doi:10.1016/j.jmb.2011.08.018 J. Mol. Biol. (2011) 413, 390–405

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

Structural and Functional Characterization of anAgonistic Anti-Human EphA2 Monoclonal Antibody

Li Peng†, Vaheh Oganesyan†, Melissa M. Damschroder,Herren Wu⁎ and William F. Dall'Acqua⁎Department of Antibody Discovery and Protein Engineering, MedImmune, One MedImmune Way, Gaithersburg,MD 20878, USA

Received 23 June 2011;received in revised form1 August 2011;accepted 9 August 2011Available online16 August 2011

Edited by I. Wilson

Keywords:cancer;epitope;structure;mimicry;mutagenesis

*Corresponding authors. E-mail addwuh@medimmune.com; dallacquaw† L.P. and V.O. contributed equalAbbreviations used: CDR, comple

determining region; CDRH3, third hECD, extracellular domain; Eph, eryproducing hepatoma; EphA, ephrinephrin type B receptor; Fab, fragmeFBS, fetal bovine serum; HEK, humHRP, horseradish peroxidase; KI, knout; LBD, ligand-binding domain; mantibody; NCS, non-crystallographiphosphate-buffered saline; PDB, Proroom temperature.

0022-2836/$ - see front matter © 2011 E

We report here the three-dimensional structure of human ephrin type Areceptor 2 (EphA2) bound to the Fab (fragment antigen binding) of anagonistic human antibody (1C1; IgG1/κ). The structure of the correspond-ing complex was solved at a resolution of 2.5 Å using molecularreplacement and constitutes the first reported structure of a human ephrinreceptor bound to an antibody. We have also defined the correspondingfunctional epitope using a mutagenesis-based approach. This studyrevealed discrete structural features that determine the fine specificity of1C1 to EphA2. Our data also provided a molecular basis for 1C1 mechanismof action. More precisely, we propose that its agonistic, internalizingproperties are the result of ligand mimicry by the third heavy-chaincomplementarity-determining region of 1C1. Because EphA2 is animportant contributor to cancer formation and progression, these findingsmay have implications for designing the next generation of anti-tumortherapies.

© 2011 Elsevier Ltd. All rights reserved.

Introduction

The Eph (erythropoietin-producing hepatoma)family of receptor tyrosine kinases and their ligands,the ephrins, play a critical role in modulating cell–

resses:@medimmune.com.ly to this work.mentarity-eavy-chain CDR;thropoietin-typeA receptor; EphB,nt antigen binding;an embryonic kidney;ock-in; KO, knock-Ab, monoclonalc symmetry; PBS,tein Data Bank; RT,

lsevier Ltd. All rights reserve

cell interactions and cell migration.1–3 Eph receptorsare type I transmembrane proteins and are dividedinto two classes. These two classes (A and B) arebased on sequence conservation and ligand-bindingpreferences1,4 and overall consist of at least 14members in mammals.5 Membrane-attached ephrinligands are also divided into two types, namely,ephrin A and ephrin B, and together consist of atleast nine members.1,6 The corresponding ligand/receptor interactions are promiscuous both withinand outside of a given class.7 Eph receptors exhibit amodular structure with the extracellular portion[extracellular domain (ECD)] consisting of anamino-terminal ligand-binding domain (LBD), acysteine-rich region, and two fibronectin type IIIrepeats.8 It is believed that a high-affinity receptor/ligand interface in LBD mediates the correspondinginteractions between cells.9,10

Several crystal structures of Eph/ephrin com-plexes have been reported [most notably ephrintype A receptor 2 (EphA2)/ephrin A5, EphA2/ephrin A1, ephrin type B receptor 4 (EphB4)/ephrin

d.

391Ligand Mimicry by an Anti-Human EphA2 Antibody

B2, EphB2/ephrin B2, EphB2/ephrin A5, EphA4/ephrin B2, and EphA4/ephrin A2].9,11–17 Thesestudies have provided crucial insights into themolecular basis of receptor/ligand interactions andof their promiscuity. They have also suggested thepresence of additional low-affinity receptor/ligandinterfaces that are thought to mediate the dimeriza-tion of two ligand/receptor dimers to form atetramer.6,15,18 These tetramers can then furthergrow into larger clusters, a prerequisite for Ephreceptor activation.18,19 Of note, the insertion of anextended ephrin loop into a channel on the Ephreceptor LBD has been identified as amajor commonstructural feature of Eph/ephrin complexes.Among all Eph receptors, EphA2 has been exten-

sively studied owing to its key role in the formationand progression of various cancers.20,21 More pre-cisely, EphA2 is highly expressed in multiple types ofcancers including ovarian, esophageal, breast, pros-tate, lung, renal, and glioblastomamultiforme.20,22–25

Overexpression of EphA2 is also often associatedwith poor prognosis of the disease.23–25 As such,EphA2 constitutes an attractive target for therapeuticantibodies. Indeed, monoclonal antibodies (mAbs)targeting EphA2 have been shown to exhibit signi-ficant anti-tumor effects.26–29 Several of these anti-EphA2 mAbs inhibited malignant cell growth invarious in vitro and in vivo preclinical models byinducing autophosphorylation, internalization, anddegradation of the receptor.26,27,29 However, amolecular understanding of their fine specificity andfunctional properties is limited by the lack of relevantstructural data.Taking advantage of the ability of EphA2 to

undergo antibody-directed internalization, we de-veloped the anti-human EphA2 fully human mAb1C1 for an antibody drug conjugate approach.EphA2 is an attractive target for such an approachdue to its low-level expression in normal tissues andbroad expression in tumors. When conjugated to themicrotubule inhibitor monomethyl auristatin phe-nylalanine, 1C1 inhibited the growth of EphA2-expressing tumors through its ability to inducerapid internalization and deliver the cytotoxicpayload.30 Our present efforts are aimed at describ-ing and understanding the molecular basis ofhuman EphA2 recognition by 1C1. For this purpose,we have solved the X-ray crystal structure of thecomplex between the Fab (fragment antigen bind-ing) of this antibody and the LBD of human EphA2.This constitutes the first reported structure of ahuman ephrin receptor bound to an antibody. Tofurther identify the molecular determinants of thisinteraction, we have mapped the correspondingepitope using a mutagenesis-based approach. Takentogether, our data have allowed us to describe indetail the 1C1/EphA2 interface. They have alsoprovided a molecular understanding of 1C1 agonis-tic properties.

Results and Discussion

General properties of 1C1 and its epitope

1C1 is a human anti-human EphA2 mAb derivedfrom the screening of a human antibodyphage libraryon human EphA2.30 Its specificity profile excludesother related human Eph receptors such as EphA1,EphA2, and EphA4.30 1C1 was selected for anantibody drug conjugate approach due to its abilityto induce rapid phosphorylation, internalization, andsubsequent degradation of EphA2. Though otheranti-human EphA2mAbs have been generated, mostdid not exhibit such a profile. As shown in Fig. 1a–c,their activity spectrum ranged from no (1C9) to weak(3F2) phosphorylation, internalization, and degrada-tion. Among these, only 1C1 efficiently mediatedthese properties (Fig. 1a–c). This difference cannoteasily be attributed to the corresponding bindingparameters (shown in Table 1) as (i) 3F2 and 1C1exhibited a nearly identical association rate; (ii) 1C1,3F2, and 1C9 exhibited similar dissociation rates(within twofold); and (iii) 1C1 and 3F2 exhibited asimilar dissociation constant (also within twofold).Thus, we speculated that 1C1 recognizes a uniqueepitope on human EphA2 that can account for itsunique functional properties. This hypothesis issupported by BIAcore binding competition studiesshowing that 1C1, 1C9, and 3F2 do not compete withone another and recognize distinct epitopes onhuman EphA2 (Fig. 1d).Interestingly, Fab 1C1 also exhibited a significant

ability to induce phosphorylation and degradationof EphA2, though not as efficiently as its IgGcounterpart on a molar basis (Fig. 1a and b). Inaddition, both monovalent Fab 1C1 (over N98.5%monomer as judged by size-exclusion chromatogra-phy; data not shown) and bivalent mAb 1C1 wereinternalized into PC-3 cells within 1 h of incubation(Fig. 1c). Binding measurements indicated that theaffinity of Fab 1C1 was ∼200-fold lower than that ofmAb 1C1, a difference primarily driven by a fasterdissociation rate for Fab 1C1 (Table 1). This likelyreflects an avidity effect for mAb 1C1. Indeed, whenthe assay format was inverted so that mAb 1C1 wasimmobilized on the surface of the ProteOn chip, thebinding kinetics of Fab and mAb 1C1 werecomparable (data not shown). The intriguing func-tional properties of 1C1 prompted us to furtherdecipher its epitope and mechanism of binding.

1C1 epitope is contained in EphA2/LBD

Human EphA2 ECD exhibits a modular structureconsisting of an LBD, a cysteine-rich region, and twofibronectin type III repeats (Fig. 2a). In order todecipher the 1C1 epitope, we carried out a compe-tition binding ELISA of this antibody to several

Table 1. Affinity measurement for the binding of mAbs1C9, 3F2, and 1C1 and Fab 1C1 to sEphA2/ECD

Molecule

Associationrate (kon)

a

(×106 M−1 s−1)Dissociation rate(koff)

a (×10−4 s−1) Kd (pM)b

1C9 3.26±0.33 2.08±0.46 63.8±12.43F2 0.60±0.07 2.43±0.31 407±751C1 0.61±0.05 4.98±0.44 809±65Fab 1C1 0.14±0.01 200c 140,000±13,000

Measurements were carried out using a ProteOn instrument asdescribed in Materials and Methods.

a Kinetic parameters represent the average of four individualmeasurements. Errors were estimated from the correspondingstandard deviations.

b Kd values were calculated from the corresponding individualkinetic parameters and averaged. Errors were estimated from thecorresponding standard deviations.

c No standard deviation could be calculated as identical valueswere measured in four individual measurements.

4°C

37°C

3F21C9 Irrelevant IgG1C1 Fab 1C1(c)

(d)

0

200

400

600

RU 1C1 vs. 1C9

1C9 vs. 1C11C1

1C9

1C1 + 1C9

Time (sec) Time (sec) Time (sec)

0

100

200

300

400

1C1

3F2

1C1 + 3F2

1C1 vs. 3F2

3F2 vs. 1C1

0

200

400

600

1C9 vs. 3F2

3F2 vs. 1C93F2

1C9

1C9 + 3F21st injection

2nd injection

1st injection

2nd injection

1st injection

2nd injection

(a)

Phosphorylated EphA2Total EphA2

[mAb] µg/ml

0 20 20 20 20 100 60 20

(b)

EphA2

GAPDH

0 20 20 20 20 6.7 20[mAb] µg/ml

RU

RU

0200

400600

8001000 0

200400

600800

1000 0200

400600

8001000

Fig. 1. Induction of EphA2 phosphorylation (a) and degradation (b) by anti-human EphA2 antibodies. mAb and Fab1C1 trigger efficient phosphorylation and degradation of human EphA2, whereas mAbs 3F2 and 1C9 exhibit only weak tono activity. The agonistic activity of these antibodies was evaluated in PC3 cells as described in Materials and Methods.Data shown are representative of two independent series of experiments. (c) Internalization of human EphA2 followingincubation with anti-human EphA2 antibodies. mAb and Fab 1C1 efficiently internalize human EphA2, whereas mAbs3F2 and 1C9 induce only weak to no internalization at 37 °C. No internalization of the antibodies was observed at 4 °C.Data shown are representative of three independent series of experiments and were generated as described in Materialsand Methods. (d) Competition binding of anti-human EphA2 antibodies to sEphA2/ECD. 1C1 binds to an epitopedistinct from that of 1C9 and 3F2. Data shown are representative of two independent series of experiments.

392 Ligand Mimicry by an Anti-Human EphA2 Antibody

ephrin ligands. As shown in Fig. 2b, mouse ephrinsA1/A2 and human ephrins A3/A4/A5 all blockedbinding of 1C1 to sEphA2/ECD. This indicated that1C1 binds to a region on human EphA2 thatoverlaps with the ephrin-binding site in EphA2/LBD. This was further confirmed by assessing thebinding of 1C1 to a truncated human EphA2 variantencoding only the LBD portion (sEphA2/LBD;Fig. 2a). Here, we found that 1C1 bound tosEphA2/LBD with a Kd of 810 pM, a value nearlyidentical with that of sEphA2/ECD (809 pM;Table 2). Finally, as shown in Fig. 2c, 1C1 did not bindto anEphA2variantwhose LBDportionwas replacedwith that of human EphA4 in the context of thewholeEphA2 extracellular portion (sEphA4/LBD-EphA2;Fig. 2a). Taken together, our data clearly indicatedthat the 1C1 epitope is contained in EphA2 LBD.

Refinement of the 1C1 epitope

In order to refine the 1C1 epitope on humanEphA2, we designed a series of so-called “knock-

out” and “knock-in” (KO and KI, respectively)variants. In these, short segments of EphA2 LBD

Table 2. Affinity measurement for the binding of 1C1 tosEphA2/ECD and sEphA2/LBD

MoleculeAssociation rate (kon)

a

(×106 M−1 s−1)Dissociation rate(koff)

a (×10−4 s−1)Kd

(pM)b

sEphA2/ECD

0.61±0.05 4.98±0.44 809±65

sEphA2/LBD

0.61±0.08 4.90±1.52 810±296

Measurements were carried out using a ProteOn instrument asdescribed in Materials and Methods.

a Kinetic parameters represent the average of four individualmeasurements. Errors were estimated from the correspondingstandard deviations.

b Kd values were calculated from the corresponding individualkinetic parameters and averaged. Errors were estimated from thecorresponding standard deviations.

(b)(a)

LBD

sEphA4/LBD-EphA2

0.0

0.5

1.0

1.5

2.0

Ab

sorb

ance

EphA4EphA2

Ephrin concentration (ng/ml)

sEphA2/LBD

Fc

sEphA2/ECD

Fc

mEphA2/LBD

mEphA4/LBD

Inh

ibit

ion

of

1C1

bin

din

g t

o E

ph

A2

(%)

0

0.01 0.1 1 10 10

010

00

1000

0

1000

00

1000

000

20

40

60

80

100

Mouse EphrinA1Mouse EphrinA2Human EphrinA3Human EphrinA4Human EphrinA5BSA

(c)

Fig. 2. (a) Schematic representation of the domain arrangement of human EphA2 and some variants thereof. (b)Competition binding of 1C1 and various human and murine ephrin ligands to sEphA2/ECD. 1C1 competes with allephrins for binding to sEphA2/ECD. Standard deviations are indicated by error bars and represent triplicatemeasurements within the same experiment. Data shown are representative of three independent series of experiments. (c)Direct binding of 1C1 to sEphA2/ECD, sEphA2/LBD, and sEphA4/LBD-EphA2 variants by ELISA. EphA2 LBD containsthe 1C1 epitope. Standard deviations are indicated by error bars and represent triplicate measurements within the sameexperiment. Data shown are representative of two independent series of experiments.

393Ligand Mimicry by an Anti-Human EphA2 Antibody

were replaced with their EphA4 counterparts (KO)and vice versa (KI). Fragments to be substituted werechosen according to the arrangement of secondarystructural elements in human EphA2 LBD (Fig. 3a).A summary of these variants is presented in Fig. 3band c. All KO and KI variants were expressed on thesurface of human embryonic kidney (HEK) 293Fcells, and binding of 1C1 (whose variable regionssequences are shown in Fig. 3d) was assessed usingflow cytometry (Fig. 4).Although binding of 1C1 to the KO A→E variant

was slightly decreased, data suggested that humanEphA2 regions spanning (i) A up to (and including)E strands and (ii) the end of K up to (and including)M strands were not majorly involved in 1C1 binding(see KO A→E, KO D→E, KO L→M, KO L, and KOM variants). The human EphA2 variant encodingthe end of E up to (and including) H strands ofhuman EphA4 (KO F→H) failed to express;however, individual variants within this segmentexpressed and bound well to 1C1 (see KO G and KO

H variants). The variant where the first part of thissegment was replaced by EphA4 residues (KO F)could not be expressed as well; however, its KIcounterpart (KI F), which substituted the EphA4 end

394 Ligand Mimicry by an Anti-Human EphA2 Antibody

of E up to (and including) F strand by EphA2residues, expressed well but was not recognized by1C1. Finally, the KO G–H variant exhibited a slightdecrease in its binding to 1C1, suggesting that thisloop might play a minor role in the correspondinginteraction. Taken together, these data allowed us topreliminary rule out the human EphA2 regionspanning the (i) A up to (and including) G strands,(ii) H strand, and (iii) end of K up to (and including)M strands as major potential epitopes for 1C1.Only upon replacing (i) the EphA2 region span-

ning the end of H up to (and including) K strands(KO J→K variant) or (ii) the EphA2 loop connectingthe J and K strands (KO J–K variant) with those ofEphA4 was the binding of 1C1 abolished. Inaddition, the region spanning the end of H up to(and including) J strands as well as the K strand didnot appear to be involved in 1C1 binding (see KO Jand KO K variants). Therefore, we concluded thatthe loop connecting the J and K strands containedresidues critical for interacting with 1C1. Additionalmutations within this loop allowed us to identifyresidues F132, E133, and A134 as critical for 1C1binding (see the KO F132→A134 variant).We noted however that knocking in the J–K

connecting loop of EphA2 into EphA4 only mini-mally restored binding of 1C1 (see the KI J–Kvariant). This suggested that the 1C1 epitope mightinclude additional regions not identified using theKO variants described in the previous paragraphs.Interestingly, analysis of publicly available three-dimensional structures of human EphA2 (see below)suggested that the J–K connecting loop is spatiallyclose to the G–H connecting loop and G and Mstrands. This prompted us to further probe theseregions by pursuing a combinatorial approachwhere two or more of these segments weresubstituted together. We found that replacing (i)the EphA2 G–H connecting loop along with its G orM strand with those of EphA4 (see KO G+G–H andKOG–H+Mvariants, respectively) or (ii) the EphA2G and M strands together with those of EphA4 (seeKO G+M) significantly reduced 1C1 binding whencompared with the variants in which each of thesesegments was individually substituted. Replacingall three segments together (KO G+G–H+M)dramatically reduced 1C1 binding when comparedwith KO G+G–H, KO G–H+M, and KO G+Mvariants. We also noted that substituting the EphA4G–H connecting loop along with its G andM strandswith those of EphA2 (KI G+G–H+M variant) didnot significantly restore 1C1 binding. However,adding EphA2 J–K loop to this variant (KI G+G–H+J–K+M variant) significantly enhanced 1C1binding when compared with the KI G+G–H+Mand KI J–K variants. Thus, we conclude that the 1C1epitope is conformational and may comprise the G–H and J–K connecting loops as well as the G and Mstrands.Within this epitope, amino acids F132, E133,

and A134 in the J–K connecting loop play a crucialrole. However, we note that the high amino acidsequence homology between human EphA2 andEphA4 may complicate our data interpretation. Forinstance, the sequence of the M strand is 73%identical between the two Eph receptors. Thus,although no significant effect on 1C1 binding wasseen upon substituting this strand (KO M variant),this piece of data alone does not formally allow us torule out the presence of an important epitope withinit. Finally, we could not exclude the possibility thatsome EphA2/A4 variants exhibited an incorrect foldin or near the 1C1 epitope region, thereby losingtheir binding capacity. An overall inability tocorrectly fold may also account for the apparentlack of expression of some constructs (such as KO Fand KO F→H). Therefore, in light of the inherentlimitations of such a mutagenesis-based approach,we embarked on a structural analysis of the 1C1/EphA2 LBD complex.

Determination of the three-dimensionalstructure of the Fab 1C1/EphA2 LBD complex

For all structural studies, recombinant EphA2LBD (amino acids 1 to 176 of the mature peptide)and Fab 1C1 were expressed, purified, complexed,and co-crystallized as previously described.32 Acomplete X-ray diffraction data set was collected,and the structure of the complex was determined toa resolution of 2.5 Å using Phaser33 and MolRep34

molecular replacement programs from the CCP4(Collaborative Computational Project 4) suite. Themodels of human EphA2 and Fab 1C1 were builtfrom Protein Data Bank (PDB35) entries 3C8X and1DFB, respectively. Prior to molecular replacement,all amino acids in the complementarity-determiningregions (CDRs) of the model were removed. Theasymmetric unit of the orthorhombic C2221 cell withparameters a=78.93 Å, b=120.79 Å, and c=286.20 Åcontained two Fab 1C1/EphA2 complexes. The firstFab 1C1 and EphA2 molecules in the asymmetricunit could be found using either Phaser or MolRep.However, the second Fab 1C1 molecule could onlybe found after splitting it into its individual FV(composed of VH and VL domains) and Fb (com-posed of CH1 and CL domains) components. Thedifference between the two Fab molecules in theasymmetric unit was approximately 17° in theirelbow angle (individually estimated at 174° and157° for the first and the second Fab molecule,respectively).36 The second human EphA2 moleculecould not be identified by either molecular replace-ment technique, despite the presence of empty spacein the crystal packing and electron density. Thus, apartial model was built without the second EphA2molecule and underwent refinement usingRefmac534 and manual rebuilding with the “O”program.37 Once completed, a traceable electron

395Ligand Mimicry by an Anti-Human EphA2 Antibody

density for the second EphA2 molecule emergedand allowed the placement of amino acids 28–58,74–96, 121–142, and 163–170. These residues

Fig. 3. (a) Amino acid alignment of human EphA2 (NationaNP004422) and EphA4 (National Center for Biotechnology Instructural elements (β-strands) are depicted as arrows.13 1C1studies are shown in blue boxes. Residues that significantlyEphA4 counterparts are highlighted in magenta. Residues inthree-dimensional structure of the corresponding complex are sadopt an α-helical conformation upon binding to Fab 1C1 are iof KO and KI variants. Thirty chimeric variants were const(orange) into EphA2 (black; KO) or of human EphA2 intoconnecting loops are represented as straight lines. Stars areintroduced. (c) Structural representation of KO and KI variantswell as portions thereof are shown in black and orange, respectCDR2, and CDR3 as defined by Kabat et al. are boxed.31

appeared proximal to the second Fab 1C1 moleculein the asymmetric unit. Density modifications, omitmap calculations, and extensive refinement with

l Center for Biotechnology Information reference sequenceformation reference sequence NP004429) LBD. Secondarypotential epitope regions as determined from mutagenesisreduced binding of 1C1 to EphA2 when changed to theirdirect contact with 1C1 CDRH3 as determined from thehown under red diamonds. J–K loop residues 124–136 thatn boldface. (b) Nomenclature and schematic representationructed by swapping various segments of human EphA4EphA4 (KI). β-Strands are depicted as arrows, whereasshown over regions where individual mutations were

. Human EphA2 (this study) and EphA4 (PDB ID 2WO3) asively. (d) Protein sequences of 1C1 variable regions. CDR1,

Fig. 3 (legend on previous page)

396 Ligand Mimicry by an Anti-Human EphA2 Antibody

Fig. 3 (legend on page 395)

397Ligand Mimicry by an Anti-Human EphA2 Antibody

TLS38,39 (translation/libration/screw) did not yielda better map. The interface between the second Fab1C1 and the second EphA2 molecule was identicalwhen compared with the first Fab 1C1/EphA2complex (within the estimated overall coordinateerror). However, the Fb portion of both Fabmolecules in the asymmetric unit exhibited a non-crystallographic symmetry (NCS) relation differentfrom that of their FV portion. Though different fromeach other, both NCSs were purely rotational andnearly twofold. To choose which fragments of thetwo Fab 1C1 molecules in the asymmetric unitshould be included in NCS restraints, we super-imposed fragments of different lengths usinglsqkab40 to identify the best analogues. The refine-ment process with imposed NCS restraints consis-tently drove the R-factors higher than when onlyTLS was employed; therefore, the structure wasrefined without NCS restraints and converged to afinal R-factor of 0.216 and a free R-factor of 0.271.The refinement statistics are shown in Table 3. Thefinal root-mean-square deviations (r.m.s.d.'s) be-tween the equivalent Cα atoms of the two Fab1C1/EphA2 complexes in the asymmetric unit weretherefore calculated separately for FV (0.9 Å over 238Cα atoms), Fb (0.8 Å over 197 Cα atoms), and EphA2(0.6 Å over 104 Cα atoms).

General description of the Fab 1C1/EphA2complex

A remarkable feature of the Fab 1C1/EphA2complex (Fig. 5a) included the observation thatonly the third heavy-chain CDR of Fab 1C1(CDRH3) made direct intermolecular contacts withEphA2 (using a distance cutoff of 3.5 Å; Fig. 5b). Theburied surface area between Fab 1C1 and EphA2 isabout 680 Å2 with a relatively low shape comple-mentarity of 0.61. For comparison purposes, theshape complementarity between an antibody and itsantigen ranges from approximately 0.64 to 0.68.42

This likely reflects the lone contribution of CDRH3to this interaction. The relatively long CDRH3 loop(19 amino acids) of Fab 1C1 extends into the ephrinbinding channel on the surface of EphA2 (Fig. 5c andd). More precisely, four of the five intermolecular

hydrogen bonds formed between Fab 1C1 andEphA2 involve the main chains of oxygen atoms ofS97, D100, V100B, and G100F in Fab 1C1 CDRH3with the side chains of R79 (G–H loop) and R135(J–K loop) in EphA2 (Fig. 5b). The fifth and shortest(2.6 Å) hydrogen bond is between theOγ atom of S97in Fab 1C1 CDRH3 and the Oɛ2 atom of E133 (J–Kloop) in EphA2 (Fig. 5b). The hydrophobic nature ofthe Fab 1C1/EphA2 interaction is contributed byamino acids G98, Y99, Y100A, V100B, A100C,V100D, A100E, and G100F in CDRH3. Theseresidues are located at the tip of CDRH3 and togetherinteract with residues M42 (strand E); M49 (E–Floop); R79, F84, and P85 (G–H loop); and F132 (J–Kloop) of EphA2. The light chain of Fab 1C1 did notprovide any direct interaction with EphA2. Theshortest distance between Fab 1C1 light chain andEphA2 is at 4.0 Å and involves amino acid acids K50and M31 in Fab 1C1 and EphA2, respectively. Thisdistance exceeds the combined van der Waals radiiof any potentially interacting atoms. This suggeststhat no direct interactions exist between EphA2 andFab 1C1 light chain. Taken together, these data are invery good agreement with our mutational studiesand pointed to the significant role played by EphA2G–H and J–K loops to bind 1C1 (Fig. 3a). Though indirect contact with Fab 1C1, EphA2 residues M42and M49 in the E strand and the E–F loop,respectively, did not appear to be energeticallysignificant when substituted to their EphA4 coun-terpart (see previous section). Conversely, althoughthe G and M strands do not directly interact withEphA2, our mutational analysis revealed theirsignificant energetic role at the 1C1/EphA2 inter-face. This effect is likely mediated through indirect,long-range effects such as maintaining an appropri-ate conformation of EphA2 G–H and/or J–K loops.Thus, a combination of functional (mutagenesisbased) and structural (crystallography based) ap-proaches constitutes an important strategy in pro-viding a complete picture of antibody epitopes andparatopes at the amino acid level.The packing interactions in the crystal are such

that two NCS-related EphA2 molecules (named “E”and “F” thereafter) do not contact one another.However, the two E molecules interact and create a

Fig. 4. Fluorescence-activated cell-sorting analysis of binding of 1C1 to human EphA2 and EphA4 variants transiently expressed on the surface of HEK 293F cells.Expression of those variants was monitored using a cocktail of antibodies recognizing both human EphA2 and EphA4. MFI, mean fluorescence intensity; SSC, sidescatter characteristics. Nomenclature of variants is explained in the text as well as in Fig. 3b and its legend.

398Ligand

Mim

icryby

anAnti-H

uman

EphA

2Antibody

Table 3. Fab 1C1/EphA2 X-ray data model refinementstatistics

Wavelength (Å) 0.9793Resolution limits (Å) 35.88–2.48Space group C2221Cell parameters (Å) a=78.93, b=120.79,

c=286.20Number of collected reflections 223,275Number of unique reflections 43,686Average redundancy 5.3 (4.9)a

Average mosaicity (°) 0.6Completeness (%) 94.5 (73.9)a

Average I/δ(I) 15.7 (2.2)a

Rmerge 0.125 (0.560)a

Rcryst (Rfree) 0.216 (0.271)b

r.m.s.d. bonds (Å) 0.016r.m.s.d. angles (°) 1.59Residues in most favored region of {φ,ψ}

spacec (%)87.3

Residues in additionally allowed region of{φ,ψ} space (%)

12.1

Residues in generously allowed region of{φ,ψ} space (%)

0.6

Number of protein atoms 8819Number of non-protein atoms 43Average B-factor: model/Wilson (Å2) 52/63Average B-factor: Fab 1C1/EphA2/waters

(Å2)51/62/42

The data set was reprocessed since our publishing of thecorresponding preliminary X-ray analysis.32 Therefore, thecurrent table shows some updated values for the X-ray datastatistics.

a Values in parentheses correspond to the highest-resolutionshell.

b Approximately 5% or 2278 reflections were used for Rfreecalculations.

c The Ramachandran plot was produced using PROCHECK.41

399Ligand Mimicry by an Anti-Human EphA2 Antibody

buried surface area of about 500 Å2 that involves sixhydrogen bonds (the so-called F molecules were notfound to make any contact due to the incompletemodel; see above). Five publicly available ligandedEphA2 structures exist and correspond to PDB entries3CZU (ephrin A1 complex with EphA2 LBD12), 3HEI(ephrinA1 complexwithEphA2LBD13), 2X11 (ephrinA5 complex with EphA2 ECD11), 3MBW (ephrin A1complex with EphA2 LBD Cys-rich region12), and3MX0 (ephrin A5 complex with EphA2 ECD12).Comparison of our EphA2 structure with thosecontaining liganded EphA2 LBD did not reveal anylarge differences with r.m.s.d.'s ranging from 0.75 (forPDB ID 3CZU, over 174 Cα atoms) to 0.90 (for PDBID 3HEI, over 173 Cα atoms). Four unligandedEphA2 structures are available and correspond to

Fig. 5. (a) Three-dimensional view of the Fab 1C1/EphA2 cmagenta and beige, respectively. Human EphA2 LBD is sintermolecular contacts between human EphA2 and Fab 1C1 Cand cyan, respectively. Nitrogen and oxygen atoms are shownincludes several hydrogen bonds shown as black dotted lines.molecule via its predominantly hydrophobic tip. Sulfur atomsidentical with that in (b). (d) The maximum likelihood weigharea of Fab 1C1 CDRH3 penetration into EphA2. Color code

PDB entries 3C8X (EphA2 LBD12), 3FL7 (EphA2ECD12), 3HPN (EphA2 LBD13), and 2X10 (EphA2ECD11). Larger deviations were detected when ourEphA2 structure was compared with those containingunliganded EphA2 LBD. Here, r.m.s.d.'s ranged from1.44 (for PDB ID 3HPN, over 173 Cα atoms) to 2.46(for PDB ID 3C8X, over 160 Cα atoms). Theseobservations are in agreement with our hypothesisthat the complex between EphA2 and Fab 1C1 is of asimilar nature with that with its ligands (seesubsequent section). For reference, a superimpositionof various relevant liganded and unliganded humanEphA2 structures is shown in Fig. 6.

Molecular basis of ephrin mimicry by 1C1

Superimposition of the Fab 1C1/EphA2 complexover that of the EphA2/ephrin A1 and EphA2/ephrin A5 complexes (PDB IDs 3MBW12 and2X1111) revealed that both ephrins and Fab 1C1bind to the same site on EphA2 (Fig. 7a). This is invery good agreement with the competition datashown in Fig. 2b. More precisely, although residuescomprising the interactive loop of ephrin A1/A5[“FTPF(S/T) LG”] are different from their counter-part in Fab 1C1 CDRH3 (“YVAVAGPA”), thecorresponding peptides adopt similar conforma-tions (Fig. 7b). In fact, the insertion of this portion ofCDRH3 into the ephrin binding channel on EphA2closely resembles the interaction of ephrin-receptor-binding loops with various Eph receptors.9,11,13–17 Inaddition, J–K loop residues 124–136 in EphA2 adoptan α-helical conformation upon binding to Fab 1C1.This particular feature is observed in all knownliganded EphA2 structures but absent (PDB ID3HPN; coil conformation) or invisible (PDB IDs2X10, 3FL7, and 3C8X) in unliganded ones. However,1C1 distinguishes itself from the more promiscuousephrins by exhibiting a strong specificity to humanEphA2, which excludes other related Eph receptorssuch as human EphA1 and EphA4; murine EphA3,EphA4, EphA6, EphA7, and EphA8; and rat EphA5.30

We propose that residues F132 and E133 mediate thisremarkable specificity due to their (i) uniqueness inhuman EphA2 sequence when compared with theseEph receptors and (ii) demonstrated functional andstructural importance in the present study.The previously described structural mimicry of

ephrin molecules by 1C1 translates functionally.

omplex. Fab 1C1 heavy chain and light chain are shown inhown in cyan. (b) Stereographic representations of theDRH3. Fab 1C1 and human EphA2 are shown in magentain blue and red, respectively. The corresponding interface(c) Fab 1C1 CDRH3 penetrates into a channel of the EphA2are shown in yellow, whereas the rest of the color code isted 2mFo-DFc electron density map is shown around theis identical to that in (b). The map is contoured at 1.5 σ.

Fig.

5(legend

onprevious

page)

400 Ligand Mimicry by an Anti-Human EphA2 Antibody

Fig. 6. Stereographic representation of the superimposition of several models of human EphA2 LBD molecules. Loopsare indicated by letters according to the nomenclature shown in Fig. 3a and its legend. EphA2 LBD of the present study isshown in green. The orange molecule is taken from PDB entry 3CZU where human EphA2 LBD was co-crystallized withephrin A1; the bluemolecule is taken from PDB entry 3HEI where human EphA2 LBDwas co-crystallized with ephrin A1;the cyan and magenta models represent unliganded structures of human EphA2 LBD and are taken from PDB entries3C8X and 3HPN, respectively. All liganded EphA2 molecules superimpose quite well with r.m.s.d.'s comparable to theestimated coordinate error of the model pairs. The most divergent model belongs to the unliganded EphA2 structure PDBentry 3C8X (whose J–K loop was not modeled due to the absence of corresponding electron density). The superimpositionwas carried out through the Cα atoms of the EphA2 molecules using lsqkab.40

401Ligand Mimicry by an Anti-Human EphA2 Antibody

Indeed, we have found that monovalent Fab 1C1 isfully capable of inducing phosphorylation, internal-ization, and degradation of EphA2. This is the firsttime that an antibody Fab fragment is shown toactivate an Eph receptor. These ephrin mimicry

Fig. 7. (a) Stereographic representation of the superimpositiID 3MBW; blue), human EphA2 with ephrin A5 (PDB ID 2X11magenta, respectively). (b) Stereographic representation of FaColor code is identical with that in (a). The superimposition wausing lsqkab.40

findings are in very good agreement with the recentobservation that soluble monovalent ephrin A1 canbe a functional ligand of EphA2.21 Interestingly, ithad long been believed that soluble ephrin ligandsare unable to induce activation of Eph receptors and

on of the complexes of human EphA2 with ephrin A1 (PDB; olive), and human EphA2 with Fab 1C1 (cyan and beige/b 1C1 CDRH3 mimicry of ephrin-receptor-binding loops.s carried out through the Cα atoms of the EphA2molecules

402 Ligand Mimicry by an Anti-Human EphA2 Antibody

transduce signals due to their inability to triggerclustering of Eph receptor/ephrin complexes.19

Therefore, additional studies will be important tofully understand the events that occur after bindingof Fab 1C1 and lead to EphA2 activation. Our resultshave important implications for the design andgeneration of novel anti-tumor therapeutics targetingEph receptors and exhibiting beneficial agonistic26 orephrin-blocking29 properties.

Materials and Methods

Reagents, conventions, and illustrations

All chemicals were of analytical grade. Restrictionenzymes and DNA-modifying enzymes were purchasedfrom New England Biolabs, Inc. (Beverly, MA). Oligonu-cleotides were purchased from Operon (Huntsville, AL).Murine ephrin A1 and A2 and human ephrin A3, A4, andA5 were obtained from R&D Systems (Minneapolis, MN).Human (1C130 and 1C9) and humanized (3F243) anti-human EphA2 mAbs were generated at MedImmune. Theantigen amino acid positions mentioned in the text wereidentified according to a consecutive numbering scheme.The antibody amino acid positions mentioned in the textwere identified according to the definition of Kabat et al.31

Soluble human EphA2 ECD (sEphA2/ECD) and LBD(sEphA2/LBD) variants were defined as human EphA2residues encompassing positions 1–510 and 1–182, respec-tively. The soluble sEphA4/LBD-EphA2 variant wasdefined as a direct genetic fusion of human EphA4residues 1–190 to human EphA2 residues 183–510. Allsoluble EphA2 and EphA4 constructs were expressed as aC-terminal Fc fusion protein where they were fused to theFc domain of a human IgG1. The membrane-boundhuman mEphA2/LBD variant was defined as a directgenetic fusion of human EphA2 residues 1–182 to 504–582. The membrane-bound human mEphA4/LBD variantwas defined as a direct genetic fusion of human EphA4residues 1–190 to 504–582. In all cases, the +1-positioncorresponded to the first residue of the mature protein. Allillustrations were prepared using PyMOL (DeLanoScientific, Palo Alto, CA).

EphA2 phosphorylation assay

Human prostate cancer cells (PC3) endogenously expres-sing human EphA2 were seeded into six-well plates at adensity of 2×105 cells/35 mm well in RPMI 1640 mediumcontaining 10% fetal bovine serum (FBS; Invitrogen,Carlsbad, CA) and incubated for 24 h at 37 °C under 5%CO2. Cells were then treated with anti-human EphA2 mAb1C9, 3F2, or 1C1 (20 μg/ml) or with the Fab portion ofmAb1C1 (Fab 1C1; 20, 60, or 100 μg/ml) in RPMI 1640 mediumcontaining 10%FBS for 15min at 37 °Cunder 5%CO2. Threewashes in phosphate-buffered saline (PBS) followed. Cellswere then lysed using Triton X-100 lysis buffer (BostonBioProducts, Ashland, MA) containing protease and phos-phatase inhibitor cocktail I and II according to themanufacturer's instructions (Sigma, Saint Louis, MO). For

each activation measurement, a cell lysate sample corre-sponding to 300 μg of total protein (as determined by thebicinchoninic acid method) was immunoprecipitated using3 μg of a mouse anti-human EphA2 antibody (clone D7;Millipore, Billerica, MA) and 50 μl of anti-mouse IgGagarose (Abcam, Cambridge, MA). Beads were thenresolved by SDS-PAGE, transferred to a nitrocellulosemembrane (Invitrogen), and probed successively with2 μg/ml of a mouse anti-phosphotyrosine antibody (clone4G10; Millipore) and a 1/10,000 dilution of a goat anti-mouse horseradish peroxidase (HRP) conjugate (JacksonImmunoResearch, West Grove, PA). HRP activity wasdetected using SuperSignal West Pico ChemoluminescentSubstrate (Thermo Scientific, Waltham, MA). The mem-brane was then stripped using stripping buffer (ThermoScientific) and successively reprobed with 3 μg/ml of amouse anti-human EphA2 antibody (Invitrogen) and a1/10,000 dilution of a goat anti-mouse IgG-HRP (JacksonImmunoResearch) to detect total EphA2. HRP activity wasdetected as mentioned above.

EphA2 degradation assay

PC3 cells were seeded into six-well plates at a density of2×105 cells/35 mmwell in RPMI 1640medium containing10% FBS (Invitrogen) and incubated for 24 h at 37 °Cunder 5% CO2. Cells were then treated with anti-humanEphA2 mAb 1C9, 3F2, or 1C1 (20 μg/ml) or with Fab 1C1(6.7 or 20 μg/ml) in RPMI 1640 medium containing 10%FBS for 72 h min at 37 °C under 5% CO2. Three washes inPBS followed. Cells were then lysed using Triton X-100lysis buffer (Boston BioProducts) containing protease andphosphatase inhibitor cocktail I and II according to themanufacturer's instructions (Sigma). Cell lysates corre-sponding to 20 μg of total protein (as determined by thebicinchoninic acid method) were then loaded onto an SDS-PAGE, transferred to a nitrocellulose membrane (Invitro-gen), and probed with 3 μg/ml of a mouse anti-humanEphA2 antibody (Invitrogen) and a 1/10,000 dilution of agoat anti-mouse HRP conjugate (Jackson ImmunoRe-search). HRP activity was detected using SuperSignalWest Pico Chemoluminescent Substrate (Thermo Scientif-ic). For protein loading analysis, the membrane wassuccessively probed with a 1/2000 dilution of a rabbitanti-human GAPDH mAb (Cell Signaling Technology)and a 1/10,000 dilution of a goat anti-rabbit IgG-HRPconjugate (Thermo Scientific, Waltham, MA). HRP activ-ity was detected as mentioned above.

Internalization assay

Approximately 106 PC3 cells were aliquoted into thewells of a U-bottomed 96-well plate in 100 μl of RPMI 1640containing 10% FBS (Invitrogen) and then treated withanti-human EphA2 mAbs 1C9, 3F2, and 1C1 at 10 μg/mlor Fab 1C1 at 30 μg/ml for 1 h at 37 °C under 5% CO2 or at4 °C. Cells were then washed three times with ice-coldPBS, fixed with 2% paraformaldehyde (Kodak, Rochester,NY) for 15 min at room temperature (RT), and permea-bilized using 0.5% Triton X-100 (Sigma) for 5 min at RT.Cells were then labeled using 1 μg/ml of an anti-humanIgG antibody conjugated to Alexa Fluor® 488 (Invitrogen)in PBS for 30 min at 4 °C. After three washes using PBS,

403Ligand Mimicry by an Anti-Human EphA2 Antibody

cells were resuspended in 500 μl of PBS and spun downonto coated slides using Shandon EZ Single Cytofunnels(Thermo Scientific) and a Shandon Cytospin 3 (700 rpmfor 7 min). Finally, cells were covered with VECTA-SHIELD® HardSet™ Mounting Medium (Vector Labs,Burlingame, CA) and a coverslip before examination andphotography using a confocal microscope Leica SP5(Leica, Buffalo Grove, IL).

Construction, expression, and purification of EphA2variants

DNA encoding the sEphA2/ECD, sEphA2/LBD,sEphA4/LBD-EphA2, mEphA2/LBD, and mEphA4/LBD variants (see nomenclature described earlier) wereassembled and amplified using PCR by overlap extensionand full-length ECD human EphA2 (positions 1–510)-containing, EphA4 (positions 1–528)-containing, and Fc-containing plasmids as templates (MedImmune). Theassembled DNA for sEphA2/ECD, sEphA2/LBD, andsEphA4/LBD-EphA2 variants were cloned into themammalian expression vector pcDNA3.1 (Invitrogen) asBamHI-EcoRI and EcoRI-XbaI fragments for the Eph andhuman Fc portions, respectively. The assembled DNA formEphA2/LBD and mEphA4/LBD variants were clonedinto the mammalian expression vector pcDNA3.1 as aBamHI-EcoRI fragment. Membrane-bound KO EphA2variants in which portion of EphA4 replaced their EphA2counterpart were assembled and amplified using PCR byoverlap extension with mEphA4/LBD and mEphA2/LBDas templates. Membrane-bound KI EphA2 variants inwhich portion of EphA2 replaced their EphA4 counterpartwere assembled and amplified using PCR by overlapextension with mEphA4/LBD and mEphA2/LBD astemplates. The assembled DNA for mEphA2/LBD,mEphA4/LBD, KO, and KI variants were cloned intothe mammalian expression vector pcDNA3.1 as a BamHI-EcoRI fragment. HEK 293 cells were then transientlytransfected with the various constructs using 293fectinand standard protocols according to the manufacturer'sinstructions (Invitrogen). Purification of sEphA2/ECDand sEphA2/LBD was performed directly from transfec-tion harvests using HiTrap protein A columns accordingto the manufacturer's instructions (GE Healthcare, Piscat-away, NJ).

ELISA measurements

Competition ELISA was carried out using humansEphA2/ECD at a concentration of 2 μg/ml coated onthe surface of a 96-well plate overnight at 4 °C. Plates werethen washed six times with PBS/0.05% Tween 20 andblocked with 10% SuperBlock blocking buffer (Pierce,Rockford, IL) at 37 °C for 1 h. Murine ephrin A1 and A2and human ephrin A3, A4, and A5 were serially dilutedfrom 100 μg/ml to 10 ng/ml in PBS, added to the wells,and incubated for 1 h at RT. Plates were then washed sixtimes with PBS/0.05% Tween 20, and 1C1 was added toeach well at 1 μg/ml for 1 h at RT. After six washes withPBS/0.05% Tween 20, bound 1C1 was detected using ananti-human Fab HRP conjugate (Sigma).Direct-binding ELISA was performed using 1C1 coated

onto the surface of a 96-well plate at a concentration of

10 μg/ml overnight at 4 °C. Wells were blocked with 10%SuperBlock blocking buffer (Pierce) for 1 h at 37 °C andwashed six times with PBS/0.05% Tween 20. Conditionedmedia containing sEphA2/ECD, sEphA2/LBD, orsEphA4/LBD-EphA2 were added to the wells at aconcentration of 5 μg/ml. Bovine serum albumin wasused as a negative control. After 1 h of incubation at RTand six washes using PBS/0.05%/Tween 20, bound Ephvariants were detected using a goat anti-human EphA2polyclonal antibody (R&D Systems) followed by an anti-goat HRP conjugate (GeneTex, Irvine, CA).

ProteOn measurements

A ProteOn XPR36 instrument (Bio-Rad, Hercules, CA)was used to determine the binding parameters of anti-human EphA2 mAbs 1C9, 3F2, and 1C1 and of Fab 1C1 tosEphA2/ECD. The binding parameters of 1C1 to sEphA2/LBD were also measured. Standard amine coupling wasused to immobilize sEphA2/ECD or sEphA2/LBD to thesurface of a GLC biosensor chip (Bio-Rad) at a density ofabout 200 resonance units for each channel. The anti-human EphA2 mAbs were formulated in PBS/0.005%Tween 20, pH 7.4. The samples were injected at 100 μl/min for 200 s at concentrations ranging from 0.125–2 nM,to 0.313–5 nM, to 0.625–10 nM, to 31.25–500 nM for mAbs1C9, 3F2, and 1C1 and Fab 1C1, respectively. Thedissociation phase was followed for 700 s. The surfaceswere regenerated by injecting 50 mM NaOH/1 M NaCl(Bio-Rad) for 30 s. All sensorgram data were processedusing ProteOn Manager 2.1 software (Bio-Rad) and fit to a1:1 interaction model.

BIAcore measurements

Competition measurements between immobilizedsEphA2/ECD and various anti-human EphA2 antibodieswere carried using a BIAcore 3000 instrument. Moreprecisely, the ability of mAbs 1C1, 3F2, and 1C9 to bind toimmobilized human sEphA2/ECD in the presence of oneanother was assessed as follows: sEphA2/ECD was firstimmobilized onto a CM5 sensor chip using a standardamino coupling chemistry according to the manufacturer'sinstructions (GE Healthcare). For a given antibody/anti-body pair, the first antibody at a concentration of 1 μM inHBS-EP buffer [10 mMHEPES (pH 7.4), 0.15MNaCl, 3 mMEDTA, 0.005% (v/v) Surfactant P20] was allowed to bind toimmobilized sEphA2/ECD. A mixture of this same anti-body with the second antibody in the HBS-EP buffer wasthen passed over the immobilized complex. Both antibodyconcentrations in themixturewere set to 1 μM.The extent ofcompetition was derived from the additional bindingdetected. This process was repeated for all six antibodypairs (namely, 1C9 versus 3F2, 3F2 versus 1C9, 1C9 versus1C1, 1C1 versus 1C9, 3F2 versus 1C1, and 1C1 versus 3F2).

Flow cytometry measurements

HEK 293 cells transiently transfected with mEphA2/LBD, mEphA4/LBD, KO, and KI variants were incubatedwith 1 μg/ml 1C1 for 1 h on ice. Cells were then washedthree times with ice-cold PBS and incubated with 1 μg/mlof an anti-human IgG antibody conjugated to Alexa

404 Ligand Mimicry by an Anti-Human EphA2 Antibody

Fluor® 488 (Invitrogen) for 30 min on ice. Samples wereanalyzed using an LSRII flow cytometer (BD Biosciences,San Jose, CA). Expression of the Eph variants wasmonitored using a cocktail of human anti-EphA4 andEphA2 antibodies (MedImmune) followed by detection bya fluorescein-isothiocyanate-labeled anti-human IgG anti-body (Invitrogen).

Protein crystallization and X-ray data collection

Detailed expression, purification, crystallization, anddata collection procedures have been previouslydescribed.32 In short, crystals of the Fab 1C1/EphA2complex diffracting to 2.5 Å were obtained using thehanging drop vapor diffusion method. The orthorhombiccrystals belonged to the C2221 space group with unit cellparameters a=78.93 Å, b=120.79 Å, and c=286.20 Å. Thecrystals exhibited a solvent content of 49.5% (Matthewscoefficient of 2.4 Å3 Da−1). There were two Fab 1C1/EphA2 complexes in the asymmetric unit of the cell.Molecular replacement, refinement, and electron densitycalculationwere carried out using the CCP4program suite.

Accession number

The atomic coordinates and experimental structurefactors of 1C1/EphA2 LBD have been deposited withthe PDB under accession number 3SKJ.

Acknowledgements

We thank Meggan Czapiga for expert technicalassistance with the confocal microscopy experiments.We are also grateful to ShenlanMao for her help withEphA2 phosphorylation and degradation assays.

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