches. The discontinuous epitope is composed of several segments from the D1 domain and a minor segment from the D2 domain that overlap with most of the regions responsible for the interactions with IL-2. Thus, basiliximab binding can completely block the interactions of IL-2 with IL-2Ra and hence inhibit the activation of the IL-2 signal pathway. The structural results also provide important implications for the development of improved and new IL-2Ra–targeted mAb drugs. The Journal of Immunology, 2010, 184: 1361–1368. http://www.jimmunol.org/

nterleukin-2 is the first cytokine to be identified, character- ceptor, IL-2Rb (p75 or CD122) is shared with IL-15 (15–18), and ized, purified, and cloned (1–5). It plays a pivotal role in the gc (p65 or CD132) is a common receptor shared by many cy- I immune responses against pathogenic infection (6–8). Dur- tokines including IL-2, IL-4, IL-7, IL-9, and IL-15 (19, 20). IL-2Ra ing the defense against pathogens, recognition and binding of the and IL-2Rb contribute to the rapid association and slow dissocia- foreign Ags by the TCRs stimulate both the secretion of IL-2 and tion of IL-2, respectively (16), whereas receptors b and g mediate the expression of IL-2Rs on the T cell surface. Subsequently, the the transmembrane signal transduction (21, 22). Structural studies IL-2/IL-2R interaction activates the intracellular Ras/Raf/MAPK, have shown that IL-2 has a four-helix bundle structure (23), IL-2Ra by guest on September 27, 2021 JAK/STAT, and PI3K/AKT signal pathways and ultimately stim- is composed of two b-strand–swapped “sushi-like” domains, and ulates the growth, differentiation, and survival of the Ag-selected both IL-2Rb and gc are composed of two fibronectin type III do- cytotoxic T cells (9–14). IL-2 also regulates the functions of B mains (24–26). In the structures of the IL-2/IL-2Rabgc quaternary cells, NK cells, and regulatory T cells (6, 7). complexes, IL-2Ra binds to a surface groove of IL-2 and makes The biological functions of IL-2 depend on its interactions with extensive interactions with IL-2 via mainly the D1 domain and a few three IL-2Rs to form a quaternary complex IL-2/IL-2Rab common residues of the D2 domain, and IL-2Rb and gc bind to IL-2 on the g chain (gc) to trigger the IL-2 signaling process. Among the re- opposite side of IL-2Ra and have minor interactions with each other ceptors, IL-2Ra (p55, Tac Ag, or CD25) is an IL-2–specific re- but no contact with IL-2Ra (24–26). The gc receptor alone has no detectable affinity to IL-2, and binding of gc to IL-2 needs the presence of IL-2Rb (27). It is inferred that binding of IL-2Ra to *State Key Laboratory of Molecular Biology and Research Center for Structural IL-2 stabilizes the binding site for IL-2Rb and that IL-2 and IL-2Rb Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biolog- ical Sciences, Chinese Academy of Sciences; †Graduate School of Chinese Academy together form a composite binding site for gc. The structural, bio- of Sciences; and ‡International Joint Cancer Institute, Second Military Medical Uni- chemical, and computational data together suggest a sequential versity, Shanghai, China binding scenario of IL-2 by its receptors: first IL-2Ra, which is Received for publication September 28, 2009. Accepted for publication November abundantly expressed on the T cell surface, captures and enriches 12, 2009. the secreted IL-2 and changes the conformation of IL-2 to a favor- This work was supported by grants from the Ministry of Science and Technology of able IL-2Rb binding state, then the formed IL-2/IL-2Ra complex China (2004CB720102, 2006AA02A313, and 2007CB914302), the National Natural Science Foundation of China (30730028 and 90713046), the Chinese Academy of approaches IL-2Rb through two-dimensional cell surface diffusion Sciences (KSCX2-YW-R-107 and SIBS2008002), and the Science and Technology to form the IL-2/IL-2Rab complex, and finally gc is recruited to Commission of Shanghai Municipality (07XD14032). form the biologically active IL-2/IL-2Rabgc complex to transduce The coordinates and structure factors presented in this article have been deposited in the signaling cascade (25–28). the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession number 3IU3. Because of the central role of the IL-2 signaling in the activation of Address correspondence and reprint requests to Dr. Jianping Ding, Institute of Bio- immune defense, blocking of this signal pathway could suppress the chemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese immune system (7). The activation of T cells through the IL-2 sig- Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China. E-mail address: naling is initiated by the binding of IL-2Ra to IL-2. IL-2Ra is [email protected] a specific receptor for IL-2, whereas IL-2Rb and g are shared by Abbreviations used in this paper: g , common g chain; PDB, Protein Data Bank. c c other cytokines. Intriguingly, IL-2a is not expressed on resting Tand Copyright 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 B cells but abundantly expressed on activated T cells, especially by www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903178 1362 STRUCTURE OF BASILIXIMAB Fab IN COMPLEX WITH IL-2Ra the T cells participating in some pathological conditions such as Crystallization and diffraction data collection organ allograft rejection (29, 30), some autoimmune diseases (31, Crystallization was performed using the hanging drop vapor diffusion 32), and T cell leukemia (31, 33). The critical role of IL-2Ra in the method at 20˚C. In a drop containing 0.5 ml of the protein complex sample IL-2 signal pathway and its specific expression pattern make it and 0.5 ml of the reservoir solution (0.2 M KCl, 0.05 M HEPES (pH 7.5), a good clinical target. It has been shown that blocking of IL-2Ra can and 45% pentaerythritol propoxylate (5/4 PO/OH) (38) equilibrated against interfere with the interactions between IL-2 and IL-2R and hence 400 ml of the reservoir solution, hexagonal crystals grew to a final dimension of 0.1 3 0.1 3 0.05 mm3 after 15 d. The crystal was directly mounted on inhibit the IL-2 signal pathway, resulting in suppression of the im- a nylon loop and flash-cooled into the liquid N2 stream (2170˚C). Dif- mune system, which provides clinical benefits to organ trans- fraction data were collected at Shanghai Synchrotron Radiation Facility plantation patients. Two mAb drugs against IL-2Ra, basiliximab beamline BL-17U1 and processed with the program HKL2000 (39). The (Simulect; Novartis Pharmaceuticals, East Hanover, NJ) and dacli- statistics of the diffraction data are summarized in Table I. zumab (Zenapax; Roche, Basel, Switzerland), have been approved Structure determination and refinement by the U.S. Food and Drug Administration for the prevention of The structure of the basiliximab Fab in complex with the IL-2Ra ecto- allograft rejection in organ transplantation, especially in kidney domain was determined by the molecular replacement method im- transplants. Basiliximab is a mouse‑human chimeric mAb with the plemented in the program Phaser (40) with the structure of the basiliximab variable domain of murine anti–IL-2Ra mAb RTF5 and the constant Fab as the search model, followed by manual fitting of the IL-2Ra ecto- domains of human IgG1(k) (34) that has had great success in the domain. The rotation function search and the subsequent translational function search with the structure of the free-form basiliximab Fab (Protein prevention of renal allograft rejection (35). This mAb binds spe- Data Bank [PDB] code 1MIM) (41) used as the search model yielded an cifically to the ectodomain of IL-2Ra. With the phage display outstanding solution in the asymmetric unit. To further locate the position method, the epitope recognized by basiliximab was mapped to of the IL-2Ra ectodomain, we used all of the three available structures of Downloaded from residues 116–122 of the D2 domain of IL-2Ra, which is part of the IL-2Ra (PDB codes 2B5I, 1Z92, and 2ERJ) (24–26) as templates with Phaser and other commonly used programs implemented with the mo- region interacting with IL-2 and thus explains in part why the lecular replacement method. However, these attempts were unsuccessful to binding of basiliximab with IL-2Ra can block IL-2 signaling (36). find a solution for IL-2Ra. After several cycles of structure refinement However, the detailed molecular mechanism of the inhibition of using the program CNS (42), the electron density for the D1 domain of IL- IL-2 signaling by basiliximab remains unclear. 2Ra was developed gradually. The structure of the D1 domain of IL-2Ra (PDB code 2B5I) (25) was manually placed into the electron density. After In this study, we report the crystal structure of the basiliximab Fab in http://www.jimmunol.org/ several rounds of structure refinement using the program Phenix (43) and complex with the IL-2Ra ectodomain. Structural analysis of this com- model building using the program O (44), the complete D1 and D2 do- plex and its comparison with the crystal structures of IL-2 in complex mains of IL-2Ra were modeled and fit well into the electron density. with IL-2Rabgc reveal the molecular basis for the high specificity and However, as in all of the other IL-2Ra structures (24–26), several flexible high affinity of basiliximab with IL-2Ra and the molecular mechanism regions of IL-2Ra have no defined electron density and thus could not be modeled, including the linker region between domains D1 and D2 (resi- for the blockage of the IL-2 signaling by basiliximab. The structural dues 62–100) and the C-terminal region (residues 159–217). The final results also have important implications for the design and development model contains 119 of 217 residues of the IL-2Ra ectodomain. There was of improved and new mAb drugs against IL-2Ra. a long stretch of electron density near residue Asn49 of IL-2Ra that could be modeled as an N-linked core trisaccharide (MANb-1, 4GlcNAcb-1, 49 Materials and Methods 4GlcNAcb-1-Asn ) without ambiguity. All of the diffraction data were by guest on September 27, 2021 Protein preparation and purification used in the structure refinement except 5% of randomly chosen diffraction data were set aside for free R factor cross-validation. The stereochemical The mAb basiliximab was purchased from Novartis Pharmaceuticals. The geometry of the final structure model was analyzed with the program mAb was diluted to a concentration of 1 mg/ml with a buffer of 1 mM EDTA Procheck (45). The statistics of the refinement and structure model also are and 100 mM sodium acetate (pH 5.5), followed by digestion with 10 mg/ml listed in Table I. Structural analysis was performed using the programs in papain (Sigma-Aldrich, St. Louis, MO) at 37˚C for 5 h. The reaction was the CCP4 suite (46) and the PISA server (47). Figures were prepared using quenched with 20 mM iodoacetamide. The Fab fragment was separated by the program Pymol (www.pymol.org). ion exchange chromatography using a Q Sepharose Fast Flow column (GE Surface plasmon resonance analysis Healthcare, Uppsala, Sweden). The pooled Fab fragment was further purified by gel filtration chromatography using a Superdex G-200 16/60 column (GE The kinetic studies of the interaction between basiliximab and the IL-2Ra Healthcare). The Fab sample was concentrated to ∼10 mg/ml and then ex- ectodomain were performed by the surface plasmon resonance method changed to a buffer of 10 mM HEPES (pH 7.0) and 50 mM NaCl. using a Biacore 3000 instrument (GE Healthcare) at 25˚C. The mAb ba- The cDNA encoding the ectodomain of human IL-2Ra (residues 1–217) siliximab was immobilized on a CM5 sensor chip (GE Healthcare) using was cloned into a modified pFastBac vector (Invitrogen, Carlsbad, CA) that an amine coupling kit (GE Healthcare). The purified IL-2Ra ectodomain fuses a gp67A signal sequence and a hexahistidine tag at the N and C termini was dialyzed against the HBS (0.01 M HEPES, pH 7.4, 0.15 M NaCl, of the target protein, respectively. The recombinant protein was expressed 3 mM EDTA, 0.005% Surfactant P20) buffer (GE Healthcare) and used as and secreted into the medium using a Bac-to-Bac baculovirus expression the analyte in the binding assay. The association was monitored for a 240 s system (Invitrogen). The harvested medium was centrifuged twice to remove period, and the disassociation was monitored by flowing the HBS buffer the remaining cells and concentrated to a suitable volume followed by di- for 600 s subsequently. An irrelevant chimeric mAb, the anti-CD20 mAb alysis against a buffer of 10 mM Tris (pH 8.0) and 500 mM NaCl. The re- Rituximab (Roche), was used as a reference. The experimental data were combinant IL-2Ra was purified by affinity chromatography using a Ni-NTA analyzed with a 1:1 Langmuir model using the program BIAevalutation Superflow column (Qiagen, Valencia, CA). Because the wild-type IL-2Ra (GE Healthcare). would form a disulfide-linked dimer with the free cysteine at position 192 (37), the purified disulfide-linked protein was reduced with 10 mM cysteine Accession code and further alkylated with 20 mM iodoacetamide as previously described The coordinates and structure factors of the basiliximab Fab in complex (27). The resultant protein was purified further by gel filtration chromatog- with the ectodomain of IL-2Ra have been deposited in the Research raphy using a Superdex G-200 16/60 column, and the fraction corresponding Collaboratory for Structural Bioinformatics PDB (www.rcsb.org/pdb/) to the monomeric IL-2Ra was collected for further structural and bio- with the accession code 3IU3. chemical studies. The recombinant IL-2Ra is heavily glycosylated, and its apparent molecular weight is ∼43 kDa as monitored by reduced SDS-PAGE, Results whereas the calculated molecular weight is ∼25 kDa. The protein samples of the basiliximab Fab and IL-2Ra ectodomain were Overall structure of the basiliximab Fab in complex with the mixed at a molar ratio of 1.5:1 at 4˚C for 12 h and then loaded onto a Superdex IL-2Ra ectodomain G-200 16/60 column. The protein complex was eluted with a buffer of 10 mM HEPES (pH 7.0) and 50 mM NaCl and then concentrated to 3 mg/ml for The crystal structure of the basiliximab Fab in complex with the IL- crystallization. The purity and homogeneity of the complex were confirmed 2Ra ectodomain was determined by the molecular replacement by SDS-PAGE and dynamic light scattering analysis. method implemented with the structure of the free-form basiliximab The Journal of Immunology 1363

Fab as the search model, followed by manual fitting of the IL-2Ra that assemble like a bent arm with an elbow angle of ∼90˚. The ectodomain. This structure was refined to a resolution of 2.9 A˚ , electron density for both D1 and D2 domains was well defined, yielding an R factor of 21.5% and a free R factor of 26.2% (Table I). especially in the regions participating in interactions with the Fab There are three Fab/IL-2Ra complexes in an asymmetric unit. The (Fig. 1B). However, again similar to that seen in the other IL-2Ra structure model has good stereochemical geometry with only Thr50 structures (24–26), the linker region between the D1 and D2 do- of each L chain located in the disallowed regions of the Ram- mains (residues 62–100) and the C-terminal region (residues 159– achandran plot, which resides in a classic g-turn (48). Thr50 is located 217) connecting the D2 domain to the transmembrane domain of in the generously allowed region of the Ramachandran plot in the IL-2Ra could not be observed in this complex. Thus, only 119 out of free-form basiliximab Fab and has slightly differed f and c angles a total of 217 residues of the IL-2Ra ectodomain were modeled, (41). Although there are some variations in the numbers of disordered reflecting the flexible nature of IL-2Ra. A detailed structure com- residues, the three complexes are very similar (superposition of all of parison indicates that the overall structure of the IL-2Ra ectodo- the Ca atoms yields a root-mean-square deviation of 1.3–1.4 A˚ for main in this complex is similar to that in its complexes with IL-2 IL-2Ra, 0.2–0.3 A˚ for the Fab, and 0.5–0.6 A˚ for the complex, re- (24–26). Superposition of the different structures yields a root- spectively), and the one with the most detectable residues and best mean-square deviation of 0.9–1.8 A˚ for the D1 domain, 1.6–2.0 A˚ electron density has been selected for structural analysis. for the D2 domain, and 1.4–1.7 A˚ for the whole IL-2Ra molecule. The basiliximab Fab in the complex consists of the H chain The major structural differences occur in two solvent-exposed re- residues 1–215 that fold into the VH and CH1 domains and the L gions (residues 109–117 and 132–144 of the D2 domain) that are not 49 chain residues 1–208 that fold into the VL and CL domains (Fig. in contact with other protein molecules. It is noteworthy that Asn

1A). The overall structures of the variable domains and constant of IL-2Ra in this complex is glycosylated and an N-linked core Downloaded from domains of the Fab in the complex are similar to those in the free- form Fab reported previously (41) (superposition of all of the Ca atoms yields a root-mean-square deviation of 0.7 A˚ for the variable domains and 0.9 A˚ for the constant domains). The conformations of the CDRs of the Fab in the complex also resemble

those in the free-form Fab, indicating that binding of IL-2Ra does http://www.jimmunol.org/ not induce a signiﬁcant conformational change of the Fab. As in the other IL-2Ra structures reported previously (PDB codes 1Z92, 2ERJ, and 2B5I) (24–26), the IL-2Ra ectodomain in the Fab/ IL-2Ra complex is composed of two typical b-strand‑swapped “sushi-like” domains D1 (residues 1–61) and D2 (residues 101–158)

Table I. Summary of diffraction data and structure reﬁnement statistics by guest on September 27, 2021

Summary of Diffraction Data Wavelength (A˚ ) 0.9796 Space group P6522 Cell parameters a = b (A˚ ) 137.1 c (A˚ ) 459.1 Resolution range (A˚ )a 50.0–2.90 (3.00–2.90) Observed reﬂections 836,837 Unique reﬂections (I/s(I) . 0) 56,779 Average redundancy 14.8 (10.0) Average I/s(I) 17.0 (2.0) Completeness (%) 98.0 (94.5) Wilson B factor (A˚ 2) 86.3 Mosaicity 0.24 b Rmerge (%) 16.5 (56.1)

Statistics of Reﬁnement and Model

Number of reflections [Fo . 0s(Fo)] Working set 53,580 Free R set 2,651 R factor/free R factor (%)c 21.5/26.2 Number of protein atoms 12,450 Number of sugar atoms 117 ˚ 2 Average B factor of all atoms (A ) 87.6 FIGURE 1. Overall structure of the basiliximab Fab in complex with the Fab/IL-2Ra/sugar 74.3/132.2/132.6 IL-2Ra ectodomain. A, A stereoview of the overall structure of the com- Root-mean-square bond lengths (A˚ ) 0.006 plex. The Fab is colored with the L chain in yellow and the H chain in Root-mean-square bond angles (˚) 1.1 green, and IL-2Ra is colored with the D1 domain in cyan and the D2 Ramachandran plot (%) 49 Most favored regions 86.4 domain in purple. The sugar chain of the glycosylated Asn of IL-2Ra is Allowed regions 12.0 shown with a ball-and-stick model. B, A stereoview of a representative Generously allowed regions 1.4 SIGMAA-weighted 2Fo 2 Fc electron density map (1s contour level) in Disallowed regions 0.2 regions of the D1 domain of IL-2Ra (residues 21–30 and 53–61) that are aNumbers in parentheses refer to the highest resolution shell. involved in the interactions with the Fab. The atomic coordinates of the b Rmerge=+hkl+i|Ii(hkl)i 2 ÆI(hkl)æ|/+hkl+iIi(hkl). residues are shown with ball-and-stick models. The disulfide bonds be- c 2 28 59 30 61 R factor = ||Fo| |Fc||/|Fo|. tween Cys and Cys and between Cys and Cys are clearly defined. 1364 STRUCTURE OF BASILIXIMAB Fab IN COMPLEX WITH IL-2Ra trisaccharide (MANb-1, 4GlcNAcb-1, 4GlcNAcb-1-Asn49) could The Ag binding site of basiliximab is comprised of all six CDR be modeled without ambiguity. This is the first observation of gly- loops that form a large, flat surface to accommodate a conforma- cosylation of this residue, because in the previously reported tional epitope of IL-2Ra (Fig. 2A,2B). The basiliximab epitope structures Asn49 is either disordered or mutated to serine to prohibit consists of several discontinuous segments of IL-2Ra, including the glycosylation of the protein (24–26). In our structure, although residues 1–6, 21–29, 38–48, and 56–57 of the D1 domain and Asn49 and the sugar chain do not have direct contact with the ba- residues 118–120 of the D2 domain. In total, 23 residues of IL- siliximab Fab, the region near Asn49 (residues 38–48) participates in 2Ra (21 residues of the D1 domain and 2 residues of the D2 the interactions with the Fab, which might contribute to the stabi- domain) are involved in the interactions with the Fab, which is lization of the glycosylated Asn49. However, the biological signifi- more than those in most Ag/Ab complexes (51). The sugar chain cance of the glycosylation is unclear. of the glycosylated Asn49 of IL-2Ra extends away from the interaction interface and has no direct contact with the Fab. These a Interactions between the basiliximab Fab and the IL-2R results are in agreement with the biochemical data that basilix- ectodomain imab has a very high binding affinity to the IL-2Ra ectodomain In the complex structure, the basiliximab Fab forms extensive hy- with an apparent Kd (KD) of 0.14 nM determined by the surface drophilic and hydrophobic interactions with IL-2Ra via a large plasmon resonance technique (data not shown). interface, including 10 hydrogen bonds, 4 salt bridges, and 138 van A detailed analysis of the interactions between the basiliximab der Waals contacts (Fig. 2, Tables II and III). Although the complex Fab and the IL-2Ra ectodomain shows that the Ag binding site of has a shape complementarity value of 0.57, which is slightly smaller basiliximab is composed of a shallow hydrophobic groove sur- than the average value (0.64–0.68) for Ag/Ab complexes (49), rounded by four hydrophilic patches (hereafter residues of the Fab Downloaded from formation of the complex buries a very large solvent-accessible H chain and L chain and IL-2Ra are designated with the chain surface area of 2255.4 A˚ 2 (1108.6 A˚ 2 on the Fab and 1146.8 A˚ 2 on identifiers H, L, and I, respectively) (Fig. 2A,2B). At the center of IL-2Ra), which is much higher than the common value seen in the the Ag binding site, there is a shallow groove of a hydrophobic other Ag/Ab complexes (50, 51). The Fab H chain contributes 768.9 nature consisting of six aromatic residues TyrL93,TyrH30,TrpH31, 2 2 H50 H98 H100 A˚ of the buried surface area, and the L chain 339.7 A˚ , consistent Tyr ,Tyr , and Tyr at the interface of the VL and VH

with the notion that the mAb H chain usually makes more con- domains (Fig. 2A,2B). Residues in this groove form extensive http://www.jimmunol.org/ tributions than the L chain in Ag binding (52, 53). hydrophobic interactions with a hydrophobic surface patch of IL- by guest on September 27, 2021

FIGURE 2. Interactions between the basiliximab Fab and the IL-2Ra ectodomain. A, The interaction interface between the Fab and IL-2Ra viewing down at the Fab. The basiliximab Fab is shown with an electrostatic potential surface with the locations of some of the residues involved in the interactions with IL-2Ra labeled in green. IL-2Ra is shown with a stick model with the D1 domain in cyan and the D2 domain in purple, and the residues involved in the interactions with the Fab are shown in ball-and-stick models and labeled in black. B, The interaction interface between the Fab and IL-2Ra viewing down at IL-2Ra in the same orientation as in Fig. 2A. IL-2Ra is shown with an electrostatic potential surface with the locations of some of the residues involved in the interactions with the Fab labeled in cyan. The Fab is shown with a stick model with the CDR loops H1, H2, and H3 in green and the CDR loops L1, L2, and L3 in yellow, and the residues involved in the interactions with IL-2Ra are indicated in ball-and-stick models and labeled in black. C,A stereoview showing the hydrogen bonding and salt bridge interactions between IL-2Ra (in cyan) and the L chain of the Fab (in yellow). The hydrogen bonds are indicated with gray dashes, and the salt bridges are indicated with orange dashes. D, A stereoview showing the hydrogen bonding and salt bridge interactions between IL-2Ra (D1 domain in cyan and D2 domain in purple) and the CDR loop H1 of the Fab (in green). E, A stereoview showing the hydrogen bonding and salt bridge interactions between IL-2Ra (in cyan) and the CDR loops H2 and H3 of the Fab (in green). The color coding of the structural elements is the same as that in Fig. 1A. The Journal of Immunology 1365

Table II. Hydrogen bonds and salt bridges between the basiliximab Fab Surrounding the hydrophobic groove there are three positively and IL-2Ra charged surface patches and one negatively charged surface patch at the Ag binding site. One positively charged patch is composed of Hydrogen Bonds several residues of CDRs L1 and L3, including ArgL29, ArgL90, L91 Fab Atom CDR Loop IL-2Ra Atom Distance (A˚ ) and Ser that form both hydrophilic and hydrophobic inter- I56 I57 L49 d2 I47 g1 actions with residues Asp and Asn of IL-2Ra (Fig. 2A,2B). Asp –O L2 Thr –O 2.3 I56 L52 z I47 g1 In particular, Asp stretches its side chain into a shallow cavity Lys –N L2 Thr –O 3.5 L29 LysL52–Nz L2 GlyI48–O 3.0 on the surface of the Fab and forms two salt bridges with Arg ArgL90–O L3 AsnI57–Nd2 2.8 and ArgL90, and AsnI57 forms two hydrogen bonds with ArgL90 L91 g I57 d1 Ser –O L3 Asn –O 3.0 L91 C H29 I120 d1 and Ser (Fig. 2 , Table II). The second positively charged Arg –O H1 His –N 3.0 patch is composed of the residues AspL49 and LysL52 of CDR L2 TyrH30–Oh H1 AspI4–Od2 2.8 TrpH31–Nε1 H1 AsnI27–Od1 3.2 that form three hydrogen bonds and several hydrophobic inter- I47 I48 AsnH53–Nd2 H2 GluI29–Oε2 2.7 actions with the residues Thr and Gly of IL-2Ra (Fig. 2A–C, AspH97–Od2 H3 TyrI43–Oh 2.7 Table II). The third positively charged patch is composed of mainly CDR H1 residues ArgH29 and TyrH30 that interact with Salt Bridges a negatively charged surface patch of IL-2Ra formed by two Fab Residue CDR Loop IL-2Ra Residue short segments (residues 1–6 and residues 116–120) (Fig. 2A, 2B). In particular, ArgH29 forms a salt bridge with AspI6 and L29 I56

Arg L1 Asp I120 H30 Downloaded from L90 I56 a hydrogen bond with His ,andTyr forms a hydrogen bond Arg L3 Asp I4 ArgH29 H1 AspI6 with Asp (Fig. 2D, Table II). The fourth surface patch is neg- AspH55 H2 ArgI36 atively charged and composed of mainly CDR H2 residues AsnH53,AspH55,andGluH63 that interact with a positively charged surface patch of IL-2Ra formed by the residues ArgI35, I36 I38 H53 2Ra consisting of the residues LeuI2, MetI25, LeuI42,TyrI43,LeuI45, Arg , and Lys (Fig. 2A,2B). Specifically, Asn forms I29 H55 IleI118, and HisI120. This part of the interface contributes 59 van a hydrogen bond with Glu , and Asp forms a salt bridge with http://www.jimmunol.org/ I36 H63 der Waals contacts and 2 hydrogen bonds in total and plays Arg (Fig. 2E, Table II). In addition, Glu has a weak hy- I38 ˚ a pivotal role in the formation of the complex (Tables II and III). drophilic interaction with Lys (4.1 A). Among the many resi- In particular, the residues MetI25, LeuI42, and TyrI43 of IL-2Ra dues of IL-2Ra involved in the interactions with the Fab, the key I47 I56 I57 intrude their side chains into the groove to form numerous hy- residues of the epitope appear to be Thr ,Asp , and Asn , drophobic contacts with the surrounding residues, and TyrI43 in- each of which makes two hydrogen bonds or salt bridges with the I42 I43 serts its side chain into a small cavity in the groove and forms Fab, and Leu and Tyr , each of which makes many hydro- a hydrogen bond with AspH97. In addition, AsnI27 forms a hydrophobic interactions with the Fab. gen bond with TrpH31. These results are in agreement with the by guest on September 27, 2021 notion that tyrosine is the most frequently observed residue in the Discussion CDRs and plays the most important role in the Ag/Ab interaction The epitope of basiliximab because it can form both hydrophobic and hydrogen bonding interactions (54–56). Previously, the basiliximab epitope was mapped to residues 116– 122 of the D2 domain of IL-2Ra using the phage display method (36). In the basiliximab Fab/IL-2Ra complex structure, only two Table III. van der Waals contacts between the basiliximab Fab and residues of this segment are involved in the interactions with the Fab. a ˚ IL-2R (#4.0 A) Specifically, IleI118 makes two van der Waals contacts with the Fab, and HisI120 forms one hydrogen bond with ArgH29 and five van der IL-2Ra Residue Fab Residue Waals contacts with the Fab (Tables II and III). These interactions GluI1 (4) TyrH98 (4) constitute only a small portion of the interactions at the third hy- LeuI2 (3) TyrH30 (2), TyrH98 (1) drophilic patch described above and play a less significant role in the I4 H26 H29 H30 Asp (17) Ser (5), Arg (8), Tyr (4) recognition and binding of basiliximab. These results indicate that AspI5 (7) ArgH29 (7) AspI6 (7) ArgH29 (7) the potential epitope identified by the phage display method is in- GluI22 (3) TyrH100 (3) complete and the functional role of the identified region in the GlyI23 (4) TyrH100 (4) recognition is undefined. A similar situation also was seen in the MetI25 (1) GlyH99 (1) I27 H31 H50 structural studies of the Rituximab Fab in complex with a peptide Asn (8) Trp (4), Tyr (4) corresponding to its epitope on CD20. With the phage display GluI29 (4) AsnH53 (4) ArgI36 (7) TrpH31 (1), AspH55 (5), SerH57 (1) method, the Rituximab binding epitope was mapped to two seg- LysI38 (3) GluH63 (3) ments 170ANPS173 and 182YCYSI186 on the CD20 extracellular SerI39 (1) SerL92 (1) loop. The structural studies of the Ag/Ab complex indicate that the I40 L91 Gly (2) Ser (2) 182YCYSI186 motif is not directly involved in the interaction with LeuI42 (10) TyrL93 (4), TrpH31 (5), SerH57 (1) TyrI43 (16) TyrL93 (7), TrpH31 (1), AspH97 (6), GlyH99 (2) the Ab; instead it plays a critical role in the formation of a disulfide 170 173 LeuI45 (1) ArgL90 (1) bond to define the proper geometry of the ANPS motif so that ThrI47 (11) TyrL31 (4), AspL49 (5), LysL52 (1), TyrH100 (1) the latter can be recognized by Rituximab (57, 58). GlyI48 (3) TyrL31 (2), LysL52 (1) AspI56 (13) SerL30 (2), TyrL31 (6), ArgL90 (5) Molecular mechanism of the inhibition of the IL-2 signal I57 L29 L90 L91 Asn (6) Arg (1), Arg (3), Ser (2) pathway by basiliximab IleI118 (2) TyrH50 (1), AsnH53 (1) HisI120 (5) ArgH29 (4), TyrH30 (1) Structural analysis of the basiliximab Fab/IL-2Ra complex and its There are a total of 138 van der Waals contacts. Numbers in parentheses refer to comparison with the crystal structures of the IL-2/IL-2Ra and IL- the number of van der Waals contacts. 2/IL-2Rabgc complexes provide insights into the molecular 1366 STRUCTURE OF BASILIXIMAB Fab IN COMPLEX WITH IL-2Ra mechanism of the inhibition of the IL-2 signal pathway by basi- lograft rejection in organ transplantation. The biological and liximab. In the crystal structures of the IL-2/IL-2Ra and IL-2/IL- structural data have shown that activation of IL-2 is initiated by the 2Rabgc complexes, IL-2Ra makes interactions with IL-2 mainly binding of IL-2Ra and further facilitated by the binding of IL-2Rb I25 I42 I43 via the D1 domain (24–26). Specifically, Met , Leu , and Tyr and gc (24–27). Among the three receptors, only IL-2Ra is IL-2– of IL-2Ra form a hydrophobic surface patch to interact with specific, whereas IL-2Rb and gc are shared with other cytokines residues Phe42 and Leu72 of IL-2 (24). Surrounding the hydro- and are less specific. Therefore, IL-2Ra is a more suitable drug phobic patch, several hydrophilic residues of IL-2Ra interact with target for blocking the IL-2 signal pathway. Basiliximab and da- IL-2 by forming numerous hydrogen bonds, salt bridges, and van clizumab are two IL-2Ra–specific mAb drugs that have been used der Waals contacts. In total, there are 21 residues of IL-2Ra in clinical applications for the prevention of allograft rejection in participating in the interactions with IL-2, forming 8 hydrogen organ transplantation. bonds, 2 salt bridges, and 100 van der Waals contacts (25). The Although basiliximab binds to IL-2Ra with a high affinity (Kd = interaction interface buries 971.2 A˚ 2 of the solvent-accessible 0.14 nM), the Fab/IL-2Ra complex has a relatively lower shape surface area on IL2-Ra. Structural comparison of these complexes complementarity value of 0.57, suggesting that the paratope of ba- with the basiliximab Fab/ IL-2Ra complex indicates that the resi- siliximab could be modified to have higher shape and chemical dues of IL-2Ra responsible for the interactions with IL-2 overlap complementarities with IL-2Ra and thus achieve a tighter binding largely with the epitope of basiliximab. Fifteen out of the 21 resi- and better specificity. In our previous structural studies of the Rit- dues (71.4%) are involved in the interactions with the basiliximab uximab Fab in complex with a CD20 peptide, we have proposed Fab, and ∼641.6 A˚ 2 (66.1%) of the buried solvent-accessible sur- some mutations on the CDR loops of the Ab that might be able to face area on IL-2Ra is covered by the Ab (Fig. 3A). Especially, increase its binding affinity (57). Recently, these suggestions have Downloaded from the hydrophobic patch formed by residues MetI25, LeuI42, and been validated, and the results have shown that some of the muta- TyrI43 of IL-2Ra plays a key role in the binding of both basiliximab tions could substantially improve the affinity (59). Structural anal- and IL-2. Furthermore, the basiliximab binding epitope comprises ysis of the basiliximab Fab/IL-2Ra complex also provides some several other residues besides those involved in the interactions hints for improving the mAb drug. For instance, MetI25 intrudes its with IL-2, and the basiliximab Fab/IL-2Ra interface comprises side chain into the hydrophobic groove at the paratope of basiliximab H99 more hydrophobic and hydrophilic interactions than the IL-2/IL- but forms only one van der Waals contact with Gly . Modeling http://www.jimmunol.org/ 2Ra interface. These results may explain in part the biochemical studies show that a substitute for GlyH99 with a slightly larger hy- data that the binding affinity of basiliximab to IL-2Ra (0.14 nM) is drophobic residue, such as alanine, valine, leucine, or isoleucine, ∼71-fold higher than that of IL-2 to IL-2Ra (10 nM) (27). could form more hydrophobic interactions with MetI25 and the sur- Therefore, the binding of basiliximab to IL-2Ra would compete rounding residues LeuI2, LeuI42, and TyrI43 without steric conflict, for IL-2 binding to the receptor. In the presence of a sufficient thus making the interaction interface gain better shape and chemical amount of basiliximab, the IL-2 binding sites of IL-2Ra would be complementarities. Moreover, because LysL52 forms a hydrogen blocked, and thus IL-2 signaling cannot be initiated and executed bond with the main-chain carbonyl of GlyI48 and a weak salt bridge due to the lack of binding of IL-2 to IL-2Ra and the formation of with GluI22 (4.3 A˚ ), mutation of LysL52 to arginine would make it

I22 L55 by guest on September 27, 2021 the functional IL-2/IL-2Rabgc complex (Fig. 3A,3B). This pro- form a more favorable salt bridge with Glu . In addition, Ser is vides the molecular mechanism of the inhibition of IL-2 signaling located near the interaction interface but has no direct contact with by basiliximab. IL-2Ra; however, substitution of this residue with a large positively charged residue such as arginine would make it form a favorable salt Implications for drug development bridge with GluI1 (within 3.5 A˚ ). In other words, mutations of the IL-2 signaling plays an important role in the activation of immune aforementioned residues at the paratope of basiliximab could in- responses against foreign intrusion. Blockage of this signal path- troduce additional favorable interactions between the mAb and IL- way could provide therapeutic benefits to reduce or eliminate al- 2a and thus improve the binding affinity and specificity of the mAb.

FIGURE 3. Molecular mechanism of the inhibition of IL-2 signaling by basiliximab. A, Overlay of the basiliximab Fab/IL-2Ra complex onto the IL-2/IL-

2Rabgc complex based on superposition of IL-2Ra. The basiliximab Fab is shown with a molecular surface with the L chain in light yellow and the H chain in light green. IL-2Ra (as in the Fab/IL-2Ra complex) is shown with a ribbon model with the D1 domain in cyan and the D2 domain in purple. IL-2Rb is colored in blue, and gc is colored in orange. IL-2 is colored in red. The binding site of IL-2Ra for basiliximab overlaps largely with the binding site for IL-2. B, A schematic diagram showing the molecular mechanism of the inhibition of IL-2 signaling by basiliximab. The IL-2 signal pathway is initiated by the binding of IL-2 to IL-

2Ra and further transmitted by the binding of IL-2Rb and gc to form the biologically active complex IL-2/IL-2Rabgc. The binding of basiliximab to IL-2Ra blocks its binding with IL-2 and prevents the formation of the IL-2/IL-2Rabgc complex, thus inhibits the IL-2 signal pathway. The Journal of Immunology 1367

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