FEBS 23047
`
`FEBS Letters 463 (1999) 115^120
`
`Selection of phage-displayed Fab antibodies on the active conformation
`of Ras yields a high a⁄nity conformation-speci¢c antibody preventing
`the binding of c-Raf kinase to Ras
`
`Ivo R. Horna, Alfred Wittinghoferb, Adriaan P. de Bru|«nea;c, Hennie R. Hoogenboomc;*
`aResearch Institute Growth and Development, Department of Pathology, University Hospital Maastricht, Maastricht, The Netherlands
`bMax Planck Institut fu«r Molekulare Physiologie, Abteilung Strukturelle Biologie, Rheinlanddamm 201, 44139 Dortmund, Germany
`cMaastricht University, P.O. Box 5800, 6202 AZ, Maastricht, The Netherlands
`
`Received 8 October 1999; received in revised form 15 November 1999
`
`Edited by Felix Wieland
`
`Abstract The Ras proteins cycle in the cell between an inactive
`state and an active state. In the active state, Ras signals via the
`switch I region to effectors like c-Raf kinase, leading to cell
`growth. Since Ras mutations in cancer are often associated with
`the presence of permanently active Ras, molecules that prevent
`downstream signaling may be of interest. Here, we show that by
`selection on the active conformation of Ras, using a recently
`described large phage antibody repertoire [de Haard et al. (1999)
`J. Biol. Chem. 274, 18218^18230], a Fab antibody (Fab H2) was
`identified that exclusively binds to active Ras, and not to inactive
`Ras. Using surface plasmon resonance (SPR) analysis, the
`interaction was demonstrated to be of high affinity (7.2 nM). In
`addition, the interaction with Ras is specific, since binding to the
`homologous Rap1A protein in BIAcore analysis is at least three
`orders of magnitude lower, and undetectable in an enzyme-linked
`immunosorbent assay. The antibody fragment prevents the
`binding of active Ras to the immobilized Ras-binding domain
`of c-Raf kinase (Raf-RBD) at an IC50 value of 135 nM. This
`to the KD of active Ras-binding to
`value compares well
`immobilized Raf-RBD using SPR, suggesting identical binding
`sites. Like the IgG Y13-259, which does not demonstrate
`preferential binding to either inactive or active Ras, Fab H2
`inhibits intrinsic GTPase activity of Ras in vitro. Mapping
`studies using SPR analysis demonstrate that the binding sites for
`the antibodies are non-identical. This antibody could be used for
`dissecting functional differences between Ras effectors. Due to
`its specificity for active Ras, Fab H2 may well be more selective
`than previously used anti-Ras antibodies, and thus could be used
`for gene therapy of cancer with intracellular antibodies.
`z 1999 Federation of European Biochemical Societies.
`
`Key words: Anti-GTP-Ras antibody; Antibody selection;
`Phage display; c-Raf kinase; Surface plasmon resonance
`analysis; Ras conformation; GTPase activity
`
`1. Introduction
`
`The Ras proteins are guanine nucleotide binding molecules,
`
`*Corresponding author. Fax: (31)-43-387 6613.
`E-mail: hho@lpat.azm.nl
`
`Abbreviations: ELISA, enzyme-linked immunosorbent assay; Fab,
`antigen binding fragment; GAP, GTPase activating protein; GAP-
`334, catalytic fragment of p120GAP ; GDP, guanosine diphosphate;
`GST, glutathione S-transferase; GTP, guanosine
`triphosphate;
`GTPQS, guanosine thiotriphosphate; Raf-RBD, Ras-binding domain
`of c-Raf kinase; scFv, single chain Fv fragment; SPR, surface plas-
`mon resonance analysis; TBS, Tris-bu¡ered saline
`
`essential for normal cellular proliferation and di¡erentiation
`[1,2]. These proteins belong to a superfamily of proteins
`termed GTPases that cycle between on and o¡ states triggered
`by binding and hydrolysis of the guanosine triphosphate
`(GTP) nucleotide [3,4]. Because of the ability to bind to di¡er-
`ent guanine nucleotides, leading to structural changes in the
`molecule, Ras serves as a molecular switch in signal trans-
`duction pathways [5,6]. The importance of Ras in normal
`cell physiology is underscored by the fact that in many types
`of human cancer the protein is mutated. In particular, onco-
`genic mutations are found in both glutamine 61 and glycine
`12 [7]. Such mutations a¡ect the intrinsic GTPase activity of
`Ras, rendering the molecule to be trapped in the active form
`[8]. The oncogenic Ras species resemble the active form of
`Ras on a biochemical as well as on a structural level [9,10],
`however, structural di¡erences account for the loss of inacti-
`vation in mutated Ras by the GTPase activating protein
`p120GAP [11].
`Crystal structures of the catalytic sites of both the active
`and inactive forms of Ras in complex with nucleotides have
`been determined as well as the structures of nucleotide bound
`Ras [10,12^16]. These studies have demonstrated that the
`switch I (residues 30^37) and switch II (residues 60^76) re-
`gions of Ras are conformationally a¡ected by exchange of the
`guanine nucleotides. Particularly switch I appears to be in-
`volved in the functional activity of Ras, since this domain
`overlaps the region generally referred to as the e¡ector loop
`(residues 32^40) [17,18]. The e¡ectors of Ras, amongst others
`c-Raf kinase, function as downstream signal transducing mol-
`ecules, which only bind to the active form of Ras.
`Extensive studies on the interaction of Raf-1 with Ras have
`demonstrated that on Raf-1 the binding residues are located
`within residues 55^131, generally referred to as Raf-RBD (Ras
`binding domain) [19]). The a⁄nity of Raf-RBD for GTP-Ras
`has been determined to be 18 nM, whereas the a⁄nity for
`guanosine diphosphate- (GDP)-Ras was shown to be in the
`micromolar range [20]. Determination of the crystal structure
`of the complex of the Ras-related protein Rap1A in the active
`form and Raf-RBD and by inference that of the complex of
`Ras with Raf-RBD has provided detailed insight into the
`switch I residues involved in the interaction [21]. Since it has
`been suggested that oncogenic Ras is insensitive to GAP ac-
`tivity, targeting the binding site of the e¡ectors may be an
`option to obtain reagents to treat Ras induced tumors [22].
`The e¡ector binding site has been suggested to be highly anti-
`genic, based on peptide studies, and therefore may be a suit-
`able target to select antibodies against Ras [23].
`
`0014-5793 / 99 / $20.00 (cid:223) 1999 Federation of European Biochemical Societies. All rights reserved.
`PII: S 0 0 1 4 - 5 7 9 3 ( 9 9 ) 0 1 6 1 7 - 8
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`I.R. Horn et al./FEBS Letters 463 (1999) 115^120
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`The neutralizing monoclonal antibody Y13-259 has been
`shown to inhibit the GTPase activity of Ras, binding to res-
`idues constituting the switch II region [24,25]. In vitro, the
`antibody either sterically hinders the interactions of e¡ector
`molecules binding to switch I, or prevents the exchange of
`GDP for GTP by compromising the conformational £exibility
`of Ras [26]. In vivo, the antibody has been demonstrated to
`inhibit Ras-mediated pathways using microinjection assays
`[27]. The variable domains of heavy and light chains of
`Y13-259 have been expressed as single chain Fv fragment
`(scFv) inside eukaryotic cells [28,29] (a technology generally
`referred to as intracellular immunization or intrabody expres-
`sion, [30,31]). Upon intracellular expression, the scFv Y13-259
`inhibits transforming activity in a number of studies [32,33],
`and even speci¢cally induces apoptosis in human cancer cells
`but not in untransformed cells [34]. Nevertheless, the Y13-259
`monoclonal antibody has been shown to be largely insoluble
`when expressed at 37‡C cells, leading to intracellular aggrega-
`tion [35].
`Phage display technology has been demonstrated to allow
`for rapid selection of human antibodies from large repertoires
`to any protein of interest (for a recent review, see [36]). Persic
`et al. used this approach to obtain human antibody scFv
`fragments to the switch II region of Ras using a peptide rep-
`resenting this domain for selection [37]. Anti-Ras antibodies
`were isolated, many of which were shown to inhibit cellular
`functions in mammalian cells (unpublished). In this study, we
`attempted to derive antibodies towards epitopes which are
`conformationally changed upon exchange of nucleotides, in
`particular the e¡ector loop, using the recently constructed
`large non-immunized phage-displayed antigen binding frag-
`ment (Fab) antibody repertoire [38]. We report here the iso-
`lation of a panel of human antibodies to Ras, some of which
`could be used either for dissecting the role of Ras-e¡ector
`interactions in cell physiology or eventually for cancer gene
`therapy, using intracellularly expressed antibodies.
`
`2. Materials and methods
`
`2.1. Proteins and chemicals
`Glutathione S-transferase fused H-Ras (GST-Ras), the catalytic
`fragment of p120Gap (GAP334) and the Raf-RBD were puri¢ed as
`described [11,20,39,40]. The monoclonal IgG antibodies Y13-259
`and F111-85 were purchased from Oncogene Sciences and polyclonal
`goat anti-GST as well as the pGEX4T2 vector were from Amersham
`Pharmacia Biotech. GST, encoded by the latter vector, was puri¢ed
`after expression in E. coli strain TG1 and subsequent puri¢cation
`using glutathione beads, according to the instructions of the supplier.
`Guanine thiotriphosphate (GTPQS) was purchased from Boehringer
`Mannheim. All other reagents and chemicals used were reagent grade
`(Sigma or Merck).
`
`2.2. Selections on conformationally restricted Ras
`Immunotubes (NUNC) were coated overnight at 4‡C with 100 Wg
`anti-GST. After washing three times with 20 mM Tris containing 150
`mM NaCl, pH 7.4 (TBS), tubes were blocked with TBS containing 2%
`w/v skimmed milk powder for 1 h at 37‡C. 1 WM GST-Ras was ¢rst
`bound to GDP or GTPQS by incubation in TBS containing 1 mM
`EDTA and the relevant nucleotide for 20 min at 37‡, followed by
`addition of 5 mM MgCl2. Subsequently, GST-Ras was captured by
`coated anti-GST for 30 min at 37‡C. Unbound GST-Ras was re-
`moved by performing three washing steps with TBS. Prior to selection
`on GST-Ras, 2U1012 phage from the non-immunized Fab repertoire
`[38], was depleted on coated goat serum for 30 min at 37‡C. Unbound
`phage was suspended into TBS containing 2% marvel, and allowed to
`bind for 90 min at room temperature to the captured GST-Ras. Sub-
`sequently, tubes were washed extensively using TBS with and without
`
`0.1% Tween 20 and bound phage was eluted using glycine-adjusted 50
`mM hydrochloric acid, pH 2.0. Phages were rescued and ampli¢ed as
`described [41], followed by another two rounds of selections. Phage
`repertoires were alternately depleted either by preincubation on
`coated goat serum or by adding 25% goat serum to the incubation
`mixture during the selection. During the third round of selection, the
`library was also depleted on anti-GST captured GST, prior to selec-
`tion on GST-Ras.
`
`2.3. Screening of the selected anti-GDP/GTPQS-Ras repertoires
`To identify GDP or GTPQS-Ras binding clones, an enzyme-linked
`immunosorbent assay (ELISA) was performed in which GST-Ras,
`after treatment with the relevant nucleotides, was captured by poly-
`clonal anti-GST. Coated wells were blocked using TBS containing 3%
`w/v bovine serum albumin and Fab-phage expressed by single colonies
`were allowed to bind for 1 h at room temperature. Subsequent ELISA
`washing and staining procedures were essentially as described [42].
`The number of unique Fabs was determined by PCR ¢ngerprinting
`using BstNI as described [41] followed by DNA sequencing.
`
`2.4. Puri¢cation of Fab fragments and surface plasmon resonance
`a⁄nity measurements
`Selected anti-Ras Fab fragments were expressed upon induction
`with isopropyl-L-D-thiogalactopyranoside (IPTG), harvested from
`the periplasmic space of E. coli TG1 cells and puri¢ed by immobilized
`metal a⁄nity chromatography. Brie£y, IPTG induced 500 ml cultures
`(4 h at 30‡C), expressing relevant anti-Ras antibodies, were spun at
`4600Ug for 20 min at 4‡C. Bacteria were subsequently resuspended in
`phosphate bu¡ered saline containing protease inhibitors (phenyl-
`methyl-sulfonyl £uoride and benzamidine) and sonicated using an
`ultrasonic disintegrator (MSE Scienti¢c Instruments). Suspensions
`were then centrifuged at 50 000Ug for 30 min at 4‡C, and supernatant
`fractions were incubated with TALON resin and eluted from the
`beads according to the instructions of the manufacturer (Clontech).
`Fab fragments were further puri¢ed by gel ¢ltration using a Superdex
`75 column (Amersham Pharmacia Biotech) connected to a Biologic
`instrument (Bio-Rad). Fab concentrations were quantitated using the
`bicinchoninic acid kit (Pierce).
`To determine equilibrium dissociation constants, anti-GST anti-
`bodies were immobilized onto a CM5 sensorchip in a BIAcore2000
`instrument (Biacore AB) by amine coupling at a high density of 120^
`140 fmol/mm2. Using TBS containing 2 mM MgCl2 and 0.01% v/v
`Tween 20 as a running bu¡er, the anti-GST was either loaded with
`GST or GST-Ras bound to GDP, GTP or GTPQS at a £ow rate of
`5 Wl/min at 25‡C, resulting in approximately 15 fmol captured protein,
`allowing for proper kinetic determinations. After increasing the £ow
`rate to 20 Wl/min, Fab antibodies were passed over the sensorchip at
`multiple concentrations around apparent KD values. Binding to either
`GST or anti-GST was subtracted from speci¢c binding responses to
`Ras. The rate constants kon and koff were obtained by direct ¢tting
`and from secondary plots (ks versus concentration), respectively, and
`¢tted to the data according to a single-site model, using the BIAeval-
`uation 2.1 software (Biacore AB). KD values that were calculated from
`kon and koff rate constants, ful¢lled the criteria for self-consistency
`[43].
`
`2.5. Competition studies using surface plasmon resonance analysis
`Raf-RBD was immobilized onto a CM5 sensorchip (Biacore AB)
`up to approximately 4500 resonance units (RU; corresponding to 4.5
`ng of protein). Prior to the analyses, Ras was loaded with GDP, GTP
`or GTPQS as described above. Using TBS containing 0.01% v/v Tween
`20 and 2 mM MgCl2 as running bu¡er using £ow conditions of 10 Wl/
`min, 100 nM of GTPQS loaded Ras, either untreated or preincubated
`with 1 WM concentrations of relevant antibodies, was passed over
`multiple Raf-RBD immobilized channels and residual binding was
`measured after 2 min association time. Fab antibodies were incubated
`over a range of concentrations. In case of no inhibition of Ras bind-
`ing, results are indicated in the ¢gure as 100% residual binding at the
`highest tested concentration.
`
`2.6. GTPase assays
`Hydrolytic activity of Ras either in the presence or absence of
`competitors was determined by performing GTPase assays according
`to the charcoal method of Bollag and McCormick [44]. In these ex-
`periments, Ras was incubated either in the presence or absence of the
`
`FEBS 23047 2-12-99
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`I.R. Horn et al./FEBS Letters 463 (1999) 115^120
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`117
`
`anti-Ras antibodies Fab H2, Fab A8 or IgG Y13-259 and GAP334 at
`concentrations of 1 WM. Experiments were performed in duplicate.
`
`2.7. Mapping studies
`Mapping studies using surface plasmon resonance analysis (SPR)
`were performed by loading the anti-GST immobilized chip with 8.3
`fmol (400 RU) of Ras-GTP, followed by injections well over KD of
`IgG Y13-259 immediately followed by saturating amounts of Fab H2.
`First, saturating amounts of either antibody was determined to be
`maximally 150 RU for IgG Y13-259 and 350 RU for Fab H2, re-
`spectively. Next, in the actual experiment, responses obtained were
`compared to these values.
`
`3. Results
`
`3.1. Selections of Fab antibodies on active and inactive
`GST-Ras
`Phage antibodies against Ras from large phage single chain
`Fv antibody libraries have been selected previously by a num-
`ber of methods, either selecting on peptides representing func-
`tional epitopes [37], or selecting on the full-length protein
`(unpublished results). However, to our knowledge antibodies
`have never been identi¢ed speci¢c for the conformational state
`of Ras, neither by hybridoma nor phage antibody technology.
`Since it has been suggested in literature [23] that particularly
`residues in the switch I region of the protein may be highly
`antigenic and that these residues appear to be either exposed
`or cryptic upon conformational changes induced by guanine
`nucleotide exchange, we have set out to identify antibodies
`with exquisite speci¢city by selection on GDP or GTP bound
`Ras. GTP speci¢c antibodies may later be used for intracel-
`lular immunization, in order to block Ras function in tumor
`cells. In addition, selection on conformational Ras and isola-
`tion may serve as a paradigm for the superfamily of GTP-
`binding proteins, the members of which are considerably ho-
`mologous and, most importantly, contain switch regions de-
`termining the speci¢city for intracellular e¡ector molecules
`[45].
`We chose to select antibodies to GST-Ras from the
`37 000 000 000 clones Fab antibody library, that has recently
`been constructed in our laboratory [38]. Ras was not immo-
`bilized to prevent
`immobilization-induced conformational
`changes but was captured by antibodies directed to the GST
`
`Fig. 1. Phage ELISA screening of Ras selected Fab phage. Binding
`to GTPQS-Ras is indicated by the black bars, binding to GDP-Ras
`by the hatched bars and binding to either captured GST or anti-
`GST by the dotted and open bars, respectively. A3, A8, A11, A12,
`B4 and G2 Fab phage were all selected on GDP-Ras, whereas A4,
`C6, C7, H2 and H3 were derived from GTPQS-Ras selections. Y13
`refers to the cloned Y13-259 scFv form [28].
`
`Fig. 2. Binding of Fab H2 and Fab A8 to captured GTP- or GDP-
`Ras using SPR. A: Dose-dependent binding of Fab H2 to captured
`Ras. The ¢gure shows initial loading of anti-GST with GST-Ras, ei-
`ther bound to GTP (1) or GDP (2), and subsequent binding of the
`antibody. B and C: Dose-dependent binding of Fab A8 to captured
`Ras, either to GTP- or GDP-Ras, respectively.
`
`moiety. By selection on GDP or GTPQS GST-Ras, while ex-
`tensively depleting for GST binders and binders speci¢c for
`anti-GST antibodies as described in Section 2, we obtained a
`panel of Fab fragments binding to Ras. During the four con-
`secutive rounds of selection, we found an increase in the ratios
`of output phage over input, indicative of enrichment for anti-
`gen binders, mounting to three orders of magnitude for both
`selections on GDP and GTPQS GST-Ras. To demonstrate
`binding of individual Ras Fabs, we performed a phage ELI-
`SA, in which we captured GST-Ras either bound to GDP or
`GTP. From both pannings, after the third round of selection,
`we obtained anti-Ras binders as indicated by this ELISA (70^
`80% positives, data not shown). To be able to demonstrate
`speci¢city for a de¢ned Ras conformation, we next performed
`an ELISA, in which we either captured GDP-Ras, GTP-Ras,
`GST or no protein at all, and bound phage antibodies were
`detected. The results are shown in Fig. 1. From both selec-
`tions, Fabs were selected binding to either form of Ras. The
`Fabs A3, A8, A11, A12, B4 and G2 were all obtained from
`the GDP-Ras selections. The Fabs A4, C6, C7, H2 and H3
`were derived from the GTP-Ras selections. The selections also
`yielded a low percentage ( 6 10%) non-speci¢c binders and
`GST binders. As can be seen in Fig. 1, Fab H2 demonstrated
`speci¢city for the active conformation of Ras. During the
`selections on GTP-Ras without depleting on captured GST,
`anti-GTP-Ras binders (Fab H2) were dominantly enriched
`(48.9% of binding Fabs). By depletion on GST, this number
`increased to 75% (see also Table 1). The percentage of GST
`binders (e.g. Fab H3) was reduced concomitantly from 40 to
`5.5%. Selections on GDP-Ras yielded Ras binders which do
`not discriminate between active and inactive Ras. Using
`BstNI ¢ngerprinting and DNA sequencing, we could identify
`nine di¡erent anti-Ras clones.
`
`Table 1
`GST depletion prior to pannings on GST-GTPQS-Ras increases the
`number of Ras-speci¢c clones after three rounds of selection
`Speci¢city
`No GST depletion %
`GST depletion %
`Anti-GTP-Ras
`48.9
`75.0
`Anti-Ras
`2.2
`5.6
`Anti-GST
`40.0
`5.5
`Negative
`8.9
`13.9
`
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`Only Y13-259, either in the IgG or scFv format, was capable
`of completely inhibiting the binding at micromolar concentra-
`tions. We used the same experimental setup to determine if
`our newly selected antibodies, directed to conformational epit-
`opes, may interfere with the Ras-Raf-RBD interaction. As
`shown in Fig. 3, the Fab H2 antibody inhibits the interaction
`in a dose-dependent fashion, at an IC50 value of 135 nM. The
`obtained BIAcore curves are shown in the inset ¢gure. A
`control antibody which demonstrates a good a⁄nity for Ras
`(anti-Ras 3, KD 52 nM, unpublished), as well as the Fab A11
`did not inhibit the interaction up to micromolar concentra-
`tions. Because of the observed inhibition of GTPQS-Ras bind-
`ing to Raf-RBD by Fab H2, we compared the DNA encoding
`VH and VL regions of Fab H2, with the primary sequence of
`Raf-RBD, but were unable to ¢nd similarities or signi¢cant
`homologies (data not shown).
`
`3.4. GTPase assays
`Since the IgG Y13-259 antibody can inhibit Ras mediated
`signal transduction in in vivo assays, preventing GTPase ac-
`tivity in in vitro systems and interacting with the conforma-
`tionally £exible switch II region, we were curious to see if the
`H2 antibody could also exert GTPase inhibitory activity.
`Therefore, we performed standard GTPase assays as described
`by Bollag and McCormick [44]. As controls, we performed
`incubations with Y13-259, either in the IgG or scFv format,
`with irrelevant antibodies (anti-MHC Fab G8, Cha“mes et al.,
`unpublished) and with GAP334. As expected, the incubations
`with irrelevant antibodies did not in£uence the rate of intrin-
`sic GTPase activity, whereas the incubations with Y13-259 or
`GAP334 completely inhibited or strongly stimulated the ac-
`tivity, respectively (Fig. 4). Upon incubation with Fab H2, the
`GTPase activity of Ras was completely blocked. scFv anti-
`Ras antibodies directed to regions which are una¡ected by
`conformational changes did neither inhibit nor stimulate
`GTPase activity (unpublished results).
`
`3.5. Mapping study
`Because of comparable features of antibody Y13-259 and
`Fab H2, we performed a mapping study using SPR. In this
`assay, we captured GTPQS-Ras with anti-GST and subse-
`
`Fig. 4. GTPase assay. Triangles (R) indicate the hydrolysis of Ras
`either in the presence or absence of irrelevant (G8) or anti-Ras Fab
`A11 antibodies. In£uences on the GTPase activity by GAP-334 and
`by IgG Y13-259 are represented by the diamonds (8) and by the
`circles (b), respectively. Incubations in the presence of Fab H2 are
`represented by the squares (F).
`
`Fig. 3. BIAcore competition experiment. Measurements of competi-
`tion of Ras binding to Raf-RBD by scFv anti-Ras 3 (R), Fab A11
`(F), or Fab H2 (b), using SPR. The binding curves, representing
`Ras binding in the absence of Fab H2 (upper curve), or increasing
`amounts up to 1 WM (lower curve), are shown in the inset ¢gure.
`
`3.2. A⁄nity determination of anti-Ras Fab antibodies
`Next, we puri¢ed the Fab antibodies H2 (active Ras specif-
`ic), A8 and A11 (both pan-Ras reactive) as three representa-
`tive clones and determined the a⁄nity of the clones for Ras
`using SPR. To compare binding to di¡erent Ras-forms, we
`developed a BIAcore assay as follows. After coupling a high
`amount of polyclonal anti-GST to a CM5 BIAcore sensor-
`chip, we captured Ras either treated with GDP or GTPQS and
`subsequently measured the interaction of the antibodies with
`the captured Ras. Fig. 2 shows two sensorgrams run synchro-
`nously in di¡erent channels. In channel 1, a low amount of
`GTPQS-Ras and in channel 2 a low amount of GDP-Ras was
`captured. After capturing per channel individually at a £ow
`rate of 5 Wl/min, £ow rates were increased to 20 Wl/min to
`allow for kinetic determinations and antibodies were passed
`synchronically over both channels and a control background
`channel. As can be seen in Fig. 2A, Fab H2 only binds to the
`active form of Ras. The calculated Kd for the interaction is 7.2
`nM (ka, 3.6U105 M31 s31, kd, 2.6U1033 s31). The speci¢city
`for Ras was demonstrated by the fact that Fab H2 does not
`interact with the related Rap1A protein (not shown). The Kd
`value of the interaction of Fab H2 with active Ras compares
`well to a calculated Kd value determined in an ELISA (6 nM,
`data not shown). Fab A8 bound to both forms of Ras
`(GTPQS-Ras, Fig. 2B and GDP-Ras, Fig. 2C), although
`with lower a⁄nity (calculated Kd values 197 nM and 211
`nM for GTPQS-Ras and GDP-Ras, respectively). The interac-
`tion of A11 with Ras was not quantitated accurately, how-
`ever, the apparent a⁄nity was in the same order of magnitude
`as that of Fab A8. The reverse experiment, in which the anti-
`bodies Fab H2 and A8 were immobilized to CM5 sensorchips
`and subsequent binding of either GDP, GTP or GTPQS-Ras
`was measured, yielded similar results (data not shown).
`
`3.3. Competition of Fab antibodies for Ras binding to
`Raf-RBD
`Recently, using SPR, we measured binding of GTPQS-Ras
`and GTP-Ras, but not GDP-Ras to immobilized Raf-RBD
`and determined possible interference of selected scFv’s on this
`interaction (unpublished results). None of the tested antibod-
`ies, selected on directly coated Ras, were capable of inhibition.
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`119
`
`bind, only contains selection dominant epitopes in the active
`conformation. The latter suggestion is strengthened by the
`fact that in addition to a panel of Ras binders, the GTP-
`Ras speci¢c H2 clone was dominantly selected (see also Table
`1) and the a⁄nities of antibodies to Ras indi¡erent of the
`conformation, were rather low (1037 M). Studies undertaken
`by Wang and colleagues, who made Ras peptides and isolated
`antibodies against them, have indicated that particularly the
`e¡ector binding region may be highly antigenic in vivo [23].
`Selection-dominant epitopes may often be overlapping with
`immunogenic epitopes, as demonstrated in a study by Hoo-
`genboom et al. [47]. The possible immunogenicity of the ef-
`fector loop may as well explain the strong selection of the high
`a⁄nity Fab H2 during the stringent selections.
`Since members of the superfamily of GTP-binding proteins
`are all characterized by the presence of the switch regions,
`which are a¡ected conformationally upon nucleotide binding,
`the method described could well serve as a general way to
`rapidly select antibodies against these conformationally £exi-
`ble regions. Particularly, if the aim would be to discriminate
`between di¡erent GTP-binding proteins, mediating di¡erent
`cellular events by binding to di¡erent e¡ectors, this method
`may be applicable, since the Fab H2 antibody demonstrates a
`much higher a⁄nity for Ras than for Rap1A. The di¡erence
`in a⁄nity is at least three orders of magnitude in BIAcore,
`and binding of Fab H2 to Rap1A in ELISA could not be
`detected at all.
`The Fab H2 antibody could be an interesting candidate for
`intracellular antibody expression studies. Work is currently
`performed to clone the antibody in a suitable eukaryotic ex-
`pression vector [29] and express it in the smaller scFv format.
`Antibodies have been intracellularly expressed in a number of
`studies [30,31]. However, attempts to express antibodies that
`were obtained by classical hybridoma technology and subse-
`quently cloned have not always been successful. The antibody
`may not fold correctly in certain compartments of eukaryotic
`cells and expression levels may be poor. This may be corrected
`by mutation and selection as was proposed by Martineau et
`al. for cytoplasmic antibody expression inside bacteria [48].
`On the other hand, a new mechanism of action may be ob-
`served upon intracellular expression of an antibody in a di¡er-
`ent molecular format. This was proven to be the case for the
`Y13-259 antibody by Cardinale and co-workers when they
`expressed the scFv format of this antibody in a eukaryotic
`cell system [35]. The scFv was highly aggregating intracellu-
`larly, thereby trapping the intracellular Ras in an insoluble
`complex which can be subsequently degraded by the cell.
`The question remains if a targeted approach is actually
`required for inactivation of intracellular targets. In a recent
`study, Lener and coworkers have shown that expression of
`non-inhibitory antibodies intracellularly may very well lead
`to aggregation resulting in inhibition of intracellular functions
`or pathways (unpublished). In this study, the measured low
`a⁄nity did not appear to a¡ect the e⁄ciency of intracellular
`target inactivation. The Fab H2 antibody, however, demon-
`strates speci¢city for Ras and can inhibit a particular protein-
`protein interaction, thereby most likely allowing us to discrim-
`inate between inhibition of certain intracellular pathways. Be-
`cause of its speci¢city, it may also be used to quantitate intra-
`cellular active Ras levels. Alternatively, molecules (either
`antibodies or alternative sca¡olds) inhibiting particular intra-
`cellular interactions or signal transduction routes, could also
`
`Fig. 5. Epitope mapping of Fab H2 binding to Ras using SPR.
`Captured GTPQS-Ras was ¢rst saturated with monoclonal antibody
`Y13-259 and subsequently incubated with Fab H2.
`
`quently determined maximal binding responses of antibodies
`binding to Ras. Taking into account the molecular mass of
`the antibody, maximal binding of Y13-259 was lower than
`expected, possibly due to a low accessibility of the epitope
`in this experimental setup. After determining the maximal
`binding responses, we saturated the Ras with IgG Y13-259
`and then injected a saturating amount of Fab H2. The results
`of this experiment are depicted in Fig. 5. As can be deduced
`from this ¢gure, Y13-259 and Fab H2 can both bind to the
`captured Ras,
`indicative of independent binding sites. The
`opposite experiment, in which Ras was ¢rst saturated with
`Fab H2 and then with Y13-259, yielded similar results. Be-
`cause of the apparent low accessibility of the Y13-259 epitope
`in the SPR assay, we performed a competitive ELISA in
`which we competed with Y13-259 for Fab H2 binding and
`reverse under conditions of half-maximal saturation. Since
`competition was observed only at high concentrations of
`Y13-259 for Fab H2 binding, whereas the reverse could not
`be shown, we conclude that the binding sites are non-identi-
`cal.
`
`4. Discussion
`
`In the present study, we have used phage display technol-
`ogy to rapidly select antibodies to the active conformation of
`Ras. In this way, we attempted to increase the chance of
`isolating antibodies capable of inhibiting the interaction of
`downstream e¡ectors like c-Raf kinase with Ras. We have
`used a large Fab antibody library, which in our lab has
`yielded a number of antibodies to diverse antigens binding
`to their targets with nanomolar a⁄nities [38]. This library
`was selected on captured GST-Ras, bound to either GDP or
`GTPQS. This approach yielded a Fab antibody that interacts
`with Ras with high a⁄nity and inhibits the intrinsic GTPase
`activity and the binding of Ras to Raf-RBD. Surprisingly, the
`selections on GDP-Ras did not yield any clones speci¢c for
`the inactive conformation. In fact, several antibodies were
`obtained that interact with Ras irrespective of the conforma-
`tional state. The reason for these ¢ndings may be the fact that
`the e¡ector loop is highly £exible in the GDP-bound state, but
`rather ¢xed in the GTP-bound state, when it exists in only
`two conformations due to the fact that the Q-phosphate inter-
`acts with a tyrosine residue in the loop [46]. It also suggests
`that the e¡ector binding domain, to which Fab H2 seems to
`
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`be selected using an in vivo approach. Screening could sub-
`sequently be performed based on for instance phenotypic
`changes, apoptosis, cell morphology or surface protein expres-
`sion. The initial proof of concept of an in vivo approach has
`been reported by Gargano and Cattaneo in a study in which
`cells expressing anti-retroviral antibodies were rescued [49].
`An in vivo selection may be done using a previously selected
`antibody repertoire, directed against a known antigen, as well
`as using a completely naive repertoire. In the latter case, the
`unknown target will be identi¢ed in a later instance, possibly
`yielding new insights in importance of molecules involved in
`signal transduction pathways or leading to the discovery of
`new signal transduction molecules. Such antibodies will per
`de¢nition be antibodies that can be expressed intracellularly,
`avoiding the possible problems associated with expression of
`in vitro selected antibodies. Well expressed antibodies, for
`example selected for high level expression inside the cell using
`procedures as described by Martineau, may later be used as a
`sca¡ol