Concurrent Binding of Anti-Epha3 Antibody and Ephrin-A5 Amplifies Epha3 Signaling and Downstream Responses: Potential As Epha3-Specific Tumor-Targeting Reagents

Research Article

Concurrent Binding of Anti-EphA3 Antibody and Ephrin-A5 Amplifies EphA3 Signaling and Downstream Responses: Potential as EphA3-Specific Tumor-Targeting Reagents

Christopher Vearing,1 Fook-Thean Lee,2 Sabine Wimmer-Kleikamp,1 Violeta Spirkoska,2 Catherine To,1 Con Stylianou,3 Mark Spanevello,3 Martin Brechbiel,4 Andrew W. Boyd,3 Andrew M. Scott,3 and Martin Lackmann2

1Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; 2Tumour Targeting Program, Ludwig Institute for Cancer Research, Heidelberg, Victoria, Australia; 3Leukemia Foundation Cancer Research Unit, Queensland Institute of Medical Research, P.O. Royal Brisbane Hospital, Brisbane, Queensland, Australia; and 4Radiation Oncology Branch, National Cancer Institute, Bethesda, Maryland

Abstract including malignant melanoma, sarcoma, lung cancer, kidney, The Eph receptor tyrosine kinases and their membrane-bound and brain tumors (2–4). Ephs probably do not function in ephrin ligands form a unique cell-cell contact–mediated mitogenesis (5) but promote cancer progression by reactivation system for controlling cell localization and organization. of their embryonal functions thus facilitating neoangiogenesis and Their high expression in a wide variety of human tumors directing tumor cell motility, adhesion, and positioning (1, 2). indicates a role in tumor progression, and relatively low Eph Many biological effects attributed to Eph function, such as and ephrin levels in normal tissues make these proteins segregation of mixed cell populations into Eph and ephrin potential targets for anticancer therapies. The monoclonal expression domains, require concurrent ‘‘forward’’ signaling in antibody IIIA4, previously used to isolate EphA3, binds with Eph and ‘‘reverse’’ signaling in ephrin-expressing cells, which subnanomolar affinity to a conformation-specific epitope occurs in vivo when cell surface–bound Ephs and ephrins interact within the ephrin-binding domain that is closely adjacent to in trans. Thus, soluble Eph and ephrin exodomains bind their the ‘‘low-affinity’’ ephrin-A5 heterotetramerization site. We corresponding partner as function-blocking antagonists and have show that similar to ephrin-A5, preclustered IIIA4 effectively been tried as inhibitors of Eph function during tumor progression triggers EphA3 activation, contraction of the cytoskeleton, and neovascularization (6–8). However, most ephrins can bind and and cell rounding. BIAcore analysis, immunoblot, and activate several Eph receptors within their subclass with compa- confocal microscopy of wild-type and mutant EphA3 with rable affinities (9, 10), a redundancy that provides a considerable compromised ephrin-A5 or IIIA4-binding capacities indicate challenge for selective targeting of Eph- or ephrin-expressing tumor that IIIA4 binding triggers an EphA3 conformation which is cells with soluble Eph or ephrin proteins. In this regard, permissive for the assembly of EphA3/ephrin-A5-type signal- monoclonal antibodies can be more specific and have been ing clusters. Furthermore, unclustered IIIA4 and ephrin-A5 Fc described for EphA3 (11), EphB2 (12), EphB6 (13), mouse ephrin applied in combination initiate greatly enhanced EphA3 B1 (14), and EphA2 (15), whereby an apparent antiproliferative signaling. Radiometal conjugates of ephrin-A5 and IIIA4 activity of the latter (15) has not been reproduced for any of the retain their affinity, and in mouse xenografts localize to, and other antibodies (12). We previously used a monoclonal antibody are internalized rapidly into EphA3-positive, human tumors. (mAb), IIIA4, raised against LK63 human acute pre-B leukemia cells These findings show the biological importance of EphA3/ to affinity isolate EphA3 (11). We later confirmed that IIIA4 binds ephrin-A5 interactions and that ephrin-A5 and IIIA4 have to the native EphA3 globular ephrin-binding domain (16, 17) with f 10 great potential as tumor targeting reagents. (Cancer Res 2005; subnanomolar affinity (KD 5 10 mol/L) reflecting a very 4 65(15): 6745-54) low apparent dissociation rate kd =3 10 /s (18). A number of recent studies have provided insight into the Introduction contact surfaces that are involved in the assembly of Eph/ephrin signaling complexes. Crystallography (10, 19) and complementing Eph receptor tyrosine kinases (Ephs) and their membrane-bound structure-function analysis (18) revealed that high-affinity, 1:1 ephrin ligands (ephrins) control cell positioning and tissue dimers of the isolated monomeric Eph/ephrin-binding domains organization during essential embryonic development programs (16, 20), through engagement of two additional interfaces, assemble (1). During adulthood, many Ephs and ephrins are expressed at high local concentration (within the crystal structure) into 2:2 preferentially in malignant tissue, where they are thought to heterotetramers. In the resulting ring-like heterotetramer, Ephs and participate in progressing invasive and metastatic cancers, ephrins are oriented so that their COOH termini point in opposing directions of each other, in agreement with an orientation that facilitates signaling into each of two opposing cells. However, the Note: Supplementary data for this article are available at Cancer Research Online assembly of active signaling complexes (21) and their internaliza- (http://cancerres.aacrjournals.org/). tion into the Eph-expressing cell (22) requires further oligomeri- C. Vearing and F-T. Lee are joint first authors. Requests for reprints: Martin Lackmann, Department of Biochemistry and zation, which is routinely achieved in vitro through preclustered, Molecular Biology, Monash University, P.O. Box 13D, Clayton, Victoria 3800, Australia. tetravalent ephrin-Fc fusion proteins (23). We postulated that the Phone: 61-3-9905-3738; Fax: 61-9905-3726; E-mail: [email protected]. edu.au. heterotetrameric conformation of EphA3 harbors the propensity I2005 American Association for Cancer Research. for Eph/Eph oligomerization via membrane-proximal Eph domains www.aacrjournals.org 6745 Cancer Res 2005; 65: (15). August 1, 2005

(18, 22), previously shown essential for Eph signaling in vivo (20). In Fluor647 (Molecular Probes); labeling efficiency and biological integrity of agreement with this assumption, we identified a third Eph/ephrin the proteins during labeling were monitored by spectral (high-performance interface within the cysteine-rich domain just outside the COOH- liquid chromatography, HPLC, diode array detection) and BIAcore binding terminal border of the crystallized ephrin-binding domain (18) and analysis as described previously (18, 22). Labeled proteins were recovered from the reaction mixture on a Phast Desalting column (Pharmacia, assigned the corresponding ephrin-A5 interface,5 both of which are Piscataway, NJ) and stored in aliquots at 80jC. essential for functional signaling complexes. Amino acid substitu- Cell culture. Human acute lymphoblastic pre-B cells (LK63), AO2, and tions in these EphA3 or ephrin-A5 surfaces weaken ephrin binding SK-Mel28 melanoma lines and human kidney epithelial 293 (HEK293, moderately but abrogate or severely reduce ephrin-induced American Type Culture Collection, Manassas, VA) cells have been described phosphorylation and CrkII recruitment. Interestingly, these same previously (11, 24, 26) and were cultured in RPMI or DME (HEK 293), EphA3 mutations in the tetramerization and cysteine-rich domains containing 10% FCS. Transfection was carried out using Fugene 6 affect binding of the IIIA4 mAb to EphA3 (18), suggesting by transfection reagent (Roche Diagnostics, Indianapolis, IN). Before each inference a continuous antibody binding site across both surfaces, experiment, cells were serum starved in culture medium containing 0.5 % linking the ephrin binding and cysteine-rich Eph domains. We FCS for at least 4 hours. Microscopy and immunocytochemistry. Immunocytochemistry and hypothesized that ephrin contact to this linker region within the time-lapse confocal microscopy on an Olympus FV500 microscope using tetrameric complex may facilitate or stabilize a receptor orienta- 100, 1.4 numerical aperature (NA) oil (fixed cells) and 60, 1.0 NA water tion that favors aggregation into oligomeric Eph/ephrin clusters, (live cells) immersion lenses, were done as described (24). Images of EGFP essential for effective signaling (22). (EphA3) and Alexa546 and Alexa647 (IIIA4) fluorescence were collected Here we show that ephrin-A5-triggered activation of EphA3 sequentially to minimize ‘‘bleed-through’’ from spectral overlap. EPFP was signaling can be mimicked by dimerized but not by monomeric, excited with the 488-nm line of a 100-mW argon ion laser; Alexa546 was nonclustered IIIA4 mAb. Moreover, in the presence of ephrin-A5-Fc, excited with the 543-nm HeNe laser line; Alexa647 with the 633 HeNe laser also nonclustered IIIA4 triggers rapid activation and results in line. dramatically accelerated and enhanced cell-morphologic responses, Sections from mouse xenografts were snap frozen, or frozen in optimal suggesting a distinct synergy between the two proteins. For their use cutting temperature compound (Tissue-Tek, Sakura Finetek U.S.A., Inc., Torrance, CA). Sections (5 Am) were fixed in ice-cold acetone and treated in tumor-targeting studies, we prepared 125Ior111In radioisotope with 0.3% H2O2 to eliminate endogenous peroxidase and nonspecific conjugates of IIIA4 and ephrin-A5 Fc, both of which retain the binding sites saturated with a protein-blocking reagent. Biotin-conjugated reactivity to cell surface EphA3 with affinities that are very similar to IIIA4 antibody was used for mouse xenografts sections, and hematoxylin- those estimated by BIAcore analysis for the unlabeled proteins. Both counterstained sections developed with streptavidin HRP and 3-amino-9- proteins target EphA3-positive tumors in mouse/human xenografts ethylcarbazole (AEC) as chromogenic substrate. Sections in Crystal/Mount and reveal significant tumor-specific retention in solid tumors (111In- dry mounting medium (Biømeda Corp., Foster City, CA), were coverslipped IIIA4) or in a leukemia model (111In-ephrin-A5 Fc). The synergy of the with DePeX mounting medium (Gurr, BDH Laboratory Supplies, Poole, two divalent proteins targeting EphA3, each of which contributes a England) for microscopic viewing. highly specific, avid, and functionally relevant tether to the complex, Immunoprecipitation and Western blotting. Phosphorylated proteins j suggests opportunities for the design of a tumor targeting approach and EphA3 were immunoprecipitated (minimum 4 hours, 4 C) from Triton X-100 lysates of stimulated or control cells (24) using 4G10 anti- with unsurpassed specificity for EphA3-positive cells. phosphotyrosine agarose (Transduction Laboratories) and with IIIA4 mAb agarose, respectively, the latter prepared by coupling the purified antibody Materials and Methods to Mini-leak agarose (Kem-En-Tec, Copenhagen, Denmark) according to Expression constructs and reagents. Expression vectors encoding full- the manufacturer’s instructions. Lysates and washed immunoprecipitates length wild-type (wt) and mutant EphA3 proteins, containing either were analyzed by Western Blot with appropriate antibodies and visualized tyrosine-to-phenylalanine substitutions of the three major phosphorylation using an enhanced chemiluminescence substrate (Pierce, Rockford, IL). sites (3 YF EphA3; ref. 24) or substitutions Phe152-to-Leu and Val133-to-Glu Labeling of IIIA4 monoclonal antibody and a control monoclonal 111 125 within the putative EphA3 heterodimerization and heterotetramerization antibody (CLB-CD19) with In and I. The IIIA4 antibody and ephrin- 00 domains with or without addition of a COOH-terminal enhanced green A5 Fc were conjugated with the bifunctional metal ion chelator CHX-A - fluorescent protein tag (EGFP, Clontech, Mountain View, CA) were prepared diethylenetriaminepentaacetic acid, allowing convenient chelation of the 111 125 as described previously (18, 22). Expression plasmids (pIgBOS) encoding resultant conjugate with In. Iodination with I was achieved by direct extracellular domains of ephrin-A5 or EphA3 (25) fusion proteins with the coupling using Chloramine-T as described previously (26). Retention of the hinge and Fc region of human IgG1 (gift from A. van der Merwe, Oxford affinity of labeled proteins was confirmed by radioligand-binding analysis, University, Oxford, United Kingdom) were prepared and Protein-A affinity whereby serial dilutions of unlabeled ephrin-A5 Fc or IIIA4 mAb (from) 125 111 purified from culture supernatants of stable Chinese hamster ovary and a constant concentration (20 ng) Ior In protein were incubated 7 transfectants as described (18, 22). with 1.5 10 SK-MEL-28 or EphA3/HEK 293 cells for 45 minutes at Anti EphA3 mAb, IIIA4 and affinity-purified rabbit polyclonal antibodies room temperature with continuous mixing. Radioactivity in washed cell were described (11, 16). Other antibodies and reagents were from pellets levels was determined on a gamma counter. The Ka (affinity) and Transduction Laboratories (Lexington, KY; anti-GFP), Upstate (Lake Placid, the number of binding sites per cell were calculated as described NY; anti-phosphotyrosine antibody 4G10 and 4G10-agarose), Jackson previously (26). Immunoresearch Laboratories [West Grove, PA; anti-human Fc and In vitro properties of protein-radiometal conjugates. The retention horseradish peroxidase (HRP)–conjugated anti-mouse antibodies], Bio- of binding affinities of radiolabeled ephrin-A5 Fc and IIIA4 mAb were Rad (Hercules, CA; HRP-conjugated anti-rabbit), and Molecular Probes confirmed by the Lindmo assay as described previously (26). Briefly, (Eugene, OR; Alexa488-phalloidin). cell-bound radioactivity in washed cell pellets of serial diluted SK-MEL- 7 7 Alexa546 and Alexa647 protein conjugates. Recombinant, purified 28 (3 10 ) or LK63 (4.7 10 ) cells, incubated (45 minutes, room 125 111 ephrin-A5-Fc and the IIIA4 mAb were labeled with Alexa Fluor546 and Alexa temperature) with 20 ng of I and In-mAbIIIA4 was determined on a gamma counter. The stability of 125I- and 111In-labeled proteins, incubated for 2 and 7 days at a constant radioactive concentration in human serum was determined by binding to SK-MEL-28 cells and 5 B. Day, et al., J Biol Chem. 2005 PMID:15901737. expressed as percentage of radioactivity at day 0.

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125I and 111In-IIIA4 or ephrin-A5 biodistribution in tumor-bearing the coordinated tethering of several distinct Eph contact surfaces mice. Human tumor cells were injected either s.c. into the underside flank by ephrin oligomers (27). We noted previously that the anti-EphA3 (EphA3/HEK293 or SK-MEL28 cells, 2 107 in 0.1 mL of PBS) or i.v. into 7 mAb IIIA4 also recognizes part of the Eph/ephrin tetramerization the tail vein (LK63 leukemia cells, 0.5-1 10 in 0.1 mL of PBS), of 3- to 4- interface, whereby ephrin-A5 Fc and IIIA4 can bind to the same week-old nude BALB/c mice (Animal Resources Centre, Canning Vale, EphA3 molecule with minimal effect on their individual interaction Western Australia). Solid tumors developed after 3 weeks, and mice were used when tumors weighed between 0.2 and 0.7 g or after 21 days, in the kinetics (18). case of LK63 xenografts. Doses of 5 Ag 125I-IIIA4 (7.4 ACi) or 111In-IIIA4 (11 These findings prompted us to examine if IIIA4 could act as ACi) or of 6 Ag 111In-ephrin-A5 (11 ACi) were given via tail vein injection. agonist to initiate EphA3 signaling and if concurrent binding of Following injection, three to five mice were harvested per time point at 4, IIIA4 and ephrin-A5 Fc would trigger assembly of EphA3 24, 48 hours and 3, 5, 7, 9, 13 days, sacrificed by cervical dislocation. signaling complexes. We first compared the capacities of a 1:1 Thirteen major tissues (as indicated), blood, and tumors were collected mixture of the monomeric proteins or of clustered ephrin-A5 Fc from each mouse, weighed and contained radioactivity determined on a or IIIA4 to activate signaling in EphA3/HEK 293 cells. SE-HPLC gamma counter. Radioactivity, as fraction (%) of injected dose per gram of of the proteins for use in these experiments allowed us to tissue, was calculated and graphed for each time point, and on a time confirm the oligomerization of the clustered proteins, and the scale represents the rate of serum clearance of 125I- and 111In-labeled absence of protein aggregates from nonclustered ephrin-A5 and protein. Gamma camera imaging. Anesthetized mice (0.2 mL of 18.7 mg/mL IIIA4 (Supplementary Fig. S1). Western blot analysis revealed that tribomoethanol, i.p.) were placed on a dual-headed Biad Trionix gamma clustered IIIA4 triggers stronger EphA3 phosphorylation (Fig. 1A- camera (Twinsbury, OH) equipped with a medium energy collimator. A B, peak D) and CrkII recruitment (Supplementary Fig. S1, C-D) standard containing 10% of the injected dose was placed within the field of than ephrin-A5 Fc, whereas neither nonclustered protein induced view. 111In images were acquired over 10 minutes and data recorded on a EphA3 activation above background levels. Interestingly, an SunSparc Station 10 computer. equimolar mixture of nonclustered ephrin-A5 Fc and IIIA4 together stimulated EphA3 phosphorylation to an even stronger degree than clustered IIIA4 on its own (Fig. 1A-B, peak M). Results Furthermore, whereas IIIA4-induced, similar to ephrin-A5-in- Parallel binding of monomeric ephrin-A5 Fc and IIIA4 to duced phosphorylation reached a plateau after 5 to 10 minutes EphA3 triggers strong, sustained signaling without need for (Fig. 1C), we observed a continuously increasing EphA3 agonist preclustering. The structures of Eph/ephrin complexes phosphorylation in IIIA4/ephrin-A5 stimulated samples over (10, 19) suggest that the assembly of Eph signaling clusters involves 30 minutes.

Figure 1. Parallel binding of ephrin-A5 Fc and IIIA4 triggers synergistic EphA3 activation without requiring agonist preclustering. A, Superose-12 SE-HPLC profile of IIIA4 and ephrinA5-Fc complexes used as EphA3 agonists. Fractionation of preclustered ephrinA5-Fc (ephrinA5-Fc-X) yields fractions of oligomeric (O), dimeric (D), and uncomplexed ephrinA5-Fc (X), the latter containing residual cross-linking antibody; retention times of protein standards (gray vertical lines) are overlaid onto the chromatogram. An analogous profile is obtained for anti-mouse-Fc preclustered IIIA4 (Supplementary Fig. 1D). Inset, monomeric IIIA4 and ephrin-A5 Fc (M) were obtained from samples lacking anti-Fc antibodies; the gray shaded area in the chromatogram illustrates nonclustered IIIA4 (M) used for experiments. B, preclustered IIIA4 (1.5 Ag/mL), or ephrinA5-Fc (1.5 Ag/mL), or a combination (comb) of the nonclustered proteins (0.75 Ag/mL each) were purified by SE-HPLC and protein fractions (denoted as in A) used for stimulation (10 minutes) of HEK293/EphA3 cells. Anti-phosphotyrosine (PY) immunoprecipitates from cell lysates were blotted with anti-EphA3 antibodies, a sample of nonstimulated HEK293/EphA3 cells (Nil) was analyzed in parallel. C, time course of EphA3 phosphorylation, triggered by treatment with combined IIIA4/ephrin-A5 Fc. Anti-phosphotyrosine (PY) immunoprecipitates of cell lysates, after HEK293/EphA3 activation for the indicated times with monomeric IIIA4, preclustered ephrinA5-Fc, or monomeric IIIA4, and ephrinA5-Fc in unison before probing with anti-EphA3 antibodies; combined monomeric agonists induce escalating EphA3 activation. Cell lysates (lysate) analysed in parallel by anti-EphA3 Western blot confirmed even gel loading. D, actin cytoskeletal changes following stimulation with clustered or nonclustered IIIA4 or ephrinA5-Fc or with their combination. HEK293/EphA3 cells, incubated (10 minutes) with reagent controls (PBS or anti-mouse Fc antibody, a-mFC), clustered ephrinA5-Fc (c ephn), nonclustered (nc IIIA4) or clustered IIIA4 (c IIIA4), and combined monomeric IIIA4 and ephrinA5-Fc (nc IIIA4+ephn) were fixed and stained with Alexa488-phalloidin to highlight the cytoskeleton for confocal microscopy.

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We went on to examine if simultaneous exposure to combined, involved in IIIA4-triggered EphA3 activation, by assessing the nonclustered ephrin-A5 and IIIA4 would trigger cytoskeletal effects of EphA3 point mutants, recently shown to diminish changes similar to those previously observed in response to ephrin-A5 and IIIA4 interactions (18), on IIIA4-triggered signaling. clustered ephrin-A5 Fc (Fig. 1D). In agreement with the observed Previously, analysis of the binding properties of these mutant EphA3 activation, exposure of EphA3/HEK293 cells to nonclustered receptors suggested that amino acid substitutions within the Eph/ ephrin-A5 Fc and IIIA4 resulted in dramatic cell rounding and ephrin high-affinity (heterodimerization) site severely impeded membrane blebbing, exceeding the responses observed with the ephrin-A5 binding, whereas mutations in the low-affinity (hetero- clustered ephrin-A5 Fc or IIIA4. As expected, nonclustered IIIA4 on tetramerization) site preferentially reduced IIIA4 binding (18). its own (Fig. 1D) did not affect the actin cytoskeleton or cell We assessed by confocal microscopy binding of Alexa647-labeled morphology. IIIA4 to cell surface expressed mutant or wt EphA3, transiently IIIA4-binding and IIIA4/ephrin-triggered EphA3 activation expressed in HEK 293 cells as fusion protein with a COOH-terminal is differentially affected in ephrin-binding domain mutants. EGFP (Fig. 2A). The Alexa647-antibody bound strongly to the wt We next examined the molecular surfaces of EphA3 that may be receptor, as well as to the 3YF EphA3-EGFP control mutant lacking three functionally relevant EphA3 tyrosine residues (18, 24), as evidenced by merged images of EphA3 (green) and IIIA4 fluorescence (blue), indicating a convincing overlap of the fluorescent proteins (cyan) at the cell membrane. EphA3 [Phe152- Leu], despite low expression in these experiments, also bound Alexa647-IIIA4 considerably, and the merged image reveals an almost complete overlap of cell surface EGFP and Alexa647staining at the cell membrane. By contrast, a mutation in the low-affinity binding site, EphA3 [Val133-Glu], on its own, or in combination with the Asn232-Ile substitution, affected IIIA4 binding notably and the merged images of these cells, despite robust expression of the mutant EphA3, reveal little of IIIA4-associated fluorescence on the cell membrane and thus little overlap in the merged images. We assessed the functional relevance of impaired IIIA4 binding by studying phosphorylation levels of IIIA4-stimulated EphA3 by immunoprecipitation analysis. Comparison of anti-phosphotyrosine levels in these samples confirms that site-directed substitution Phe152-Leu within the high-affinity ephrin-A5 binding site does not affect IIIA4 induced activation of EphA3. By contrast, tyrosine phosphorylation of the Val133-Glu low affinity binding site mutant, and of EphA3 bearing a Val133-Glu/Asn232-Ile double mutation were reduced to background levels, confirming this EphA3 interface as the principle IIIA4 binding region. In agreement with the synergistic activity of ephrin-A5 and IIIA4, the phosphorylation level in wt EphA3-transfected cells was notably increased after stimulation with an equimolar mix of both agonists. Interestingly, the relative EphA3 phosphorylation level was modulated differently in the EphA3 mutants, likely reflecting the combined contributions of ephrin-A5 Fc and IIIA4, which are differently affected by these mutations (18), to the formation of these signaling complexes.

Figure 2. Compromised IIIA4 binding and IIIA4/ephrin-triggered phosphorylation in ephrin-binding domain EphA3 mutants suggests proximity of interaction sites. A, confocal microscope images of HEK293 cells transiently transfected with chimeric proteins of NH2-terminal EGFP and wt EphA3, or mutant EphA3 as indicated: 3XYF, V133-E, F152-L, N232-I, V133/N232; cells were exposed (10 minutes) to preclustered, Alexa647-labeled IIIA4, and nonbound IIIA4 removed before fixation of cells and microscopy. Images of GFP fluorescence (green), Alexa647 fluorescence (blue), and merged images (cyan) of GFP and Alexa647 fluorescence. Fluorescence signals inside the cells represent cytosolic pools of GFP-EphA3 (green). B, activation of kinase activity in mutant EphA3 receptors. Protein-G immunoprecipitates from lysates of HEK293 cells transiently transfected with a vector control, wt or mutant EphA3 (as indicated), were stimulated with preclustered IIIA4 or with combined, nonclustered ephrin-A5 Fc and IIIA4 and analyzed with anti-PY Western blots (WB). Analysis of cells expressing EphA3 mutated at the principle Eph autophosphorylation sites (3YF), was done as control. To assess even loading and equivalent transfection levels, the Western blot membrane was reprobed with a polyclonal anti-EphA3 antibody. Overlays of a-PY and anti-EphA3 Western blots indicate that only the prominent upper band in the anti-EphA3 blot is phosphorylated. Bar, 20 Am.

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The coordinated interaction of IIIA4 and ephrin-A5 Fc binding of ephrin-A5 and IIIA4 leads to rapid and effective triggers accelerated EphA3 activation and cell-morphologic assembly of active signaling complexes. responses. Our finding, that combined non clustered IIIA4 and Trafficking of cell surface EphA3-bound ephrin-A5 and IIIA4 ephrin-A5 trigger EphA3 signaling to a scale exceeding that of the is accelerated when applied in combination. To examine the clustered individual agonists, prompted us to examine the trafficking of ephrin-A5 Fc and IIIA4 after their binding to assembly and dynamics of IIIA4/ephrin-elicited EphA3 clusters by confocal microscopy (Fig. 3A). In agreement with earlier time- lapse experiments (22), binding of clustered Alexa546 ephrin-A5 Fc or clustered Alexa647 IIIA4 to EphA3-transfected AO2 melanoma cells or HEK293 cells (data not shown) is clearly visible 1 minute after addition. Already at this early time point clustering of the fluorescent agonists, in particular of IIIA4, is apparent. As expected from previous studies (24), the cells respond within 10 minutes. with pronounced cytoskeletal contraction, cell rounding and membrane blebbing, which is notably stronger in IIIA4- rather than in ephrin-A5 Fc-stimulated cells. Nonclustered ephrin-A5 Fc or IIIA4 on their own do not induce a cellular response, but interestingly, after extended exposure assemble into small punctuate aggregates on the cell membrane (Fig. 3A, arrows). We assessed if this aggregation, or ‘‘patching,’’ might be an intermediate step during the formation of active signaling clusters, and treated EphA3/HEK293 cells for increasing times with nonclustered ephrin-A5 Fc or IIIA4, before addition of the complementary agonist (IIIA4 or ephrin-A5, respectively) for 1 minute only. However, increasing the duration of this cluster ‘‘preassembly’’ on the cell membrane did not increase EphA3 phosphorylation above the level observed if both reagents were added together (Fig. 3C, 0-minute clustering time). This suggests an instantaneous formation of active signaling complexes once both agonists are available. In agreement, a dramatic contraction of the cytoskeleton, cell rounding and extensive membrane blebbing was apparent within 1 minute when nonclustered ephrin-A5 and IIIA4 were added together (Fig. 3A,nc IIIA4+ephn). The merged image suggest an almost complete overlap of red (ephrin-A5), blue (IIIA4) and green (rhodamine, polymerized actin) fluorescence, indicative of ephrin-A5 and IIIA4 coassembly into common clusters. Likewise, Western Blot analysis of parallel samples reveal coinciding, rapid and sustained EphA3 phosphorylation in EphA3/HEK293 cells or in EphA3- transfected AO2 cells (Fig. 3B), confirming that cooperative

Figure 3. Synergistic binding of IIIA4 and ephrin-A5 to EphA3-expressing cells triggers accelerated clustering and cytoskeletal contraction. A, confocal microscopic images of EphA3-expressing A02 (A02/EphA3) melanoma cells, left untreated (nil), or stimulated for indicated times with clustered (c), Alexa546-labeled ephrinA5-Fc (c ephn), clustered Alexa647-labeled IIIA4 (c IIIA4), or SE-HPLC-fractionated, nonclustered (nc) ephrinA5-Fc (nc ephn), or IIIA4 (nc IIIA4), or their combination (nc IIIA4+ephn). Washed and paraformaldehyde-fixed A02/EphA3 cells were stained with Alexa488-phalloidin to accentuate the actin cytoskeleton (green fluorescence). Individual channels recording Alexa546 (ephrin, red) and Alexa647 fluorescence (IIIA4, blue), as well as merged images, indicating colocalization of actin with Alexa546 ephrin-A5 Fc (yellow), Alexa647 IIIA4 with actin (cyan) and colocalization of all three fluorescent labels (white). Nonstimulated cells (nil) reveal the cell morphology in the absence of EphA3 activation. Arrows indicate latent and assembled cluster. Bar, 20 Am. B, comparison of EphA3 activation in (preclustered) ephrinA5-Fc-stimulated HEK293/EphA3 or A02/EphA3 cells. EphA3 phosphorylation levels in anti-EphA3 immunoprecipitates from A02/EphA3 or HEK293/EphA3 cell lines were monitored by anti-PY Western blots (WB). Parallel anti-EphA3 Western blots confirm equivalent loadings. C, sequential EphA3 activation with ephrin-A5 Fc or IIIA4 does not affect the net phosphorylation level. HEK293/EphA3 cells were incubated with nonaggregated EphA3 agonist (clustering molecule, IIIA4, top; or ephrinA5-Fc, bottom) for specified times (minutes) to induce formation of latent EphA3 clusters. Receptor activation was initiated by the addition of the complementary agonist for 1 minute and analyzed on anti-EphA3 Western blot of anti-PY immunoprecipitates (IP). www.aacrjournals.org 6749 Cancer Res 2005; 65: (15). August 1, 2005

Figure 4. Concurrent exposure to ephrin-A5 Fc and IIIA4 mAb accelerates their internalization. A, confocal microscopy of HEK293/EphA3 cells, stimulated for indicated times with nonaggregated IIIA4 mAb or Alexa546-ephrin-A5 Fc or (B) their combination. Formaldehyde-fixed and Triton X-100 permeabilized cells were stained with Alexa488-conjugated anti-mouse antibodies to localize IIIA4. The illustrated optical sections were taken at 50% cell height, whereby images illustrating green (IIIA4) or red fluorescence (ephrin-A5) on their own are illustrated in gray scale for greater resolution, whereas the merged are represented in their respective colors. Bar, 10 Am. C, model of the molecular assembly of EphA3 signaling complexes triggered by binding of clustered ephrin-A5 Fc, IIIA4, or the combination of nonclustered agonists. The extracellular part of EphA3, consisting of the ephrin-binding domain (ephn binding, pink), the cysteine-rich linker domain (linker, brown), and the fibronectin III repeats (FNIII, red) as well as ephrin-A5 Fc (green) and IIIA4 (gray) are illustrated (not to scale). A putative Eph/Eph interaction site within the cysteine-rich EphA3 domain is depicted as pink surface. The illustrated aggregate of two tethered Eph/ephrin heterotetramers is indicative for an active signaling complex. Black arrows highlight the low- and high-affinity as well as the third Eph/ephrin interaction sites. transmembrane EphA3, we compared confocal microscopic shared by two other mAbs derived from the same immunization sections (at 50% cell height) of EphA3/HEK293 cells that had been protocol (Supplementary Fig. S2), is due to its ability to bind to exposed to the agonistseitheraloneor in combination (Fig. 4A andB). EphA3/ephrin-A5 complexes in a manner that promotes the We showed previously, that assembly of cell membrane ephrin-A5/ assembly of higher-order signaling clusters (Fig. 4C). In this regard, EphA3 clusters precedes internalization (22). Aggregation of IIIA4 binding replaces anti-Fc-mediated ephrin clustering that is ephrin-A5 or of IIIA4 proceeds slowly if either of the two normally required for the assembly of oligomeric complexes from nonclustered proteins is applied on its own (Fig. 3A). In agreement, Eph/ephrin heterotetramers. cytosolic ephrin-A5 Fc-associated fluorescence becomes noticeable Together, our findings lead us to explore the concept that rapid only after 40 minutes, or in the case of IIIA4, only after 60 minutes EphA3-mediated agonist uptake (22) could be exploited to develop (Fig. 4A). By contrast, exposure to combined, nonclustered ephrin- effective targeting reagents for EphA3-positive human tumors. A5 Fc and IIIA4 significantly accelerates the internalization kinetics Biodistribution of 111In-ephrin-A5 and IIIA4 and ;-camera of both proteins. Already after 10 minutes, clusters of cytosolic imaging in solid tumor-bearing xenografts. We analyzed the ephrin-A5-associated fluorescence are abundant, whereas cytosolic potential tumor targeting properties of ephrin-A5 Fc and IIIA4 in IIIA4-associated fluorescent vesicles are clearly obvious after 20 three different xenograft models, including SK-MEL28 cells, a minutes, suggesting a notable difference in the internalization malignant melanoma line that develops solid xenograft tumors in kinetics of these two agonistic proteins (Fig. 4B). However, after 40 nude BALB/c mice (26). EphA3-overexpressing HEK293 cells have minutes, the majority of the ephrin-A5 and IIIA4-associated been extensively characterized for their EphA3 signaling and fluorescence is cytosolic and, as suggested by the merged image biological responses (22, 24), whereas LK63 acute pre-B leukemia (yellow pseudocolor), a large proportion seems to colocalize within cells were used as original source for the isolation of EphA3 (11). the same cytosolic compartment. For the targeting studies we prepared radio conjugates of IIIA4 and Overall, our experiments show that exposure of EphA3-positive ephrin-A5 using the bifunctional metal ion chelator, CHX-A00- cells to IIIA4 mAb and ephrin-A5 Fc in combination dramatically diethylenetriaminepentaacetic acid, as stable chelation of radio- accelerates and enhances EphA3-specific cellular responses and isotopes proved to yield higher retention of biological activities trafficking of both agonistic proteins into common cytosolic compared with conventional methods, yielding proteins with 50% compartments. It seems that this unique property of IIIA4, not to 75% binding capacity to EphA3-positive cells. In agreement with

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. EphA3-Specific Tumor-Targeting Reagents the binding properties of nonderivatized proteins (18), the targeting experiment in mice that had received saturating amounts Scatchard and Lindmo analysis revealed subnanomolar affinities of hu3S193, a humanized antibody against the nonrelevant Lewis Y 10 of iodine or indium-labeled ephrin-A5 Fc (KD = 7.3-7.5 10 ), [Le(y)] carbohydrate antigen (28) to block anti-human Fc binding 10 111 and IIIA4 (KD = 4.2-5.5 10 ), and an estimated 20,000 to 40,000 sites. An unchanged biodistribution profile of In-ephin-A5 Fc in IIIA4-binding sites on SK-MEL 28 cells, in agreement with strong these mice (Fig. 6B) indicated that Fc receptor–mediated uptake IIIA4 staining of SK-MEL 28 tumor or control sections from was not contributing notably to the distribution of ephrin-A5 Fc in xenograft-bearing mice. We initially assessed biodistribution and these mice. Comparison of the relative 125I and 111In levels 4 hours clearance of IIIA4 and ephrin-A5 Fc in SK-Mel28 xenograft-bearing after injection of ephrin-A5 Fc, or 24 hours after injection of IIIA4, BALB/c Nude mice; however, due to a rapidly decreasing blood revealed in all organs that had preferentially accumulated the concentration of 125I- or 111In-labeled ephrin-A5 Fc, the retention targeting proteins, a notably lower 125I concentration (Fig. 6A, white in SK-MEL28 tumor was difficult to assess (Fig. 5A). By contrast, 111In-IIIA4 has a longer, f48-hour blood half-life and a significant uptake and retention in EphA3/HEK293 xenografts, that was lasting for 8 to 10 days (Fig. 5B). A survey of the biodistribution of 111In-IIIA4 revealed rapid accumulation within 4 days of tumor- associated radioactivity to 27% of injected dose per gram (ID/g), whereas the 111In concentration in blood and all other tissues dropped below 10% ID/g within 24 hours (Fig. 5C). Likewise, the 125I concentration in all tissues, but also in the xenografted tumor decreased to less than 5% ID/g within 72 hours (Fig. 5C). This observation is in line with the notion of IIIA4 internalization into tumor cells and its degradation to release 125I radio-halogen, whereas the radiometal 111In is retained within the cell for much longer, as has been shown in previous studies (see ref. 26 and references within). To visualize the deposition of 111In-labeled IIIA4 in the grafted EphA3/HEK293 tumors, we did whole body gamma camera imaging of a tumor-bearing mouse at various times after receiving the injection of the radio-metal antibody conjugate. As shown in Fig. 5D, the blood pool in the heart was clearly visible at all time points, whereas accumulation of 111In-IIIA4 within the tumor on the left dorsal flank was barely visible at 4 hours after injection. However, concentration of 111In-IIA4 was clearly apparent at 24, 48, and 96 hours after injection, whereas the whole-animal images confirm the absence of 111In-labeled IIIA4 deposition in other organs, such as liver or spleen. 111In-ephrin-A5 Fc and IIIA4 biodistribution in leukemia- xenografted BALB/c nude mice. We were interested to assess if 125I- or 111In-labeled ephrin-A5 Fc, despite its rapid clearance from the circulation, could be used to target haematopoietic tumors. BALB/c nude mice, following i.v. injection of 2 107 LK63 leukemia cells, were injected with radiolabeled ephrin-A5 Fc or IIIA4, and relative levels of either protein in the blood and the various organs determined (Fig. 6A). We observed a rapid accumulation of ephrin- A5 in spleen, lung, and liver, reaching after 4 hours relative concentrations of 24%, 33%, and 35% ID/g, respectively. To assess if this was reflecting ephrin-A5 Fc binding to an Eph receptor or was due to anti-human Fc immunoreactivity, we carried out the

Figure 5. Biodistribution of 111In and 125I-labeled IIIA4 mAb in EphA3/HEK293 xenograft-bearing BALB/c nude mice. A, mice bearing SK-Mel28 or (B) HEK293 tumor xenografts were injected with a mixture of 111In (green, black graphs) and 125I(red, blue graphs)-conjugated ephrin-A5 Fc (A) or IIIA4 (B), and at indicated times after injection five animals per group were sacrificed and radioactivity in the blood (red, black) and tumor (green, blue) determined. The 111In (green, black) and 125I(red, blue) radioactivity is expressed as mean fraction of the injected dose/gram of tissue or blood (% ID/g). C, the concentration of 111In and 125I-labeled IIIA4 (as indicated) in individual tissues of tumor-bearing mice was assessed at indicated times as described above. D, whole body gamma camera images taken from EphA3/HEK293 xenograft-bearing BALB/c nude mice (dorsal left flank) at indicated times following injections of 111In-IIIA4. A standard containing 10% of the injected dose was placed into each field of view (white arrowheads). The yellow arrows indicate the approximate position of the tumor xenograft. www.aacrjournals.org 6751 Cancer Res 2005; 65: (15). August 1, 2005

Figure 6. Tissue distribution of 111In and 125I-labeled ephrin-A5 and IIIA4 mAb in LK63 xenograft-bearing BALB/c nude mice. A, the distribution of 111In-labeled ephrin-A5 Fc and IIIA4 (as indicated) in individual tissues of LK63 leukemia-bearing mice was assessed in groups of five animals at indicated times after injection of the targeting reagents and is expressed as mean % ID/g. The concentration of 125I- ephrin-A5 Fc at 4 hours (top) and of 125I- IIIA4 at 24 hours is shown for comparison (white boxes). B, two parallel groups of mice received injections of hu3S193, a humanized anti Lewis Y (Le-y) antibody, before injection of the targeting reagents, and biodistribution in blood and organs determined after 1 and 2 hours (green and purple, respectively). C, expression of EphA3 in liver and spleen of LK63 xenograft-bearing BALB/c nude mice was examined by immunohistochemical analysis of frozen tissue sections with biotinylated IIIA4 mAb. Parallel control sections were stained with a nonrelevant, isotype-matched control antibody. D, four weeks after inoculation with LK63 cells, mice were killed, single cell suspensions recovered from the organs, including spleen, and analyzed by flow cytometry using anti-human and anti-mouse CD45 and IIIA4 mAb as primary antibodies. The fraction (%) of cells in each quadrant is shown.

columns) suggesting similar to the solid tumor model, an active with elevated levels of the cognate ephrin-A1 (Supplementary uptake and degradation into LK63 cells colonizing spleen, lung, and Fig. S3A). In agreement, extraction of endogenous Ephs from liver. The 111In-IIIA4 localized to the same organs, whereby the normal mouse liver with Protein-A Sepharose-bound ephrin-A5 relative IIIA4 concentration in the spleen amounted here to some confirmed the presence of EphA2 but not of EphA3 as candidate 70% ID/g 4 hours after injection. Indeed, detection of the infiltrating receptor binding ephrin-A5 Fc (Supplementary Fig. 3B), explaining human cells by immunohistochemistry (data not shown) or flow the localization of ephrin-A5 Fc to these organs. cytometry using anti-human CD45 and Alexa488-labeled IIIA4 for In summary, these tumor targeting studies show that in tumor detection revealed abundant population with CD45 and EphA3- xenograft-bearing mice radiometal conjugates of ephrin-A5 and positive tumor cells of spleen (Fig. 6D), liver and bone marrow (data IIIA4 effectively localize to, and are internalized, into EphA3- not shown). In agreement with the biodistribution data, immuno- positive human tumor cells. We also observe that ephrin-A5 Fc and histochemical analysis of EphA3 expression in sections of these IIIA4 act strongly synergistic on EphA3-positive tumor cells, mice confirmed IIIA4-specific immunoreactivity in the spleen above together providing the basis for ongoing studies to develop that observed in the liver (Fig. 6C). We noted however an 111In-IIIA4 effective tumor targeting strategies that combine the specificity concentration in liver and lung that was substantially lower than and avidity of these two EphA3 agonists. that of 111In-ephrin-A5, possibly suggesting that an endogenous Eph receptor other than EphA3 is targeted by ephrin-A5 Fc. Whereas there is no known expression of EphA3 in either organ, quantitative Discussion reverse transcription-PCR indicated substantial expression of IIIA4 monoclonal antibody is an ephrin-A5 mimetic. One of EphA1 and EphA2 in normal liver samples, interestingly coincident the typical features of Eph signaling is the formation of oligomeric

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Eph/ephrin signaling clusters that are necessary for biological That addition of IIIA4 to cells, already binding nonclustered ephrin responses (21–23), and in vivo may involve preformed aggregates of (or vice versa), results in almost instantaneous EphA3 activation cell surface associated ephrins, achieved experimentally by cross- and cell rounding suggests that the additional tether immediately linking of soluble ephrins into clusters containing four Eph binding converts the latent microclusters into functional signaling com- modules (Fig. 4C). In the present study, we have shown that IIIA4, a plexes. It is plausible, that due to the tight binding of ephrin-A5 to mAb which had been raised for the isolation of EphA3 (11) and the high-affinity interface but much weaker binding to the low- which is specific for the ephrin-binding domain of EphA3 (20), affinity interface (to which IIIA4 binds with very high affinity), will mimics this ephrin-A5 property and in dimerized form but not as lead to the formation of complexes where each EphA3 is tethered monomeric nonclustered antibody triggers robust EphA3 phos- by an ephrin-A5 and a IIIA4 (Fig. 4C). This allows for the assembly phorylation, CrkII recruitment, and cell rounding. and propagation of clusters that is limited only by the Whereas agonistic antibodies against the related EphA2 receptor concentration and diffusion rate of components on the cell have been reported, the authors of these studies suggested that the membrane and in solution. Interestingly, whereas IIIA4 was mAb epitope is distinct from the ephrin-A1 binding site, present isolated together with two other mAbs with very similar binding only in EphA2-positive cancer cells, and only in these facilitates epitopes, but differing considerably in their affinities, only IIIA4 EphA2 phosphorylation, degradation, and inhibition of tumor mAb in combination with ephrinA5-Fc can induce receptor growth in vitro and in vivo (15, 29). By contrast, a recently activation effectively, suggesting that in addition to the binding described anti-EphB2 mAb, which in agreement with lacking site, also the high affinity dominated by a very low dissociation rate mitogenic activity of Ephs does not affect tumor growth, effectively may be essential for its agonistic properties. blocks ephrin-B2 binding and EphB2 phosphorylation in colon Ephrin-A5 and IIIA4 as tumor targeting reagents. The carcinoma cells in vitro, thus suggesting overlapping ephrin and pronounced synergistic mode of ephrin-A5 and IIIA4 interacting mAb binding sites (12). In the case of IIIA4, earlier structure- with cell surface EphA3, leading to almost quantitative internali- function studies revealed close proximity of one of the three zation of resulting signaling complexes, suggests their potential as ephrin-A5-binding sites and the IIIA4 interaction surface (Fig. 4C): tumor-targeting reagent for EphA3-positive cancer cells. Whereas specifically, amino acid substitutions within the low-affinity Eph/ ephrin-A5 on its own, despite its preferential interaction with ephrin interface (19) affected ephrin-A5 as well as IIIA4 binding EphA3, will bind most EphA receptors, as well as EphB2 (10, 32), (18). It is established from detailed structure/function studies (10, IIIA4 binds exclusively to EphA3 (data not shown) and in 18, 19) that high-affinity Eph/ephrin complexes are held together combination with ephrin-A5 does not activate EphB2 (Supplemen- by three distinct interfaces forming a ring-like heterotetrameric tary Data). In contrast to the reports on direct antitumor effects of structure (Fig. 4C) and likely serve as nucleating unit for the anti-EphA2 mAbs (15, 29), we have not observed an effect of EphA3 formation of oligomeric signaling clusters (22). It is of note that activation, either with ephrin-A5, IIIA4, or their combination, on the low-affinity Eph/ephrin interface (Fig. 4C) is not apparent in cell viability in vitro or in vivo. However, the efficient receptor the crystal structure of the EphB2/ephrin-A5 complex, which, mediated uptake of both agonists, and the specificity, in particular compared with the EphB2/ephrin-B2 or EphA3/ephrin-A5 com- of the combined proteins, for EphA3-expressing cells advocates plexes, is of lower-affinity and has reduced signaling capacity (10). their use as tumor-targeting reagents suited to specifically deliver a We argue that this contact might function to stabilize the complex conjugated cytotoxic cargo to EphA3-positive tumor cells. Thus, for effective signaling, whereas ephrin-binding is maintained in radiometal conjugates of ephrin-A5 and IIIA4 retain their binding particular by the high-affinity ephrin-A5/EphA3 interface (18). This specificities, and in mice bearing solid or hematopoietic tumor rationale would provide an explanation, why in contrast to xenografts are effectively taken up into EphA3-positive cancer cells. competitive blocking by antagonistic antibodies (12, 30), high- In its current form, the rapid clearance of ephrin-A5 Fc from the affinity IIIA4 binding to this EphA3 surface has little effect on the circulation hindered assessment of its properties as targeting overall affinity of ephrin-A5 interaction but provides a molecular reagent in solid tumors, whereas the longer-lasting retention of tether for the formation of active signaling clusters (Fig. 4C). IIIA4-associated radioactivity in EphA3-positive, kidney endothelial Concurrent binding of nonclustered IIIA4 and ephrin-A5 is tumor xenografts for 14 days form the basis for development of a more efficient than aggregation. Our demonstration of signifi- specific tumor targeting strategy. Despite its short blood half-life, cantly elevated and accelerated EphA3 activation and internaliza- ephrin-A5, similar to IIIA4, effectively targets hematopoietic tumor tion by two distinct, nonclustered agonists which share a common xenografts. As expected, its targeting properties are determined by the expression pattern of endogenous binding partners; in contrast binding site on EphA3, superficially seem counter-intuitive to the to marginal EphA3 expression we show here significant endoge- known, well-established Eph activation mechanism. The individual, nous EphA2 mRNA and protein in normal mouse liver, emphasizing monomeric IIIA4 and ephrinA5-Fc proteins seem to facilitate the conceptional difficulties of using ephrin-Fc fusion proteins as protracted assembly of small ‘‘microclusters’’ of transmembrane specific Eph agonists in targeting studies. Experiments, exploiting EphA3, that remind of ‘‘capped’’ immune receptors (31) but have the possibility to combine ephrin-A5 and IIIA4 to increase the no apparent cell morphologic effect and clearly do not trigger targeting of the combined therapeutic reagent, are ongoing. notable EphA3 activation. It is of note that Eph activation without the need for cross-linking of either ephrin (4) or agonistic antibody (15) has been reported. However, our experiments clearly show that Acknowledgments in the case of IIIA4, this activity is due to small amounts of Received 3/4/2005; revised 4/21/2005; accepted 5/23/2005. antibody aggregates that are frequently present in the antibody Grant support: National Health and Medical Research Council of Australia grant 234707 (F-T. Lee, M. Lackmann, and A.M. Scott) and the Anti-Cancer Council of preparation. Indeed, we observed the presence of higher molecular Victoria (M. Lackmann and S.H. Wimmer-Kleikamp). weight aggregates also in some of our ephrin-A5 Fc preparations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance To avoid potential ambiguities arising from these aggregates, we with 18 U.S.C. Section 1734 solely to indicate this fact. routinely used SE-HPLC-purified protein in all our experiments. We thank Melissa Ciccomancini for help with xenograft studies. www.aacrjournals.org 6753 Cancer Res 2005; 65: (15). August 1, 2005

References 12. Mao W, Luis E, Ross S, et al. EphB2 as a therapeutic 23. Davis S, Gale NW, Aldrich TH, et al. Ligands for EPH- antibody drug target for the treatment of colorectal related receptor tyrosine kinases that require membrane 1. Poliakov A, Cotrina M, Wilkinson DG. Diverse roles of cancer. Cancer Res 2004;64:781–8. attachment or clustering for activity. Science 1994;266: eph receptors and ephrins in the regulation of cell 13. Luo H, Wan X, Wu Y, Wu J. Cross-linking of EphB6 816–9. migration and tissue assembly. Dev Cell 2004;7:465–80. resulting in signal transduction and apoptosis in Jurkat 24. Lawrenson ID, Wimmer-Kleikamp SH, Lock P, et al. 2. Dodelet VC, Pasquale EB. Eph receptors and ephrin cells. J Immunol 2001;167:1362–70. Ephrin-A5 induces rounding, blebbing and de-adhesion ligands: embryogenesis to tumorigenesis. Oncogene 2000; 14. Stuckmann I, Weigmann A, Shevchenko A, Mann M, of EphA3-expressing 293T and melanoma cells by CrkII 19:5614–9. Huttner WB. Ephrin B1 is expressed on neuroepithelial and Rho-mediated signalling. J Cell Sci 2002;115: 3. Nakamoto M, Bergemann AD. Diverse roles for the cells in correlation with neocortical neurogenesis. 1059–72. Eph family of receptor tyrosine kinases in carcino- J Neurosci 2001;21:2726–37. 25. Coulthard MG, Lickliter JD, Subanesan N, et al. genesis. Microsc Res Tech 2002;59:58–67. 15. Carles-Kinch K, Kilpatrick KE, Stewart JC, Kinch MS. Characterization of the Epha1 receptor tyrosine kinase: 4. Brantley-Sieders D, Parker M, Chen J. Eph receptor Antibody targeting of the EphA2 tyrosine kinase inhibits expression in epithelial tissues. Growth Factors 2001;18: tyrosine kinases in tumor and tumor microenvironment. malignant cell behavior. Cancer Res 2002;62:2840–7. 303–17. Curr Pharm Des 2004;10:3431–42. 16. Lackmann M, Mann RJ, Kravets L, et al. Ligand for 26. Lee FT, Rigopoulos A, Hall C, et al. Specific 5. Kullander K, Klein R. Mechanisms and functions of EPH-related kinase (LERK) 7 is the preferred high localization, gamma camera imaging, and intracellular Eph and ephrin signalling. Nat Rev Mol Cell Biol 2002; affinity ligand for the HEK receptor. J Biol Chem 1997; trafficking of radiolabelled chimeric anti-G(D3) gangli- 3:475–86. 272:16521–30. oside monoclonal antibody KM871 in SK-MEL-28 6. Cheng N, Brantley D, Fang WB, et al. Inhibition of 17. Lackmann M. Isolation and characterization of melanoma xenografts. Cancer Res 2001;61:4474–82. VEGF-dependent multistage carcinogenesis by soluble ‘‘orphan-RTK’’ ligands using an integrated biosensor 27. Himanen JP, Nikolov DB. Eph signaling: a structural EphA receptors. Neoplasia 2003;5:445–56. approach. Methods Mol Biol 2001;124:335–59. view. Trends Neurosci 2003;26:46–51. 7. Brantley DM, Cheng N, Thompson EJ, et al. Soluble 18. Smith FM, Vearing C, Lackmann M, et al. Dissecting 28. Scott AM, Geleick D, Rubira M, et al. Construction, Eph A receptors inhibit tumor angiogenesis and prog- the EphA3/Ephrin-A5 interactions using a novel func- production, and characterization of humanized anti- ression in vivo. Oncogene 2002;21:7011–26. tional mutagenesis screen. J Biol Chem 2004;279:9522–31. Lewis Y monoclonal antibody 3S193 for targeted 8. Dobrzanski P, Hunter K, Jones-Bolin S, et al. Anti- 19. Himanen JP, Rajashankar KR, Lackmann M, Cowan immunotherapy of solid tumors. Cancer Res 2000;60: angiogenic and antitumor efficacy of EphA2 receptor CA, Henkemeyer M, Nikolov DB. Crystal structure of an 3254–61. antagonist. Cancer Res 2004;64:910–9. Eph receptor-ephrin complex. Nature 2001;414:933–8. 29. Coffman KT, Hu M, Carles-Kinch K, et al. Differential 9. Gale NW, Holland, SJ, Valenzuela DM, et al. Eph 20. Lackmann M, Oates, AC, Dottori M, et al. Distinct EphA2 epitope display on normal versus malignant cells. receptors and ligands comprise two major specificity subdomains of the EphA3 receptor mediate ligand Cancer Res 2003;63:7907–12. subclasses and are reciprocally compartmentalized binding and receptor dimerization. J Biol Chem 1998; 30. Weinreb PH, Simon KJ, Rayhorn P, et al. Function- during embryogenesis. Neuron 1996;17:9–19. 273:20228–37. blocking integrin avh6 monoclonal antibodies: distinct 10. Himanen JP, Chumley MJ, Lackmann M, et al. 21. Stein E, Lane AA, Cerretti DP, et al. Eph receptors ligand-mimetic and nonligand-mimetic classes. J Biol Repelling class discrimination: ephrin-A5 binds to and discriminate specific ligand oligomers to determine Chem 2004;279:17875–87. activates EphB2 receptor signaling. Nat Neurosci 2004; alternative signaling complexes, attachment, and as- 31. Anderson CC, Sinclair NR. FcR-mediated inhibition 7:501–9. sembly responses. Genes Dev 1998;12:667–78. of cell activation and other forms of coinhibition. Crit 11. Boyd AW, Ward LD, Wicks IP, et al. Isolation and 22. Wimmer-Kleikamp SH, Janes PW, Squire A, Bastiaens Rev Immunol 1998;18:525–44. characterization of a novel receptor-type protein PI, Lackmann M. Recruitment of Eph receptors into 32. Flanagan JG, Vanderhaeghen P. The ephrins and Eph tyrosine kinase (hek) from a human pre-B cell line. signaling clusters does not require ephrin contact. J Cell receptors in neural development. Annu Rev Neurosci J Biol Chem 1992;267:3262–7. Biol 2004;164:661–6. 1998;21:309–45.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Concurrent Binding of Anti-EphA3 Antibody and Ephrin-A5 Amplifies EphA3 Signaling and Downstream Responses: Potential as EphA3-Specific Tumor-Targeting Reagents

Christopher Vearing, Fook-Thean Lee, Sabine Wimmer-Kleikamp, et al.

Cancer Res 2005;65:6745-6754.

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