Structural Biology and Crystallization Communications Crystallization and Preliminary X-ray Diffraction Analysis of the Complex between a Human Anti-interferon Antibody Fragment and Human Interferon A-2a

Recombinant human interferon-2A (rhIFN-2A) has been crystallized in complex with the recombinantly produced Fab fragment of a therapeutic mono-clonal antibody (MEDI545; IgG1/) which targets several human interferon subtypes. This constitutes the first reported crystals of a human type I interferon bound to an antibody. The orthorhombic crystals belonged to either space group I222 or I2 1 2 1 2 1 , with unit-cell parameters a = 134.82, b = 153.26, c = 163.49 A ˚. The diffraction of the crystals extended to 3.0 A ˚ resolution. The asymmetric unit contained two Fab–rhIFN-2A complexes. This corresponded to a crystal volume per protein weight (V M) of 3.02 A ˚ 3 Da À1 and a solvent content of 59.3%. The corresponding three-dimensional structure is expected to shed light on the mechanism of action of MEDI545 and the molecular basis of its specificity.


Introduction
Type I interferons (IFNs) are a family of cytokines that are expressed from over 15 functional genes and include IFN-, IFN-, IFN-, IFN-, IFN-", IFN-and IFN-!. Interferons, in particular IFN-and IFN-, have historically been characterized in terms of their role in modulating the responses of mammalian hosts to infections (Isaacs & Lindenmann, 1957;Gresser et al., 1976;Alsharifi et al., 2008). However, it is becoming increasingly clear that they also play a key role in immune regulation and autoimmune diseases Theofilopoulos et al., 2005). The IFN-family alone comprises at least 13 different subtypes (Foster & Finter, 1998) and is thought to play a role in systemic lupus erythematosus (SLE), type I diabetes, Sjö gren's syndrome and thyroid diseases (Selmi et al., 2006). This attractive targeting potential led to the development by MedImmune of MEDI545, a fully human monoclonal antibody which exhibits binding to multiple IFN-subtypes. MEDI545 is currently undergoing several human clinical trials for various indications such as lupus, chronic plaque psoriasis, dermatomyositis and poliomyelitis (http://www.clinicaltrials.gov).
In an effort to better understand the molecular basis of the recognition of IFN-molecules by MEDI545, we set out to solve the X-ray crystal structure of the complex between the Fab fragment of this antibody and human IFN--2A. Three type I human interferon structures have previously been determined using either X-ray crystallography or NMR. These correspond to human IFN--2a, IFN--2b and IFN- (Radhakrishnan et al., 1996;Karpusas et al., 1997;Klaus et al., 1997). Here, the first reported diffracting crystals of a human type I interferon bound to an antibody are described and characterized.

Expression and purification of MEDI545 Fab and rhIFN-a-2a
The heavy and light chains of the Fab fragment of MEDI545 (IgG1/) were cloned into a mammalian expression vector. More precisely, each chain was placed under the control of its own human cytomegalovirus major immediate early (hCMVie) promoter and enhancer (described in Boshart et al., 1985). Each of those two genes also incorporated an SV40 poly-A sequence to allow proper processing of its 3 0 end. In this system, a human 1 chain truncated at position Lys235 (according to the Kabat numbering convention; Kabat et al., 1991) is secreted along with a human chain (Oganesyan et al., 2008). The construct was then transiently transfected into human embryonic kidney (HEK) 293 cells using Lipofectamine (Invitrogen, Carlsbad, California, USA) and standard protocols. MEDI545 Fab was typically harvested 72, 144 and 216 h post-transfection. Conditioned media containing the soluble Fab fragment were then dialyzed against 50 mM sodium acetate pH 5.2, 20 mM NaCl and purified using a HiTrap HP SP column in a NaCl gradient according to the manufacturer's instructions (GE Healthcare, Piscataway, New Jersey, USA). Purified MEDI545 Fab, typically >95% homogeneous as judged by SDS-PAGE, was then concentrated to approximately 30 mg ml À1 .
High-quality nearly 100% homogeneous Escherichia coli-expressed recombinant human interferon -2A (rhIFN--2A) was purchased directly from Potomac Affinity Proteins LLC (Rockville, Maryland, USA). The cytokine's mature sequence corresponded to residues 24-188 of entry No. P01563 of the Swiss-Prot protein database (http:// www.expasy.org) and included two extra threonine and serine residues on its N-terminal end. MEDI545 Fab and rhIFN--2A were mixed in a 1.2:1 molar ratio. The resulting mixture was then adjusted to approximately 10 mg ml À1 . The complex was further purified by size-exclusion chromatography using a Superdex S200 column (GE Healthcare) equilibrated with 20 mM sodium acetate pH 5.2, 20 mM NaCl at a flow rate of 0.5 ml min À1 . After separation from excess MEDI545 Fab, the complex was concentrated to 6.5 mg ml À1 using an Amicon YM-30 concentrator (Millipore, Billerica, Massachusetts, USA).

Crystallization screening and optimization
Sitting-drop crystallization experiments were initially set up in 96-well plates with conical flat-bottomed drop compartments (Corning 3785; VWR, West Chester, Pennsylvania, USA) using a Phoenix crystallization robot (Art Robbins, Sunnyvale, California, USA). Favorable conditions were initially identified using the following commercially available crystallization screens: Crystal Screen HT, Index Screen, SaltRx (Hampton Research, Aliso Viejo, California, USA) and ProPlex (Molecular Dimensions, Apopka, Florida, USA). For further optimization purposes, a commercially available kit was used (Additive Screen HT; Hampton Research). More particularly, in screening mode the reservoirs and drop compartments of the 96-well plates were filled with 99 and 0.3 ml, respectively, of the various screen solutions using the Phoenix robot. 0.3 ml of the MEDI545 Fab-rhIFN--2A complex at a concentration of 6.5 mg ml À1 was then added to the drop compartment. In optimization mode, the reservoirs were first filled with 80 ml screen solution. 20 ml of the various additives were then added and the reservoirs were subjected to five cycles of mixing by aspiration/dispensing. 0.3 ml of each reservoir solution was then mixed with 0.3 ml of the MEDI545 Fab-rhIFN--2A complex at a concentration of 6.5 mg ml À1 in the drop compartment. Diffraction-quality crystals were grown in hanging drops once optimal conditions had been identified (see x3).

X-ray diffraction data collection and processing
Diffraction data were collected from a single crystal at the Center for Advanced Research in Biotechnology (CARB, University of Maryland Biotechnology Institute, Rockville, Maryland, USA) using a MicroMax-007 rotating-anode generator fitted with an R-AXIS IV ++ imaging plate (Rigaku/MSC, The Woodlands, Texas, USA). The crystal was harvested using a litho-loop and flash-cooled in a liquidnitrogen stream using an X-stream 2000 cryogenic cooler (Rigaku/ MSC). Prior to this step, cryoprotection was achieved by soaking the crystal in 200 mM NaI, 8 mM NiCl 2 .6H 2 O, 80 mM Tris-HCl pH 8.5, 16%(w/v) polyethylene glycol monomethyl ether 2000, 20% glycerol. 240 consecutive images were collected using an oscillation range of 0.5 , a crystal-to-detector distance of 250 mm and an exposure time of 600 s. The diffraction images were integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997).

Results and discussion
Crystals of less than 10 mm in size and of tetragonal bipyramidal shape appeared during the screening process with Crystal Screen HT under condition H9 [0.01 M NiCl 2 .6H 2 O, 0.1 M Tris-HCl pH 8.5, 20%(w/v) polyethylene glycol monomethyl ether 2000] as seen in Fig. 1(a). During the optimization process, crystals of similar morphology appeared in almost all of the conditions of the Additive Screen. However, the largest crystals were obtained when using additives B8 ( Fig. 1(b).
SDS-PAGE analysis of these crystals confirmed that they indeed contained the expected complex formed by MEDI545 Fab and the recombinant human interferon -2A (see Fig. 2). Incidentally, the SDS-PAGE profile of MEDI545 Fab also demonstrated the presence of the expected interchain disulfide bond between C/Cys214 and C H 1/Cys233 (Kabat numbering;Kabat et al., 1991) owing to its differential migration pattern under reducing and nonreducing conditions (see Fig. 2).
The diffraction limit of the crystal was 3.0 Å (see Fig. 3). The possible space groups were determined to be either I222 or I2 1 2 1 2 1 , with unit-cell parameters a = 134.82, b = 153.26, c = 163.49 Å . The asymmetric unit contained two MEDI545 Fab-rhIFN--2A complexes. This corresponded to a V M of 3.02 Å 3 Da À1 and a solvent content of 59.3%. Data-collection statistics are shown in Table 1. Structure determination using molecular replacement is currently under way.

Figure 2
SDS-PAGE profile of a dissolved crystal of the MEDI545 Fab-rhIFN--2a complex under reducing and nonreducing conditions. The 4-20% gradient gel (BioRad, Hercules, California, USA) was stained with Bio-Safe Coomassie Stain (BioRad). It shows rhIFN--2a migrating at around 15 and 20 kDa under nonreducing and reducing conditions, respectively. This difference is likely to be attributable to the presence of two internal disulfide bonds, the reduction of which alters the compactness (and thus the migration) of the molecule. The same argument can be applied to the MEDI545 Fab fragment, which migrated slightly under its expected molecular weight of 50 kDa under nonreducing conditions. As expected, its heavy and light chains both migrated at 25 kDa under reducing conditions.