WO2010108824A1

WO2010108824A1 - L-plastin antibodies and their use

Info

Publication number: WO2010108824A1
Application number: PCT/EP2010/053387
Authority: WO
Inventors: Jan Gettemans; Veerle Delanote; Evelyne Friederich
Original assignee: Vib Vzw; Universiteit Gent; Universite Du Luxembourg
Priority date: 2009-03-23
Filing date: 2010-03-16
Publication date: 2010-09-30

Abstract

The present invention relates to antibodies, especially camelid antibodies, binding to the tandem actin binding domain in L-plastin, but not to the single actin binding domains. It further relates to the use of those antibodies to limit cell invasion and metastasis in cancer.

Description

L-PLASTIN ANTIBODIES AND THEIR USE

The present invention relates to antibodies, especially camelid antibodies, binding to the tandem actin binding domain in L-plastin, but not to the single actin binding domains. It further relates to the use of those antibodies to limit cell invasion and metastasis in cancer. Reorganization of the actin cytoskeleton accompanies several physiological processes including cell motility, differentiation and endo-/exocytosis, but is also more and more recognized as being essential for cancer cell motility and invasion (Vignjevic & Montagnac, 2008; Yamaguchi & Condeelis, 2007). Actin remodeling is controlled by activation of signaling pathways in which small-GTPases are involved resulting in activaton of downstream actin binding proteins (ABPs), of which many are expressed in mammalian (cancer) cells. Aberrant expression of ABPs in cancer cells, as well as their direct involvement in malignant cell behavior, have been demonstrated in many studies (Hara et al, 2007; Thompson et al, 2007; Wang et al, 2004; Wang et al, 2006). L-plastin (also called L-fimbrin) is an actin bundling protein consisting of an N-terminal Ca2+ binding module, and two C-terminal actin binding domains. The Ca2+ binding modules are made up of 2 EF-hand structures, whereas each actin binding domain is built up of two calponin homology (CH) domains (reviewed in Delanote et al, 2005b; Hara et al, 2007).

F-actin binding and bundling activities of L-plastin are negatively regulated by Ca2+ (Namba et al, 1992). L-plastin is also localized in cell structures such as microspikes and podosomes that are involved in motility, adhesion and invasion (Evans et al, 2003; Janji et al, 2006; Messier et al, 1993). In addition, L-plastin shuttles between the nucleus and cytoplasm of cells in contrast to its close relative, T-plastin (Delanote et al, 2005a). In adults, L-plastin is exclusively found in hematopoietic cells. However, 'ectopic' expression of the protein is observed in epithelial and mesenchyme-derived cancer cells of solid tumors (Park et al, 1994). L-plastin expression is significantly correlated with the progression of colorectal cancer staging (Otsuka et al, 2001 ), and has been suggested as a potential metastatic marker. Futhermore, a clear causal relationship between plastin expression modulation, either by down-regulation or overexpression, and invasion/metastasis is well established (Janji et al, 2006; Klemke et al, 2007; Zheng et al, 1997; Zheng et al, 1999). However, the exact mechanism by which L-plastin interaction with the actin cytoskeleton contributes to malignant cell behavior remains unclear. Conventional antibodies have proven useful in the development of diagnostics and drugs for treatment of various diseases including cancers. By immunomodulation, the in vivo function of selected proteins can be regulated by intracellular expression of antibodies (intrabodies). Immunomodulation has advantages over RNA interference (RNAi) since it is possible to specifically target individual protein -protein interactions (Visintin et al, 2008). Furthermore, RNAi is limited by incomplete RNA cleavage, inaccessible RNA sequences, unspecific targeting and difficult to achieve for target proteins with a long half-life (Cao & Heng, 2005; Persengiev et al, 2004). lntrabodies can be directed against unique protein domains. This great selectivity allows modulation of the function of one domain by the specific intrabody at any time without interfering with other activities of the target protein.

Such in vivo "protein domain knockout" has obvious advantages over depleting the cell of the whole protein (Lichtlen et al, 2002). Intracellular expression of multidomain antibodies (single chain variable fragments or scFv) has proven problematic due to incorrect assembly of the VH and VL chains, low solubility and proteolytic degradation of the linkers (Cattaneo & Biocca, 1999; Worn et al, 2000; Worn & Pluckthun, 2001 ). A promising alternative are the naturally occurring heavy chain antibodies, which are devoid of light chains, first discovered in Camelidae (Hamers-Casterman et al, 1993). Their antigen-binding domains, called variable domains of heavy chain antibodies, VHHs or nanobodies, are extremely stable and soluble entities (Dumoulin et al, 2002; Perez et al, 2001 ). VHH antigen binding regions are limited to single immunoglobulin (IgG) domains, which makes them relatively easy to clone. These robust proteins represent the smallest in vivo matured functional antigen binding domains. In addition, VHHs recognize unique conformational epitopes due to their enlarged complementarity determining region 3 (CDR3) (Desmyter et al, 2001 ). Several applications of VHHs as intrabodies have been reported. Fluorescent protein-tagged nanobodies can be used as tracers in living cells (Rothbauer et al, 2006), VHHs can inhibit the proapoptotic protein bax in living cells (Gueorguieva et al, 2006) and Jobling et al., (2003) showed that VHHs can inhibit enzyme function more efficient than anti-sense approaches.

Classical antibodies - but no nanobodies - against L-plastin have been described (US65310653, Frederick et al., 1996; Shinomya et al., 2003; Toyooka et al., 2006). Said antibodies can be used for diagnostic purposes. However, none of those antibodies has been expressed or was used as intrabody. Moreover, none of these antibodies are capable of inhibiting actin bundling. Indeed, as these antibodies have been developed for diagnostic purposes, the fact that they can be used in Westerns blot indicates that these antibodies recognize linear epitopes, whereas the recognition of a conformational epitope is essential for the inhibition of actin bundling and repression of the biological function of L-plastin, and/or its role in cell invasion, metastasis and/or cancer. In this study, VHHs were generated against human L-plastin. Surprisingly the antibodies generated recognized conformational epitopes, and several of those antibodies could modulate cancer cell morphology, migration and invasion when expressed as intrabodies. These results substantiate not only the role of L-plastin in cancer cell motility and invasion, but point to single domain VHHs as a new instrument to probe and curb the activity of pro-invasive proteins.

A first aspect of the invention is an anti-L-plastin antibody, binding both actin binding domains of said L-plastin. Preferably, the antibody is not binding the N-terminal end of L-Plastin, more preferably it is not binding to the EF-hand structures. Preferably, said antibody recognizes a conformational epitope. A preferred embodiment is an antibody according to the invention whereby the binding of the antibody to L-plastin is Ca²⁺ independent.

Even more preferably, said antibody is a nanobody. Preferably, said nanobody comprises SEQ ID N°1 (Table 3), even more preferably the CDR1 loop of said nanobody consists of SEQ ID N°1. Most preferably, said nanobody comprises, preferably consists of SEQ ID N° 2 (Table 3; Figure 1 : Nb 5). A still further preferred embodiment is an antibody according to the invention whereby said antibody is expressed as an intrabody. Intrabody, as used here, is an antibody that is present as a functional antibody within a living cell and exerts its function and binding in said cell. Said intrabody can be obtained by expressing the coding sequence of the antibody in the cell, whereby said expression results in a functional antibody. Alternatively, the antibody may be internalized in the cell, preferably as a fusion product, such as a fusion of said antibody with a protein transduction domain, whereby said antibody is functional after internalization in the living cell. Another aspect of the invention is the use of an antibody according to the invention to suppress cell invasion and/or metastasis. Suppressing, as used here, means that there is significantly less cell invasion and/or metastasis with the use of the antibody according to the invention compared with a control where no antibody or an irrelevant antibody is used. Suppressing of cell invasion and/or metastasis can be realized in vitro or in vivo. Therefore, another aspect of the invention is the use of an antibody to treat cancer. In vivo treatment of cancer implies that the antibody can be active in the cancer cells. Expression of the antibody can be realized by gene therapy; methods for gene therapy are known to the person skilled in the art. Alternatively the antibody according to the invention is fused to a protein transduction domain, and internalized in the cancer cells. Protein transduction domains are known to the person skilled in the art and comprise, but are not limited to TAT (disclosed in WO0119393), pep-1 (disclosed in US2003119725) and penetratin (disclosed in US5888762). Alternatively, as L-plastin is mainly overexpressed in epithelial derived tumors, the antibody according to the invention may be fused to an EGFR1 binding protein, such as an antibody directed to EGFR1. By binding to the receptor, the construct will be internalized and the antibody according to the invention will bind to L-plastin and suppress cell invasion and/or metastasis.

Brief description of the figures

Figure 1. Amino acid sequences of anti-human L-plastin Nanobodies. All Nanobodies originate from VHH germline sequences. The complementarity determining regions (CDRs) are shown in bold. The gaps were introduced in order to align the sequences. The differences between some of the Nanobodies are limited to only one or few amino acids. Figure 2. lmmunoprecipitation of endogenous L-plastin with specific nanobodies in PC-3 cells. PC-3 cells were transfected with V5-tagged nanobodies which were recovered from the lysate by binding on anti-V5 agarose. Endogenous L-plastin co-immunoprecipitates with all L- plastin nanobodies but to varying extent (upper panel). Lower panel; western blot with mouse anti-V5 antibody showing expression of the VHHs in PC-3 cells. (Nb = Nanobody). Members of the same nanobody family are boxed.

Figure 3. Nanobodies bind different conformations of L-plastin. (A) SDS-PAGE and Coomassie staining of purified recombinant His6-tagged L-plastin nanobodies used in gelfiltration and ITC analysis. (B) Schematic representation summarizing thermodynamical parameters for different nanobodies used in the ITC experiments. (C) Superdex gelfiltration of

L-plastin-Nb complexes. Black line: L-plastin (75 μg) without Ca2+ or nanobody; light grey line:

L-plastin (75 μg) with nanobody (50 μg), without Ca2+; dark grey line: L-plastin (75 μg) including nanobody (50 μg) and Ca2+. Left panel: addition of recombinant nanobody 5. Ca2+ has no influence on binding of nanobody 5 to L-plastin. Right panel: addition of recombinant nanobody 9. Nanobody 9 only binds L-plastin when Ca2+is included. AU = absorbance units.

Figure 4. Nanobodies do not affect F-actin-L-plastin interaction in vitro. (A) SDS-PAGE analysis of a high speed co-sedimentation assay. Binding of L-plastin to F-actin: G-actin (7 μM) was copolymerized with recombinant L-plastin (3μM) and different recombinant nanobodies up to a concentration of 30 μM. Coomassie-staining patterns of pellets and supernatants in the absence or presence of nanobodies are shown for comparison. p= pellet, s=supernatant. (B)

Quantification of binding of L-plastin to F-actin in the presence or absence of nanobodies. Amou nts of L-plastin in pellets and supernatants were quantified by densitometry of

Coomassie-stained protein bands. Each bar is the mean of three independent experiments ± s.d.

Figure 5. Nanobodies inhibit F-actin bundling by L-plastin. (A) Low speed co-sedimentation assay: G-actin (7 μM) was copolymerized with recombinant L-plastin (3μM) and different recombinant nanobodies at a concentration of 30 μM. Supernatants and pellets were analyzed by SDS-PAGE. Coomassie-staining patterns of pellets and supernatants in the absence or presence of nanobodies are shown. Notice that Nbs 5-8 reverse the pattern of the amount of actin in the pellet (p) and supernatant (s). (B) Quantification of bundling of F-actin by L-plastin and nanobodies. Amounts of actin in pellet and supernatant were quantified by densitometry of

Coomassie-stained protein bands. Each bar is the mean of three independent experiments ± s.d. (C) Quantification of the amount of nanobody in the pellet fraction and supernatant by densitometry of Coomassie-stained protein bands. Co-sedimentation of nanobodies 2 and 9 is significantly higher compared to the other nanobodies, implicating that there is binding between these nanobodies and L-plastin in the absence of Ca2+ but presence of actin. (D) The low speed co-sedimentation assay as in (A) was repeated with increasing molar concentration ratios of L-plastin to nanobody as indicated in the horizontal axis. p=pellet; s=supernatant.

Figure 6. Binding of VHHs to L-plastin EF-hands or CH domains (epitope mapping). (A) Schematic diagram of the different recombinant GST-tagged L-plastin fragments used in the GST-pull down assay. GST was included as a control. LPL= L-plastin, ABD= actin binding domain, CH= calponin homology domain EF= EF-hand domain. GST=glutathione S- transferase. N= NH2-terminus; C= COOH-terminus (B) SDS-PAGE of GST pull down assay using different recombinant GST-tagged L-plastin fragments with addition of recombinant nanobody. Nb 5 binds only with the combination of the two ABDs. Nbs 2 and 9 bind with the EF-hand motifs. Last lane = nanobody input. (C). SDS-PAGE analysis of chemical crosslinking (X-link) of nanobodies with GST-EF. Cross-linking was carried out with 8μM plastin construct and 32 μM nanobody. Reaction products were analyzed by SDS-PAGE showing that nanobodies 2 and 9 bind to the EF-hands of L-plastin.

Figure 7. MOM recruitment assay and effect of nanobodies on filopodia. Through binding with the MOM-tagged Nb, L-plastin localizes to mitochondria. (A-D) Fluorescence images of fixed PC-3 cells expressing different MOM-tagged nanobodies. Staining of the nanobodies was performed with anti-V5 antibody (red). Endogenous L-plastin was visualized with anti-Lplastin antibodies (green). Expression of the negative control anti-GFP nanobody does not direct endogenous L-plastin to the mitochondria (A). (B-D) Colocalization of MOM-tagged nanobody (MOM Nb 2, 5 and 9) and endogenous L-plastin at mitochondria. Bar = 10 μm. (EH) Intracellular distribution of L-plastin and intrabodies in PC-3 cells. PC-3 cells were transfected with the empty GFP vector or with GFP-tagged L-plastin nanobodies. White arrows point at specialized surface structures. Endogenous L-plastin was visualized with anti-L-plastin antibody (red). GFP tagged nanobody is shown in green. There is colocalization in filopodia and in membrane ruffles (F-H). There is no specific localization between empty GFP and L- plastin (E). Bar = 10 μm. (I) Cells expressing nanobody 5 have less filopodia compared to cells transfected with nb 2 or nb 9. Cells with long filopodia were counted for GFP expression. Each bar is the mean of three independent experiments ± s.d.

Figure 8. Effect of L-plastin siRNA or L-plastin intrabody expression on cell migration of PC-3 cells in a woundhealing assay. Cells were transfected with (A) negative control siRNA or L- plastin siRNA. Downregulation of L-plastin by siRNA compared with the control siRNA is shown in the upper part (B) Transfection of PC-3 cells with different nanobodies. Anti-GFP nanobody is the negative control. Efficiency in wound closure as a function of time is shown. Motility as measured by the rate of migration of cells into a wound was lower in L-plastin siRNA (A) or L-plastin nanobody transfected cells (B) in comparison with the corresponding negative controls.

Figure 9. lmmunomodulation of L-plastin on matrigel invasion of PC-3 cells. PC-3 cells were transfected with a nanobody. Different types of L-plastin intrabodies decrease invasion of PC-3 cells into matrigel. Anti-GFP nanobody was used as a negative control. RFU, relative fluorescence units are shown in the y-axis. Each bar is the mean of 3 independent experiments ± s.d..

Examples

Materials and methods to the examples

Reagents and antibodies

Monoclonal anti-V5 antibody and HiFi Platinum Taq polymerase were purchased from Invitrogen (Merelbeke, Belgium). Polyclonal anti-V5 antibody was purchased from Sigma (St Louis, USA). An L-plastin-specific antibody was purchased from Neomarkers (Fremont, CA, USA); rabbit anti-GFP was from Santa Cruz, goat anti-GST from Amersham Biosciences. β-G- actin was purchased from Cytoskeleton (Boechout, Belgium). Lipofectamine reagent was purchased from Invitrogen. All commercial antibodies were used at the dilution recommended by the manufacturer.

Cell culture

PC-3 cells were maintained at 37 °C in a humidified 10% CO2 incubator and grown in RPMI 1640 (Gibco-BRL Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 IU/ml penicillin. The cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Generation of VHHs and purification

Immunization. VHH antibodies were obtained in collaboration with the VIB nanobody service facility (VUB, Brussels, Belgium). An alpaca was injected subcutaneously on days 0, 7, 14, 21 , 28, 35 with -500 μg human L-plastin per injection. L-plastin was purified as described (Arpin et al., 1994). On day 39, anti-coagulated blood was collected for the analysis of the immune response and for the preparation of lymphocytes. IgG subclasses were obtained by successive affinity chromatography on protein A and protein G columns. Total serum and three purified IgG subclasses were tested by ELISA to assess the immune response to human L-plastin. IgGI and lgG2 responses were similar, while lgG3 response was the highest.

Construction of a nanobody library. Total RNA from peripheral blood lymphocytes was used as template for first strand cDNA synthesis with oligodT primer. Using this cDNA, the VHH encoding sequences were amplified by PCR, digested with Pstl and Notl, and cloned into the Pstl and Notl sites of the phagemid vector pHEN4. A VHH library of about 7 x 107 independent transformants was obtained. About 86% of these transformants harbored the vector with the right insert size.

Isolation of anti-plastin nanobodies. Three consecutive rounds of panning were performed on solid-phase coated recombinant plastin (10 μg/well). The enrichment for antigen-specific phages was further evaluated by polyclonal phage ELISA. Antigen-specific phages were enriched after each round of panning. In total, 144 individual colonies were obtained and analyzed by ELISA for the presence of antigen-specific VHHs in their periplasmic extracts. Out of these 144 colonies, 42 colonies scored positive in this assay. Based of Hinfl RFLP analysis, 17 clones were selected for sequence analysis. These 17 clones represented 9 different nanobody cDNAs. The specificities of these VHHs were again confirmed by ELISA in an independent experiment using soluble nanobodies. cDNA cloning. The expression plasmids pHEN6c (kindly provided by Dr. Gholamreza Hassanzadeh Ghassabeh, VIB nanobody service facility), pEGFP-N1 (clontech) and pcDNA3.1A/5His/TOPO (invitrogen) were used to subclone nanobodies, which were amplified by PCR with the following primers: GFP Nb Fwd : 5' TCG AAT TCG CCA CCA TGC AGG TGC AGC TGC AGG AG 3'; GFP Nb Rev : 5' ATG ACT GAT GGA TCC TTG CTG GAG ACG GTG ACC TG 3'; pHEN6 Nb Fwd: 5' GAT GTG CAG CTG CAG GAG TCT GGA/G GGA GG 3'; pH EN6 Nb Rev: 5' GGA CTA GTG CGG CCG CTG GAG ACG GTG ACC TGG GT 3'; pcDNA3.1 V5/His Nb Fwd: 5' GGT TTA AAC GCC ACC ATG GCC CAG GTG CAG CTG CAG GAG TCT GGG 3'; pcDNA3.1 V5/His Nb Rev: 5' GCC ACT AGT TGC TCG GCC GGA ACC GTA GTC CGG 3'. The cDNA of the anti-GFP nanobody was a kind gift of Dr. Gholamreza Hassanzadeh Ghassabeh, VIB nanobody service facility.

For the analysis of intracellular recruitment of L-plastin by VHHs, a vector was constructed containing the transmembrane sequence of the mitochondrial outer membrane protein TOM 70 (Pfanner and Meijer, 1997; Hill et al., 1998; Schatz, 1998) according to Kaufmann et al., 2000. Two pairs of nucleotide sequences coding for the 29 amino acids of the TOM70 (EMBL X05585) peptide identified earlier as the mitochondrial anchor sequence (McBride et al., 1992) were annealed, phosphorylated with T4 kinase (Invitrogen), ligated with T4 ligase and inserted into the pcDNA3.1A/5His/TOPO vector to create the vector pMOM. Primers used are: fwα"l : CTC GAG ATG AAG TCA TTC ATC ACT CGT AAC AAG ACT GCA ATC CTA GC; fwd2: TAC GGT CGC TGC AAC TGG TAC CGC TAT CGG AGC TTA CTA TTA CTA TA; rev1 : GAT TGC AGT CTT GTT ACG AGT GAT GAA TGA CTT CAT CTC GAG A; rev2: ATA GTA ATA GTA AGC TCC GAT AGC GGT ACC AGT TGC AGC GAC CGT AGC TAG. This vector was then used to clone the different VHHs after amplification with the following primers: MOM V5 pcDNA3.1 Nb Fwd: 5' CGT ACC GGT GCC CAG GTG CAG CTG CAG GAG TCT GG 3'; MOM V5 pcDNA3.1 Nb Rev: 5' CGT ACC GGT CTA GCT GGA GAC GGT GAC CTG GGT C 3'. Nanobody purification. pHEN6c-nanobody His6 plasmids were transformed in E. coli WK6 and a freshly transformed colony was grown overnight in LB medium containing ampicilin (100μg/ml) and 1 % glucose. The next day, 1 ml of pre-culture was added to 330 ml of TB medium supplemented with ampicillin, 2mM MgCI2 and 0.1% glucose and grown at 37°C with shaking until an OD600 of 0.6-0.9 was reached. Nanobody expression was induced by addition of IPTG to a final concentration of 1 mM and incubated at 28°C with shaking overnight. Recombinant His6 - tagged nanobodies were purified from E. coli WK6 cells by binding to Ni2+-chelating beads (Probound nickel resin, Invitrogen) and were recovered from the beads by elution with 50OmM imidazole pH 8.25. The nanobodies were further purified by gel filtration on a Superdex 75 column equilibrated in 20 mM Hepes pH 7.5, 100 mM KCI.

Expression and purification L-plastin(domains).

Wild-type human L-plastin, as well as its subdomains were produced in E. coli BL21 pLysS Star cells as GST-fusion proteins. The L-plastin fragments were subcloned in the pGEX-2T vector and fusion proteins were cleaved with factor Xa.

lmmunoprecipitation and immunoblotting.

Cells were disrupted in ice-cold lysis buffer (20 mM Tris-HCI, pH 7.5, 137 mM NaCI, 0,5 % Triton X-100, 1 mM PMSF, and a protease inhibitor cocktail mix) followed by centrifugation (20,000 xg for 10 min at 4 °C). Protein concentrations were determined by the method of Bradford (Bradford, 1976) using bovine serum albumin as a standard. 1 mg of total protein was incubated with 15 μl V5 agarose at 4°C. The beads were washed 4 times with lysis buffer, boiled for 5 min in Laemmli sample buffer and proteins were fractionated by SDS-PAGE. Western blotting was performed as described (Towbin et al, 1992).

Gelfiltration.

L-plastin (75 μg) alone, or with a nanobody (50 μg) was analyzed by gelfiltration on a Superdex 200 HR 10/30 column at a constant flow rate of 0.3 ml/min. The column was equilibrated in 50 mM sodium phosphate buffer pH 7.0, 100 mM KCI, 1 mM MgCI2, 1 mM ATP with 0.5 mM EGTA or 1 mM Ca2+. Proteins eluting from the column were detected by fluorescence (280 nm emission; 330 nm excitation).

lmmunostaining and microscopy.

Cells were washed with PBS, fixed with 3% paraformaldehyde for 25 min at room temperature and permeabilized with 0.1 % Triton X-100 in PBS. Cells were then blocked in PBS-1% BSA for 10 min at room temperature and incubated with primary antibody for 1 h at 37 °C (anti-L-plastin antibody or anti-V5 antibody). Cells were washed in PBS, incubated with secondary antibody (Alexa 488-conjugated goat anti-mouse) and Alexa Fluor 594 phalloidin for 30 min at room temperature. Following immunostaining, samples were analyzed with an ApoTome Zeiss Axiovert 200 epifluorescence microscope (63^χ objective) equipped with an Axiocam cooled CCD camera and processed using Axiovision software (Zeiss, Gottingen,, Germany).

Isothermal titration calorimetry (ITC) experiments.

5-1 0 μ M of L-plastin was titrated with 50-80 μ M of L-plastin nanobodies in Hepes buffer (2OmM) with 7OmM KCI with or without 100 μM Ca2+ in a Microcal VP-ITC MicroCalorimeter (Microcal Inc., Northampton, MA). Time between injections was set at 4 min to allow for reaching the baseline. Data were integrated and fitted using the Microcal Origin software, assuming a single class of site.

Actin binding and bundling assays.

Nanobodies and L-plastin were subjected to high speed centrifugation at 200,000 g before initiating the experiment. G-actin was polymerized overnight at 4°C or for 4 hours at room temperature in the presence of L-plastin and different nanobodies in polymerization buffer (100 mM KCI, 1 mM MgCI2, 1 mM ATP, 0.5 mM EGTA, 50 mM sodium phosphate buffer, pH 7.0) as indicated in figure legends. Sedimentation of actin filaments and L-plastin was achieved by high-speed centrifugation at 200,000 xg for 30 minutes. To sediment actin bundles, samples were centrifuged for 15 minutes at 12,000 xg. Proteins in pellets and supernatants were separated by SDS-PAGE. Coomassie stained protein bands were scanned and densities were quantified with ImageJ.

GST-pull down assay.

To 20 μl of 50% glutathione-sepharose (Amersham-biosciences), equilibrated in PBS with 0.5 % Triton, 10 μg GST-fusion protein was added. After incubation at 4 °C for 1 h, 5 μg nanobody were added and incubated for two more hours at 4 °C. Glutathione beads were then recovered by centrifugation and washed four times with binding buffer. Proteins bound to the beads were eluted by SDS sample buffer and resolved by SDSPAGE followed by Coomassie Blue staining.

Chemical crosslinking experiments. Eight μM of GST-EF hand domain was incubated with a 4* molar excess of recombinant nanobodies together in K-phosphate buffer including 75 mM NaCI and 100 μM Ca2+ during 20 min. Then, 5 mM EDC (zero cross linker) and 5 mM sulfo-NHS (stabilizer) were added and incubated during 20-40 min at RT. The mixture was then analyzed by SDS-PAGE.

Wound healing assays.

PC-3 cells (1 x 106) were seeded into 6-well cell culture plates. The following day, cells were transfected either with pcDNA3.1 V5/His vector containing a nanobody or with an L-plastin siRNA. Fourty eight hours after transfection, a wound was made by scratching a line in a confluent monolayer. Cell debris was removed by washing the cells with serum-free medium. Migration of cells into the wound was then observed at different time points. At hourly intervals, the width of the wound was measured at the same location. Cells were followed for 24 h.

Invasion assays.

Cell invasion assays were performed with the QCM™ 24-well Matrigel Invasion assay (Chemicon® International, Temecula, CA) as described (van den Abbeele et al, 2007). Data are expressed as relative fluorescence units (RFU). Assays were performed in triplicate.

Example 1: L-plastin single domain VHHs (nanobodies) bind their target in vivo

Panning of a phage display library with recombinant L-plastin yielded 9 different nanobody cDNAs. The sequences of the nanobodies are shown in Figure 1. The characteristics of the nanobodies are summarized in Table 1 and Table 2. Six of these evolved from the same B-cell clone since few amino acid substitutions were found (Nb 1 , 4, 5, 6, 7, 8). They presumably have the same epitope. The three remaining VHH cDNAs (2, 3 and 9) have different CDRs and are clonally independent. These nanobodies were subsequently evaluated in their ability to function as intrabodies. To this end, pcDNA3.1A/5-nanobody cDNAs were transfected into

PC-3 prostate cancer cells. Following expression, nanobodies were retrieved from cell extracts by use of anti-V5 antibody conjugated to agarose. Endogenous L-plastin coimmunoprecipitated with all different nanobodies, although in varying amounts, possibly due to differences in their stability and/or affinity (Fig. 2A top panel). Nanobodies 5, 6, 8 and 9 were particularly efficient in binding endogenous L-plastin. By contrast, nanobodies 1 , 3 and 4 bound with lower affinity, and they were excluded for this reason from further research. All nanobodies were expressed at comparable levels (Fig. 2B), but they did not detect endogenous or overexpressed L-plastin on western blot, suggesting that they recognize conformational epitopes.

Example 2: L-plastin nanobodies recognize different L-plastin conformations with different affinities

Selected nanobodies were recombinantly produced and purified (Fig. 2A) by immobilized metal ion affinity chromatography (IMAC) and gelfiltration. Next, we analyzed the binding characteristics with L-plastin in more detail using isothermal titration calorimetry (ITC). Table 1 summarizes all parameters obtained from ITC measurements. Surprisingly, for VHH 2 and 9 no significant binding was found although both nanobodies bind to and immunoprecipiate endogenous L-plastin (Fig. 3A). However, as L-plastin is known to bind Ca2+ through its EF- hand structures (Namba et al, 1992), we included 100 μM Ca2+ in the buffer. In both cases strong binding was observed in the presence of Ca2+, indicating that VHH 2 and 9 recognize a conformational epitope in L-plastin. For other nanobodies, the effect of Ca2+ on binding to L- plastin was only minor. The dissociation constant (Kd) ranges from -1.04 μM (Nb2) to -40 nM (Nb5). Of note, the high negative entropy (parameters for different nanobodies are summarized in Figure 3B) observed for Ca2+-loaded L-plastin and nanobodies 2 and 9 suggested that binding involves a conformational change. We observed a high change in enthalpy upon binding of these nanobodies which counteracts an unfavorable change in entropy. Large entropy variations are typical for induced fit mechanisms (Boniface et al, 1999). On the other hand, reaction parameters for binding of Nb5 to L-plastin implies a key and lock mechanism consistent with recognition of two rigid molecules that do not undergo large conformational changes upon binding (Karlsson & FaIt, 1997). The higher entropy observed in binding between nanobodies 7 and 8 to their epitope might originate from the exclusion of water molecules surrounding the hydrophobic surfaces, thus omitting the unfavorable interaction between hydrophobic surfaces and water.

Complexes between L-plastin and Nb5 or Nb9 were gel filtered on a superdex 200 HR column with or without Ca2+. Two peaks were observed for L-plastin at 85 kDa and 160 kDa, indicating that L-plastin may form dimers in vitro (Fig. 3C). For Nb5 we observed an increase in relative fluorescence of the L-plastin peaks in the presence of nanobody, irrespective of Ca2+. In addition the L-plastin-Nb5 complex eluted as a larger protein as compared to Lplastin alone (Fig. 3C, asterisks). A significant increase in relative fluorescence applied also to binding of Nb9 to L-plastin, but this occured only in the presence of Ca2+ (Fig. 3C, right). Here L-plastin also eluted as a slightly higher MW protein when Ca2+and Nb9 were present (Fig. 3C). The protein peaks were analyzed by SDS-PAGE confirming the presence of nanobody and/or L- plastin. Example 3: Nanobodies do not significantly inhibit F-actin-L-plastin binding in vitro

To study a possible effect of nanobodies on L-plastin function we first investigated if nanobodies have an effect on F-actin-L-plastin binding. Therefore we used a high speed cosedimentation assay with F-actin. G-Actin was allowed to polymerize in the presence of recombinant L-plastin, with or without different nanobodies. By centrifugation at high speed (200,000 xg), only filaments are precipitated. Pellet and supernatans were then separated and analyzed by SDS-PAGE followed by Coomassie blue staining (Fig. 4A) and band intensity was quantified. While some variation was observed in the amount of L-plastin retrieved in the pellet fraction in the presence of a nanobody (Fig. 4B), these changes were not drastic. Up to a molar ratio of 10:1 nanobody versus L-plastin, no significant inhibition in binding of Lplastin to F-actin was observed.

Example 4: Nanobodies inhibit actin bundling by L-plastin

L-plastin harbours two closely spaced F-actin binding sites allowing the protein to crosslink actin filaments into thight bundles. The effect of nanobodies on the bundling activity of L-plastin was assessed in a low-speed centrifugation assay (12,000 xg) allowing sedimentation of F- actin bundles but not single filaments (Glenney et al, 1981 ). L-plastin promotes actin bundle formation resulting in high actin concentrations in the pellet fraction following sedimentation (Fig. 5A-B). For nanobodies 2 and 9, no difference in bundling of F-actin by L-plastin was detected. However, in the case of nanobodies 5, 6, 7 and 8, originating from the same B-cell clone, a strong inhibition of bundling by L-pastin was observed (Fig. 5A-B) when the molar ratio of L-plastin:nanobody was 1 :10. The amount of nanobody cosedimenting with L-plastin in the pellet fraction was much lower for Nbs 5-8 as compared to Nb2 and 9 (Fig. 5C). As these experiments were carried out in the presence of EGTA, no binding of Nb2 and Nb9 to L-plastin was expected (Table 1 ) because L-plastin activity is inhibited at elevated Ca2+ concentrations (Namba et al, 1992). As nanobodies 2 and 9 cosedimented with L-plastin under these conditions we deduce that they can also bind to Ca2+-deprived L-plastin when L-plastin is bound to actin. Lower molar ratios of L-plastin/Nb induced similar effects. In fact, nearly complete inhibition of L-plastin bundling activity was also noticed at a plastin to nanobody molar ratio of 1 :1 (Nbs 5-8, as compared to Nbs 2 and 9). (Fig. 5D).

Example 5: Nanobodies bind different epitopes in L-plastin

L-plastin is characterized by a modular structure consisting of 2 amino-terminal EFhands and two tandem actinbinding domains (ABD), each divided into two calponin homology (CH) domains (de Arruda et al, 1990). In order to map the epitopes of selected nanobodies in L- plastin we performed an in vitro GST pull down assay. Recombinant GST-Lplastin and L- plastin modular domains (Fig. 6A) were purified and incubated with a nanobody. Following immobilization on glutathione-Sepharose beads complex formation was analyzed by SDS- PAGE. Nanobody 5 was found to bind to GST-full length L-plastin (GST-LPL) as well as to both ABDs combined (GST-ABD1 +2), but not to ABD1 or 2 alone or to the EFhands (GST-EF) (Fig. 6B). By contrast, nanobodies 2 and 9 both bind the EF-hands of Lplastin and full length L- plastin, but not to any other domain. Other VHHs were not investigated since their similar CDRs indicate that they all presumably share the same epitope. These findings were further confirmed by in vitro cross-linking experiments with the zero cross linker EDC. When either Nb2 or 9 was cross-linked to GST-EF (-35 kDa), a unique cross-linked product of -50 kDa could be observed (Fig. 6C, arrowheads), in agreement with the predicted molecular mass of the cross-linked product. This band was not observed in the GST-EF/Nb5 cross link. Thus nanobodies 2 and 9 target the same epitope. In addition we investigated whether these L- plastin subdomains encompass the entire epitope. We therefore compared the affinities of the VHHs for full length GST-tagged L-plastin with the affinity for GST-tagged L-plastin domains. This was done by ITC, and the values obtained (3.2 ± 0.2 μM for Nb2, 98.0 ± 9.4 nM for Nb9, and 46.5 ± 3.7 nM for Nb5) are very close to those obtained for full length L-plastin. These findings suggest that the entire epitope of the different nanobodies is present in the L-plastin fragments.

Example 6: Nanobody 5 obstructs filopodia formation of PC-3 cells To convincingly demonstrate an interaction in PC-3 cells between the VHH intrabodies and endogenous L-plastin, we performed a mitochondrial outer membrane (MOM) recruitment assay (Kaufmann et al, 2000). A nanobody was tagged with a V5 epitope and the mitochondrial outer membrane (MOM) anchor sequence from TOM70 (McBride et al, 1992). This sequence targets proteins to the MOM, and binding partners can piggyback with the protein to this compartment. Tagged nanobodies were expressed in PC-3 cells which were subsequently stained for V5 and for endogenous L-plastin. A GFP-specific VHH (Rothbauer et al, 2006) was included as a negative control. No colocalization was observed between the negative control and endogenous L-plastin (Fig. 7A) which stains homogenously in the cytoplasm and nucleus as shown earlier (Delanote et al, 2005a). However, L-plastin nanobodies 2, 5 and 9 were able to recruit L-plastin to mitochondria as evidenced by their colocalization, thereby confirming a genuine intracellular interaction (Fig. 7B-D). No significant difference in efficacy was seen for nanobodies 2 and 9 (Ca2+-dependent binding to L-plastin) as compared to Nb5 (Ca2+ -independent binding). This might be explained by the fact that Nb2 and 9 not only recognize the Ca2+-loaded form of L-plastin, but also the actin-bound conformation of L-plastin (Fig. 5A). Apart from its localization in the cytoplasm and nucleus, L- plastin is usually located at peripheral cell membrane protusions. Immunofluorescence was used to examine the effect of L-plastin nanobodies on cell morphology. We expressed nanobodies as GFP-fusion proteins in PC-3 cells and stained the cells for endogenous L- plastin. Colocalization between L-plastin and different nanobodies can be seen in specialized surface structures such as membrane ruffles and microvilli as well as in and filopodia (Fig. 7F- H). Similar observations were made with V5-tagged intrabodies. Strikingly, a large subpopulation of cells, particularly in the case of nanobody 5, were deprived of long filopodia (Fig. 7H). Conversely, cells with long plasma membrane cell surface projections never showed expression of nanobody. Thus Nb5 impedes formation of filopodia in PC-3 cells, which was not or much less observed with the empty GFP control vector or Nb 2 and 9, respectively. Quantification of this phenomenon by counting cells with long filopodia clearly showed a very strong effect exerted by Nb5 (Fig. 7I) which, unlike to Nb2 and 9, inhibits L- plastin mediated bundling of actin filaments.

Example 7: L-plastin nanobodies counteract cell migration and Matrigel invasion of PC- 3 cells As L-plastin has been linked to cell migration (Foran et al, 2006; Klemke et al, 2007; Zheng et al, 1999), we investigated if nanobodies could affect cell migration of PC-3 cells in a woundhealing assay. We first investigated if downregulation of L-plastin expression by siRNA transfection negatively regulates migration of these cells. PC-3 cells were transiently transfected with a control siRNA or an L-plastin siRNA. Fig. 8A shows that downregulation of L-plastin expression retards cell motility. We next repeated these experiments by using plastin nanobodies instead of siRNAs. Cells were transfected either with a negative control (anti-GFP nanobody) or one of the L-plastin nanobodies. From Fig. 8B it can be deduced that PC-3 cells expressing a nanobody showed reduced woundhealing efficiency as compared to the control (Fig. 8B). Together these findings demonstrate that depletion or immunomodulation of L- plastin slows down cell migration. It has been shown earlier that down-regulation of L-plastin reduces invasion of PC-3 cells through matrigel (Zheng et al, 1999). In view of our results we addressed the question if nanobodies could have a similar inhibitory effect on invasion of prostate cancer cells. In our setup however expression of the protein is not affected. Again, the anti-GFP nanobody was used as a negative control. PC-3 cells transiently transfected with one of the 3 different nanobodies showed a markedly diminished capacity to invade through matrigel compared to PC-3 cells transfected with the control GFP VHH (Fig. 9). This shows that intrabodies against L-plastin can counteract cell invasion similar to expression regulation of L-plastin. Table 1 : Thermodynamic parameters of Isothermal Titration Calorimetry (ITC) measurements between different nanobodies and L-plastin

* with T(Kelvin)=303,15 « ΔG = ΔH-TΔS

Table 2 €K eiΛ ievt suinmaiizing the diffeient piopeities of L-pϊasfni πaiiobodiεa

Table 3: Sequences

SEQ ID N°1: GGTFGRVGVG

SEQ ID N°2: QVQLQESGGGLVQAGDSLRLSCAVSGGTFGRVGVGWFRRAPGKEREFVAAVNWSGISTFY ADSVKGRFTISRDDNKHTVDLRMNSLKPEDSAWFCATDFRFNVPMNGTEYDYWGQGTQVT VSS References

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Claims

1. An anti-L plastin antibody binding both actin binding domains.

2. The antibody of claim 1 , whereby said antibody is not binding the EF-hand structures.

3. The antibody according to claim 1 or 2, whereby said binding is Ca²⁺ independent.

4. The antibody according to any of the claims 1-3, whereby said antibody is a nanobody.

5. The antibody according to claim 4, whereby said antibody comprises SEQ ID N° 1.

6. The antibody according to claim 4 or 5, whereby said antibody comprises SEQ ID

N° 2.

7. The antibody according to any of the claims 1-6, whereby said antibody is an intra body.

8. The use of an antibody according to any of the claims 1-7 to suppress cell invasion and/or metastasis.

9. The use of an antibody according to claim 8, whereby said antibody is coupled to a protein transduction domain.