Chemotrap-1: an Engineered Soluble Receptor That Blocks Chemokine-Induced Migration of Metastatic Cancer Cells in Vivo

Published OnlineFirst August 24, 2010; DOI: 10.1158/0008-5472.CAN-10-0175

Tumor and Stem Cell Biology Cancer Research Chemotrap-1: An Engineered Soluble Receptor That Blocks Chemokine-Induced Migration of Metastatic Cancer Cells In vivo

Silvia Lanati1, Darryl B. Dunn1, Myriam Roussigné2,3, Maxine S. Emmett1, Virginie Carriere2,3, Denis Jullien4, Jessica Budge1, Justin Fryer1, Monique Erard2,3, Françoise Cailler4, Jean-Phillippe Girard2,3, and David O. Bates1

Abstract Cancer and dendritic cells recognize and migrate toward chemokines secreted from lymphatics and use this mechanism to invade the lymphatic system, and cancer cells metastasize through it. The lymphatic-secreted chemokine ligand CCL21 has been identified as a key regulatory molecule in the switch to a metastatic phenotype in melanoma and breast cancer cells. However, it is not known whether CCL21 inhibition is a potential therapeutic strategy for inhibition of metastasis. Here, we describe an engineered CCL21-soluble inhibitor, Chemotrap-1, which inhibits migration of metastatic melanoma cells in vivo. Two-hybrid, pull-down, and coimmunoprecipitation assays allowed us to identify a naturally occurring human zinc finger protein with CCL21 chemokine-binding properties. Further analyses revealed a short peptide (∼70 amino acids), with a predicted coiled-coil structure, which is sufficient for association with CCL21. This CCL21 chemokine-binding peptide was then fused to the Fc region of human IgG1 to generate Chemotrap-1, a human chemokine-binding Fc fusion protein. Surface plasmon resonance and chemotaxis assays showed that Chemotrap-1 binds CCL21 and inhibits CCL21-induced migration of melanoma cells in vitro with subnanomolar affinity. In addition, Chemotrap-1 blocked migration of melanoma cells toward lymphatic endothelial cells in vitro and in vivo. Finally, Chemotrap-1 strongly reduced lymphatic invasion, tracking, and metastasis of CCR7-expressing melanoma cells in vivo. Together, these results show that CCL21 chemokine inhibition by Chemotrap-1 is a potential therapeutic strategy for metastasis and provide further support for the hypothesis that lymphatic- mediated metastasis is a chemokine-dependent process. Cancer Res; 70(20); 8138–48. ©2010 AACR.

Introduction When they metastasize specifically through the lymphatic system, tumor cells locate and recognize lymphatic endothe- Tumor metastasis occurs through either the lymphatic or lial cells (LEC). The secreted lymphatic chemokine CCL21 is the vascular systems. Lymphatic metastasis is the most com- released from LEC (1) and used by dendritic cells (DC) to mon route for melanoma, breast, prostate, and other cancers. guide their invasion into lymphatics by acting on its receptor CCR7, expressed on DCs (2). CCR7 expression is increased in Authors' Affiliations: 1Microvascular Research Laboratories, Bristol patients with metastatic melanoma (3); prostate (4), head and Heart Institute, Department of Physiology and Pharmacology, School of neck (5), and squamous cell carcinoma (6); and breast cancer Veterinary Sciences, University of Bristol, Bristol, United Kingdom; 2CNRS, Institut de Pharmacologie et de Biologie Structurale (IPBS); (7) compared with nonmetastatic cancers. Thyroid, lung, and 3Université de Toulouse, UPS, IPBS, Toulouse, France; and 4Endocube oral squamous cell carcinoma; melanoma; and breast cancer SAS, Prologue Biotech, Labège, France cells that upregulate receptors for lymphatic-secreted chemo- Note: J-P. Girard and D.O. Bates are joint senior authors. S. Lanati and kines such as CCL21 are more likely to metastasize (3, 8–10). It D.B. Dunn are joint first authors. has therefore been hypothesized that CCR7 is used by meta- Author contributions: F. Cailler, J-P. Girard, and D.O. Bates designed the research; D.B. Dunn, M.S. Emmett, M. Roussigné, V. Carriere, S. Lanati, static melanoma cells to detect locally secreted lymphatic- D. Jullien, J. Budge, J. Fryer, D.O. Bates, and M. Erard performed the specific chemokines, such as CCL21, and migrate toward it research; D.B. Dunn, M.S. Emmett, M. Roussigné, V. Carriere, M. Erard, to find areas of increased lymphatic density (11). One potential J. Fryer, J. Budge, F. Cailler, J-P. Girard, and D.O. Bates analyzed the data; and J-P. Girard and D.O. Bates wrote the paper. therapeutic strategy to prevent metastasis would be to inhibit Corresponding Authors: Jean-Philippe Girard, IPBS-CNRS-Université this chemokine-mediated entry of cancer cells into lympha- de Toulouse, 205 route de Narbonne, 31077 Toulouse, France. Phone: tics. Therapeutic strategies based on soluble human receptors 33-5-61-17-59-67; Fax: 33-5-61-17-59-94; E-mail: Jean-Philippe.Girard@ prepared as Fc fusion proteins, termed Traps, have been suc- ipbs.fr or David O. Bates, Microvascular Research Laboratories, University of Bristol, Southwell Street, Bristol BS2 8EJ, United Kingdom. Phone: cessfully developed for several cytokines, including tumor 44-117-928-9818; Fax: 44-117-928-8151; E-mail: Dave.Bates@bris. necrosis factor α (etanercept; refs. 12, 13), interleukin-1 ac.uk. (IL-1-Trap; ref. 14), and vascular endothelial growth factor doi: 10.1158/0008-5472.CAN-10-0175 (Aflibercept; ref. 15). These are generated using the fixed ©2010 American Association for Cancer Research. chain (Fc) domain of the human IgG fused to a protein-binding

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domain to provide a stable, dimeric protein with long plasma and BamHI and cloned in-frame downstream of the Gal4 ac- half-life. The protein-binding domain has been either the tivation domain in the pGADT7 two-hybrid vector (Clontech). ligand-binding domain of the receptor (e.g., etanercept; ref. 16) or a fusion protein of different ligand-binding domains of Y2H assays multiple receptors (e.g., aflibercept; ref. 15) to improve the Two-hybrid screening of a high endothelial venule (HEV) affinity of the Trap. To determine whether CCL21 could be cDNA library (17) was performed using human CCL21 as bait. a potentially useful antimetastatic target, we identified a pGBT9-hCCL21 was cotransformed with the pGAD424-HEV naturally occurring human protein with CCL21-binding cDNA library in yeast strain Y190 (Clontech). Transformants properties and used its chemokine-binding domain to gener- (1.5 × 107) were screened, and positive protein interactions were ate a human IgG1-Fc fusion protein that binds CCL21 with selected by His auxotrophy. Two-hybrid interactions were high affinity and inhibits its activity. We then determined confirmed in strain AH109 using Matchmaker Two-Hybrid whether this protein, Chemotrap-1, could inhibit melanoma System 3 (Clontech). AH109 cells were cotransformed with growth toward areas of high lymphatic density. pGBKT7-THAP1, pGADT7-hCCL21, pGADT7-mCCL21, pGADT7-hCCL27, or pGADT7-hCCL21ΔCOOH expression vec- Materials and Methods tors. Transformants were selected on medium lacking histidine and adenine. Two-hybrid interaction between THAP1 mutants Cell culture and transfection and chemokine CCL21 or wild-type THAP1 was tested by cotrans- Melanoma cells, purchased from the American Type Culture formation of AH109 with pGADT7-THAP1-C1, pGADT7-THAP1-C2, Collection, from Bioware, or supplied by I.J. Fidler (University of pGADT7-THAP1-C3, pGADT7-THAP1-N1, pGADT7-THAP1-N2, Texas M.D. Anderson Cancer Center, Houston, TX), were main- pGADT7-THAP1-N3, pGBKT7-CCL21, or pGBKT7-THAP1. tained in 10% fetal calf serum (FCS)–DMEM (A375) or 10% FCS–Eagle's MEM (mouse B16-Luc). Human dermal LECs GST pull-down and coimmunoprecipitation assays (CC-2814, Cambrex) were maintained in EGM-2 MV medium GST and GST-THAP1 fusion proteins were produced and (CC-3202, Cambrex). For transfection, cells at 80% to 90% con- purified as previously described (17). In vitro–translated fluency were seeded in six-well plates and transfected with 1 or hCCL21, mCCL21, hCCL27, and hCCL21ΔCOOH were gener- 1.5 µg of DNA and 4 μL of Lipofectamine 2000 (Invitrogen) in ated with the TNT Coupled Reticulocyte Lysate System Opti-MEM according to the manufacturer's instructions. Anti- (Promega) using pGADT7-hCCL21, pGADT7-mCCL21, biotic selection was started on day 2 following transfection. pGADT7-hCCL27, or pGADT7-hCCL21ΔCOOH as template. 35S-labeled chemokines (25 μL) were incubated with immo- Plasmid constructions bilized GST-THAP1 or GST proteins overnight at 4°C in pGBT9-hCCL21 vector was generated by inserting a cDNA 10 mmol/L NaPO4 (pH 8.0), 140 mmol/L NaCl, 3 mmol/L MgCl2, encoding the mature form of human CCL21 (Genbank acces- 1mmol/LDTT,0.05%NP40,0.2mmol/Lphenylmethyl- sion no. NP_002989; amino acids 24–134) into the BamHI site sulfonyl fluoride, 1 mmol/L sodium vanadate, 50 mmol/L of Matchmaker Yeast Two-Hybrid (Y2H) System 2 vector β-glycerophosphate, 25 μg/mL chymotrypsin, 5 μg/mL apro- pGBT9 (Clontech). pGBKT7-hCCL21 vector was generated tinin, and 10 μg/mL leupeptin. Beads were washed five times by subcloning the BamHI CCL21 fragment from pGBT9- in 1-mL binding buffer. Bound proteins were eluted with 2× hCCL21 into the BamHI site of pGBKT7 (Clontech). Laemmli SDS-PAGE sample buffer, fractionated by 10% SDS- pGADT7-hCCL21 and pGADT7-hCCL21ΔCOOH expression PAGE, and visualized by fluorography using Amplify (Amer- vectors were generated by subcloning the BamHI fragment sham Pharmacia Biotech). For coimmunoprecipitation (encoding CCL21 amino acids 24–134) or the BamHI-PstI experiments, U2OS cells were transfected with plasmids en- fragment (encoding CCL21, amino acids 24–102) from coding CCL21-Flag and/or HA-THAP1 using a calcium phos- pGBKT7-CCL21 into pGADT7 expression vector (Clontech). phate precipitation procedure. Two days after transfection, pGADT7-mCCL21 and pGADT7-CCL27 expression vectors cellular extracts were prepared [15 mmol/L Tris-HCl (pH were generated by cloning the cDNAs encoding mature 7.5), 0.4 mol/L NaCl, 5 mmol/L MgCl2, and 0.1% Tween 20 forms of mCCL21 (Genbank accession no. NP_035254; amino containing protease and phosphatase inhibitors (Roche)]. acids 24–133) or hCCL27 (Genbank accession no. NP_006655; After three freeze/thaw cycles, cellular extracts were centrifuged amino acids 25–112) into pGADT7. Construction of pGBKT7- at 12,000 × g for 10 minutes and then precleared at 4°C for 1 hour THAP1 and pEGFP.C2-THAP1 expression vectors has been in a rotating wheel with 20 μLproteinG–Sepharose beads previously described (17). The glutathione S-transferase (Amersham). Beads were precipitated by centrifugation, and (GST)–THAP1 expression vector was generated by cloning supernatants were incubated overnight at 4°C with 5 μgofFlag the full-length coding region of human THAP1 (amino acids M2 antibody (Sigma). Immune complexes were captured with 20 1–213) in the pGEX-2T prokaryotic expression vector (Amer- μL of protein G–Sepharose beads at 4°C for 1 hour and washed sham Pharmacia Biotech). THAP1 deletion mutants THAP1- four times with 0.4 mol/L NaCl. Bound proteins were eluted and C1 (amino acids 90–213), THAP1-C2 (amino acids 120–213), subjected to immunoblotting with an anti-HA antibody. THAP1-C3 (amino acids 143–213), THAP1-N1 (amino acids 1–90), THAP1-N2 (amino acids 1–166), and THAP1-N3 (amino In silico sequence analysis and molecular modeling acids 1–192) were amplified by PCR using pEGFP.C2-THAP1 Coiled-coil predictions were performed at PAIRCOIL as template. The PCR fragments were digested with EcoRI (http://paircoil.lcs.mit.edu/cgi-bin/paircoil) and MULTICOIL

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(http://multicoil.lcs.mit.edu/cgi-bin/multicoil) Web sites. (BIAcore) program was used for calculations of association/ The structure of the THAP1 dimeric coiled-coil was built dissociation kinetic constants (Kd = kd/ka). by homology with the X-ray structure of the GCN4 parallel coiled-coil (PDB code: 1KD8). The energy of the interaction ELISA interface was minimized by using the Affinity module within The CCL21 DuoSet ELISA was carried out as per the manu- Insight II (Accelrys). facturer's instructions. To determine the effect of Chemotrap-1 on CCL21 release from LEC, cells were cultured for 24 hours in Generation of Chemotrap-1 expression vector 0.5% EGM-2, and 5-mL medium was transferred to a T25 flask The sequence encoding residues 140 to 213 of human of B16-Luc melanoma cells for a further 24 hours. Medium was THAP1 was amplified by PCR with oligonucleotides then collected, spun to remove cells, and then concentrated THAP1-XhoI-5′(5′-ccgctcgaggatacaatgcacc-3′)andTHAP1- through an Amicon 10000 spin column. BamH1-3′(5′-gcgggatccgctggtacttcaactatttcaaag-3′). The resulting XhoI-BamHI fragment was used to replace the In vitro cell migration XhoI-BamHI fragment encoding L-selectin in the pCDM8– Migration assays were performed in a modified Boyden L-selectin–IgG1 plasmid (18, 19). A linker encoding the Igκ chamber consisting of a cell culture insert with an 8-μm pore chain signal peptide (SP; 21 amino acids) from plasmid polycarbonate membrane (Millipore) seated in each well of a pSecTag2 (Invitrogen) was ligated into the XhoIsiteof 24-well plate as previously described (11). For dose response, pCDM8-THAP1140–213-IgG1 to obtain the expression vector migration assays were performed as above in 150 ng/mL pCDM8-SP-THAP1140–213-IgG1. The whole construct was in- CCL21 and increasing concentrations of Chemotrap. The serted into the pcDNA3.1 vector (Invitrogen) to generate number of cells that migrated through were normalized to pcDNA3.1–Chemotrap-1. pcDNA3.1–Chemotrap-189 expression the lowest concentration of Chemotrap (10 pmol/L) and to vector was obtained by replacing the XhoI-THAP1140–213-BamHI 0.1% FCS alone (no CCL21), and EC50 was determined by fragment of pcDNA3.1–Chemotrap-1 by an XhoI-THAP1189–213- nonlinear curve fitting with fixed top and bottom parameters BamHI fragment generated by PCR. (100% and 0%, respectively) using a sigmoidal dose-response curve. Chemotrap-1 production and purification pcDNA3.1Zeo-Chemotrap or pcDNA3.1–Chemotrap-189 In vivo metastatic chemotaxis model expression vectors were transfected into Chinese hamster Experiments were carried out as previously described (11). ovary (CHO) cells using JetPEI (Ozyme). Cells were grown Briefly, nude mice were injected with 1 × 106 tumor cells s.c. with zeocin (100 mg/mL; Invivogen) to establish clonal lines. with a Monastral blue–coated needle. The mice were then Supernatants were diluted in ImmunoPure-Gentle Ag/Ab injected with 1 × 105 human LEC ∼10 mm caudal to the mel- Binding Buffer (Pierce) and mixed with protein A–Sepharose anoma injection site. Mice were left until tumors reached CL-4B (Amersham Biosciences) for 1 hour at room tempera- 8 to 10 mm when measured through the skin. The animals ture under gentle agitation. The packed resin was rinsed were then killed, tumors were excised, and the skin was pro- using 15 volumes of binding buffer before elution with nated and photographed. Directional growth of melanoma ImmunoPure-Gentle Elution Buffer (Pierce). The fractions was measured as the plan area of the tumor below the per- containing Chemotrap were concentrated on an Amicon pendicular axis (i.e., closer to the endothelial cell injection Ultra-4 (molecular weight cutoff, 10,000) and analyzed by site) and expressed as a percentage of the total area of the immunoblotting with goat α-human IgG horseradish perox- tumor. idase (1:5,000) and an enhanced chemiluminescence kit (Amersham Bioscience). In vivo bioluminescent imaging of tumors CD1 mice were injected s.c. with 1 × 106 B16-Luc cells. To Surface plasmon resonance experiments image bioluminescence, mice were injected with 0.15 mg/g Purified recombinant THAP11–213-Fc and Chemotrap-1 luciferin i.p. at 20 mg/mL. Five minutes later, they were anes- (THAP1140–213-Fc) were covalently bound from their amino thetized by isofluorane (5% in 95% O2) and imaged using an groups to the gold sensor chip surface (CM5 sensor chip). IVIS Lumina (Caliper Life Sciences). Lesion maximum width Purified recombinant human CCL21 (Chemicon) was used and length were measured, and the ratio was used to calcu- as analyte protein at a fixed concentration in the fluid phase late directional growth. Once tumors reached 16 mm in size, [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 3 mmol/L the mice were imaged as above and then killed, and the flank EDTA, 0.005% P20 surfactant, as a running buffer] and at a was dissected to investigate in-transit metastases. Areas of constant flow rate of 20 μL/min in a BIAcore 3000 system black tracking along lymphatics were photographed using a (BIAcore AB). Association and dissociation curves were es- Nikon Coolpix digital camera. tablished for 12.5, 25, 50, 100, and 200 nmol/L CCL21. Che- mokine was injected during the association phase for Results 4 minutes (20 μL/min). The dissociation phase was carried out over 5 minutes, and flow cells were regenerated by injec- Identification of a CCL21 chemokine-binding protein tion of 0.05% SDS (30 seconds at a flow of 20 μL/min) be- While searching for CCL21-interacting proteins in a two- tween each phase of association-dissociation. BIAevaluation hybrid screen of a HEV cDNA library, we identified the cDNA

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encoding a novel nuclear factor, which we designated THAP1 purified from cell supernatants and immobilized on a sensor (17). Eight positive clones (of 5 million transformants) were chip. Addition of purified recombinant human CCL21 identified with the CCL21 bait, and all corresponded to the revealed association with immobilized THAP11-213-Fc with – COOH-terminal part of THAP1 (amino acids 90 213; Fig. 1A). a Kd of 87 nmol/L (Fig. 1C). Together, our observations indi- To confirm the interaction in yeast, we performed in vitro cated that the human THAP–zinc finger protein THAP1 is a GST pull-down assays. Full-length THAP1-GST protein was chemokine-binding protein that binds CCL21 with nanomo- incubated with radiolabeled in vitro–translated CCL21. Both lar affinity. human and mouse CCL21 proteins bind to GST-THAP1 but not to GST (Fig. 1A). The interaction was also observed when Mapping the CCL21 chemokine-binding domain the basic COOH-terminal extension of CCL21 was deleted, To identify sequences mediating CCL21 binding, a series of indicating that THAP1 interacts with the core chemokine do- THAP1 deletion mutants were generated. These truncated main of CCL21 (Fig. 1A). No binding of THAP1 was observed versions were used as Gal4 DNA-binding domain fusion pro- to CCL27, another CC chemokine. We next addressed wheth- teins (baits) in the Y2H system, together with preys er THAP1 is able to interact with CCL21 in cells. We corresponding to mature CCL21 (amino acids 24–134) in performed immunoprecipitation experiments in cells coex- fusion with the Gal4-activating domain. Schematic represen- pressing epitope-tagged CCL21 (Flag-CCL21) and THAP1 tation of all THAP1 deletion mutants and summary of two- (THAP1-HA). We observed specific immunoprecipitation of hybrid results are presented in Fig. 2A. We found that neither THAP1 with anti-Flag antibodies in cells coexpressing Flag- the THAP1-N1 mutant, corresponding to the DNA-binding CCL21, whereas no precipitation of THAP1 with anti-Flag THAP–zinc finger (20), nor THAP1-N2 and THAP1-N3, which antibodies was observed in control cells (Fig. 1B). These contain the THAP–zinc finger and the proline-rich linker re- findings showed that THAP1 interacts with CCL21 both gion, interact with CCL21. In contrast, cells coexpressing in vitro and in cells. To determine the kinetic parameters CCL21 with NH2-terminal deletion constructs, THAP1-C1, of the THAP1/CCL21 complex, we performed surface plas- THAP1-C2, or THAP1-C3, exhibited strong growth on selec- mon resonance (SPR). THAP11-213-Fc fusion protein was tive medium, indicating that the COOH-terminal domain of

Figure 1. Interaction between CCL21 and THAP1. A, two-hybrid and GST pull-down interaction assays show interaction between human CCL21 (hCCL21) and human THAP1. This interaction was also observed with mouse CCL21 (mCCL21), indicating evolutionary conservation. Schematic representation of results obtained in two-hybrid and GST pull-down assays using human THAP1 together with human CCL21, mouse CCL21, human CCL21 deleted from its COOH-terminal extension (hCCL21DCterm), and human CCL27, another CC-chemokine used as a control. B, coimmunoprecipitation of THAP1-HA with Flag-CCL21 in cellular extracts (0.4 mol/L NaCl) followed by immunoblot with anti-HA antibody. Stars represent Ig subunits, and arrows indicate the HA-tagged THAP1. C, SPR analysis of THAP1/CCL21 association. Full-length THAP1 protein (amino acids 1–213) was immobilized on the sensor chip as an IgG1-Fc fusion protein and incubated with increasing concentrations of soluble CCL21 protein. The Kd for the THAP1/CCL21 association had a calculated value of 87 nmol/L.

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Figure 2. The CCL21 chemokine-binding domain of THAP1 has characteristics of a coiled-coil domain. A, summary of two-hybrid results obtained with THAP1 deletion mutants and CCL21 or wild-type (wt) THAP1 (homodimerization). B, GST pull-down interaction assays show interaction between human CCL21 and human THAP1 residues 126 to 213 and 142 to 213, less so for 166 to 213, and not for 186 to 213. C, THAP1 contains a predicted coiled-coil domain between residues 142 and 190. The 213–amino acid sequence of THAP1 was analyzed using the PAIRCOIL program. The probability of each residue within the THAP1 sequence contributing to a coiled-coil structure is plotted (maximal probability score = 1). D, molecular modeling of the THAP1 coiled-coil domain based on the crystallographic structure of the GCN4 homodimeric parallel coiled-coil. Molecular modeling was performed as described in Materials and Methods.

THAP1 (amino acids 143–213) is necessary and sufficient for motifs found in proteins (23). The coiled-coil structure con- CCL21 binding. To confirm the Y2H results, we performed sists of two α-helices wrapped around each other with a in vitro GST pull-down assays with GST-THAP1 deletion con- slight superhelical twist. Its most characteristic feature is a structs that showed the COOH-terminal part of THAP1 (ami- heptad repeat pattern of primarily apolar residues that con- no acids 142–213) sufficient for interaction with radiolabeled stitute the dimer/oligomer interface. Both PAIRCOIL and in vitro–translated CCL21 (Fig. 2B), but that it required ami- MULTICOIL programs predicted a parallel left-handed no acids 142 to 166, as deletion of amino acids 142 to 166 coiled-coil domain with seven heptad repeats (49 residues), reduced binding and deletion abolished it completely. These extending between amino acids 142 and 190 of human THAP1. results show that the CCL21 chemokine-binding domain of The probability for formation of the coiled-coil structure of THAP1 corresponds to the last 70 amino acids (amino acids this region was 1 (maximum score) with both programs, indi- 143–213). cating that this region of THAP1 has a very high probability of existing as a coiled-coil structure (Fig. 2C). Molecular model- The CCL21 chemokine-binding domain has ing of the THAP1 coiled-coil domain was performed using the characteristics of a coiled-coil X-ray crystal structures of known coiled-coil domains as Two-hybrid assays with THAP1 bait (Fig. 2A) revealed that a template. The resulting three-dimensional model of the the CCL21 chemokine-binding domain (amino acids 143– THAP1 coiled-coil is shown in Fig. 2D. The parallel homodi- 213) also mediates interaction of THAP1 with itself (homodi- mer is stabilized by a perfect zipper based on hydrophobic merization or oligomerization). The PAIRCOIL (21) and interactions between identical residue side chains from each MULTICOIL (22) algorithms revealed that the CCL21 chemo- monomer. The CCL21-binding domain also includes the last kine-binding domain of THAP1 has characteristics of a 23 COOH-terminal amino acids of THAP1 (amino acids 191– coiled-coil, one of the principal dimerization/oligomerization 213), not required for homodimerization/oligomerization

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(Fig. 2A) but, with residues 142 to 166, essential for CCL21 growth, 1 × 106 A375 cells transfected with pcDNA3.1–Che- binding. motrap-1, pcDNA3.1–Chemotrap-189, or pcDNA3 (control vector) were implanted s.c. into mice 8 to 10 mm from a Chemotrap-1—an engineered soluble inhibitor based simultaneous implantation of 1 × 105 human LEC. After 14 on the CCL21 chemokine-binding domain days, tumors were apparent in all mice. Whereas tumors in The CCL21 chemokine-binding domain of THAP1 could be mice injected with the pcDNA3– and pcDNA3.1–Chemotrap- useful to engineer a high-affinity soluble inhibitor capable of 189–transfected cells grew significantly back toward the LEC blocking CCL21 chemokine activity in vivo. We thus generat- depot, the pcDNA3.1–Chemotrap-1–transfected cells grew in ed a fusion protein between the Fc region of human IgG1 and the location where they were injected (Fig. 4B). Tumors were the CCL21 chemokine-binding domain of human THAP1 excised and pronated, and the area of tumor on each side of (Fig. 3A). This fusion protein—Chemotrap-1—was then ex- the injection site was quantified. Figure 4C shows that pressed in CHO cells and purified. As a negative control, we Chemotrap-1 expression resulted in a statistically significant generated a fusion protein of human IgG1 with a 26–amino inhibition of tumor growth toward LEC to a level equivalent acid region (amino acids 189–213) of THAP1 that does not to nondirected growth (toward the site of a saline injection). bind CCL21(Fig. 2B). Chemotrap-1 bound to CCL21 with the same affinity as the full-length THAP1-IgG1 fusion pro- Chemotrap-1 prevents in-transit metastasis of tein (∼100 nmol/L), as determined by SPR (Fig. 3B). In con- melanoma cells in lymphatics in vivo trast, Chemotrap-189 did not bind CCL21 (data not shown). CCR7 overexpression increases lymph node metastasis of To determine whether Chemotrap-1 could inhibit CCL21 B16 mouse metastatic melanoma cells, resulting from uptake chemokine-mediated migration of cancer cells, we seeded into and tracking along the local lymphatics (24). To deter- A375P-CCR7–transfected melanoma cells on polycarbonate mine whether Chemotrap-1 could inhibit this tracking, in a inserts and measured migration across those inserts to CCR7-dependent manner, we imaged tumors growing in CD1 CCL21, or CCL21 and Chemotrap-1. Chemotrap-1 (1 μg/mL) mice by bioluminescence. B16 melanomas formed tumors significantly inhibited migration of these melanoma cells that were elongated (Fig. 5A). The maximum major to minor toward 150 ng/mL CCL21 (Fig. 3C). To determine the poten- axis ratio was 4.1 ± 1.1 (mean ± SEM, n = 6; Fig. 5A, i). In cy of Chemotrap-1 in vitro, we performed a dose-escalating contrast, tumors expressing Chemotrap-1 had a mean ratio study using 150 ng/mL CCL21 as a promigration agent. of 2.5 ± 0.2 (Fig. 5A, iii), which was significantly lower than Figure 3D shows that Chemotrap-1 dose dependently inhib- that observed with tumors expressing Chemotrap-189 (3.9 ± ited CCL21-mediated migration with an IC50 of 77 pmol/L, 0.5; Fig. 5A, ii). Moreover, on excision, tracking of the black significantly more potent than to immobilized CCL21 on a melanomas (Fig. 5A, iv) could be seen along lymphatics in chip in vitro. To determine the specificity of Chemotrap-1 one third of the B16 melanomas and in 60% of the cells ex- in vitro, we investigated its effect on migration of A375P- pressing Chemotrap-189, whereas this tracking was rarely CCR7 cells induced by three different chemokines: CCL19 seen with cells expressing Chemotrap-1 (17%; Fig. 5A, v). (binds CCR7), CXCL12 (binds CXCR4), and CXCL10 (binds CCR7/Chemotrap-189–cotransfected cells all showed CXCR3). Any of the four chemokines (150 ng/mL) induced evidence of tracking (Fig. 5B, i, ii, and iv). In contrast, migration of melanoma cells, but only the CCR7 ligands CCR7–Chemotrap-1–transfected cells showed a significant CCL21 and CCL19 were inhibited by Chemotrap-1 (Fig. 3E). inhibition of tracking (Fig. 5A, iii and v) to 33% (P <0.01, χ2 test; Fig. 5C), indicating that coexpression of Chemo- Chemotrap-1 blocks migration of metastatic melanoma trap-1 completely inhibited the effect of CCR7 overexpres- cells toward lymphatics in vitro and in vivo sion. To determine whether recombinant Chemotrap could We previously showed metastatic melanoma cells migrat- prevent metastasis in vivo, we used a CCR7-dependent model ing toward LEC conditioned medium (CM) in a CCL21- of lymph node metastasis in syngeneic mice. B16-Luc cells dependent manner (11). To determine whether this were stably transfected with CCR7, and 50 μLof1×105 cells could be inhibited by Chemotrap-1, A375 cells were were injected into the footpad of 10 C57/BL6 mice. Three an- transfected with expression vectors for Chemotrap-1 imals were treated twice weekly with 50 μg Chemotrap-1 by (pcDNA3.1– Chemotrap-1) or Chemotrap-189 (pcDNA3.1– i.p. injection. After 22 days, the mice were injected with lucif- Chemotrap-189). Expression of the Fc fusion protein was erin and tumors were imaged. Mice were killed, and lymph tested by immunoblotting, and the transfected A375 cells nodes were exposed and imaged. Figure 5D shows lumines- were then seeded into the top of Boyden chambers. LEC cence of the primary tumor and of a lymph node metastasis CM was placed on the bottom part of the chamber, and in the popliteal lymph node. Metastases were seen in all sev- A375 migration was measured over 24 hours. Figure 4A en control mice, but in only one of three treated mice shows that expression of Chemotrap-1, but not Chemotrap- (P <0.02,χ2 test), suggesting that systemic administration 189, resulted in complete inhibition of migration of A375 could reduce lymph node metastases in this mouse model. cells toward LEC CM. We have also previously shown that To confirm that Chemotrap-1 was being secreted from the when implanted into nude mice 8 to 10 mm from a depot transfected cells, we used a commercial DuoSet CCL21 of human LECs, metastatic, but not nonmetastatic, melano- ELISA. Figure 5F shows that the melanoma cells do not ex- mas grow toward the LEC injection depot. To determine press CCL21 at levels detectable on the CCL21 DuoSet kit whether Chemotrap-1 could inhibit directed metastatic [confirmed by reverse transcription-PCR (RT-PCR); data

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Figure 3. Chemotrap-1 binds CCL21 and blocks CCL21-induced melanoma cell migration in vitro. A, the chemokine-binding peptide of THAP1 was fused to the Fc region of human IgG1 to generate Chemotrap-1, a fully human chemokine-binding Fc fusion protein. B, SPR analysis of Chemotrap-1/CCL21 association. Chemotrap-1 was immobilized on the sensor chip as an IgG1-Fc fusion protein and incubated with increasing concentrations of soluble

CCL21 protein. The Kd for the Chemotrap-1/CCL21 association had a calculated value of 102 nmol/L. C, Chemotrap-1 blocks CCL21-induced melanoma cell chemotaxis in vitro. Cell migration of medium containing cells transfected with 150 ng/mL CCL21 or 150 ng/mL. Columns, mean; bars, SE. ***, P < 0.01, compared with medium and Chemotrap-189. ANOVA, Student-Neuman-Keuls test. D, Chemotrap-1 has a high affinity for inhibition of CCL21-mediated migration. A375P-CCR7 cells were subjected to migration assay toward 150 ng/mL CCL21 or increasing concentrations of Chemotrap-1.

The IC50 was 77 pmol/L. E, Chemotrap-1 inhibition of migration is restricted to CCR7 ligands. Migration of A375P-CCR7 melanoma cells across polycarbonate filters toward 150 ng/mL of CCL21, CCL19, CXCL12, or CXCL10 was measured in the presence or absence of 1 μg/mL Chemotrap-1. +, P < 0.05, compared with control (value of 1); *, P < 0.05; ***, P < 0.001, compared with cytokine alone, one-way ANOVA, Dunnet's test.

not shown]. LEC expressed CCL21 protein (1.79 pg/mL reduction in available CCL21, indicating binding of CCL21 per flask per hour) and mRNA as determined by RT-PCR to Chemotrap-1 to below the detection limit of the assay. (data not shown). When LEC CM was mixed with medium In contrast, when LEC CM was added to Chemotrap-189, from Chemotrap-1–expressing cells (B16-CT1), there was a there was no reduction in CCL21.

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Chemotrap-1 Blocks Chemokine-Induced Metastasis

Discussion apparent, yet spread months or even years after excision. For melanoma, prognosis is poor once metastasis has occurred Metastatic mechanisms are potential therapeutic targets, and there are no widely accepted therapies. We show here as cancers can be detected and excised before metastasis is that one mechanism of metastasis, chemokine-mediated

Figure 4. Chemotrap-1 inhibits directed growth of melanoma toward LECs. A, migration of melanoma cells or melanoma cells expressing empty vector (pcDNA3), negative control Fc (Chemotrap-189), or Chemotrap-1 was measured in vitro to LEC CM. B, melanoma cells as above were injected into nude mice 8 to 10 mm from a depot of LEC, and direction of tumor growth was measured. The red highlighted area is growing toward, and the blue away, from the LEC. C, the % growth toward the LEC is reduced in A375 cells expressing Chemotrap-1 to the same level as in the absence of LECs (PBS). Columns, mean; bars, SE. ***, P < 0.001; **, P < 0.01; *, P < 0.05, compared with pcDNA3 control (ANOVA).

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Figure 5. Chemotrap-1 inhibits tracking of melanoma cells into and along lymphatics. A, B16-Luc melanoma cells (i), transfected with Chemotrap-189 (ii) or Chemotrap-1 (iii), were injected into CD1 mice, and circularity of tumor growth was measured by bioluminescence. On excision, tracking (arrow) of the tumor was seen in control (iv) but not in Chemotrap-1–transfected (v) tumors. B, effect of overexpression of CCR7 (i) or CCR7 and Chemotrap-189 (ii) increased nonuniform growth but CCR7–Chemotrap-1 expression again results in more circular growth (iii). Tracking was increased in CCR7-transfected tumors (iv) but reduced in CCR7- and Chemotrap-1–expressing tumors (v). C, percentage of tumor tracking. **, P < 0.01; *, P < 0.05, χ2 test, compared with control. D, C57/BL6 mice were injected with B16-Luc–CCR7 melanoma cells in the footpad. Twenty-two days later, the tumors were imaged by bioluminescence (i) and then the mouse was killed and the lymph node was exposed and imaged (ii). E, Chemotrap-1 treatment significantly reduced the incidence of lymph node metastasis (P < 0.02, χ2 test). F, CCL21 DuoSet ELISA kit was used to determine the concentration of CCL21 in the LEC CM and LEC CM incubated with B16 cells expressing Chemotrap-1 or Chemotrap-189. *, P < 0.05, Dunnet's test.

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Chemotrap-1 Blocks Chemokine-Induced Metastasis

LEC targeting, is potentially therapeutically amenable, as in- CCL21-mediated recruitment of lymphocytes in vivo (28). hibition of the CCL21/CCR7 axis can prevent melanoma cells A major advantage of Chemotrap-1 is that it is a fully hu- from recognizing and migrating toward areas of high lym- man recombinant protein. The M3 protein also binds and phatic density and invading those lymphatics. blocks the activity of other chemokines in vivo (28), and it Chemokine receptors, including the CCL21 receptor CCR7 will be important in future studies to widen the chemo- and the CXCL12 receptor CXCR4, have been associated with kines against which Chemotrap-1 has been tested to un- metastatic melanoma in humans and increased metastatic derstand the full binding potential of this protein. growth in mouse and human tumors in experimental animals Most human melanomas metastasize through the local (3, 25). Two nonmutually exclusive mechanisms have been lymphatics and, through the lymph node, gain access to proposed for CCR7-CCL21–mediated metastasis: metastatic the circulation and reach distant organs such as the lung, chemotaxis toward areas of high lymphatic density (11) and brain, and liver. We therefore used a model in experimen- autologous chemotaxis (26). Current experiments did not de- tal animals whereby lymphatic metastasis is generated termine the relative contributions of these two mechanisms from s.c. injection of primary tumor cells, in contrast with toward invasion of melanoma cells into the lymphatic system. experimental models using i.v. injection of tumor cells. Although we showed that secreted Chemotrap-1 bound This is not the usual route for melanoma metastasis. In to CCL21 secreted from LEC, and that recombinant patients, melanomas can form secondary lesions either lo- Chemotrap-1 could inhibit metastasis, we do not exclude cally within the skin (local metastasis), along the lympha- the potential for synthesis of Chemotrap-1 acting intracellu- tics (in-transit metastasis), or distantly (lymph node, larly or autocrinely on the tumor cells. We show here that primarily, followed by brain, lung, liver, etc.). We show CCR7-dependent lymphatic recognition by metastatic mela- here that chemokine-binding agents can prevent melano- noma is inhibited by the CCL21-binding agent Chemotrap-1. ma migration toward areas of high lymphatic density, in- Chemotrap-1 is a human IgG1-Fc fusion protein containing vasion into lymphatics, and in-transit metastasis. the CCL21 chemokine-binding domain of human THAP1, a The findings here indicate that inhibition of chemokines physiologic regulator of endothelial cell proliferation (17, 27). acting on tumor cells can prevent tumor migration toward CCL21 contains a functional nuclear localization sequence in and into draining lymphatics. This suggests that chemokine its COOH-terminal extension and may localize to the nucleus antagonists may be potential therapeutic agents. Further under certain conditions,5 but the physiologic relevance of evidence for this would be required before moving to clinical the THAP1/CCL21 interaction has not yet been shown. studies, particularly in terms of generating pharmacologically Chemotrap-1 is the first example of a human recombinant relevant inhibitors, and protein therapeutics, but the results chemokine-binding Fc fusion protein. The THAP1 here show for the first time that an endogenous human pro- chemokine-binding domain, predicted to adopt a coiled-coil tein sequence can be used to block lymphatic metastases. helical structure, presents no sequence or structural homolo- gies with CCR7 and other chemokine receptors. Determina- Disclosure of Potential Conflicts of Interest tion of the three-dimensional structure of the complex will M. Roussigné and J-P. Girard are inventors on a patent associated with be therefore required to understand the molecular basis for this work. specific recognition of the chemokine core domain of CCL21 by the THAP1 chemokine-binding peptide. Inhibitors of Acknowledgments chemokine receptors have been shown to be effective in animal models of melanoma, specifically the CXCR4 receptor We thank Dr. Thomas Clouaire for help with the generation of THAP1 inhibitor AMD3100 (25). However, there are no specific small- deletion mutants, Dr. Luc Aguilar for expert advice on Chemotrap-1 studies, – and the BIAcore technological platform of the IFR150-CHU Toulouse for SPR molecule inhibitors of CCR7, and CCR7-IgG1 soluble recep- experiments. tors cannot be prepared because of the seven-transmembrane nature of chemokine receptors. Grant Support Chemotrap-1 blocked CCR7-mediated tracking of metastatic melanoma cells into and along lymphatics, indicating Ligue Nationale contre le Cancer (“Equipe Labellisée Ligue 2009”;J-P. Girard), Wellcome Trust grant 083583 (D.O. Bates and S. Lanati), Skin Cancer that Chemotrap-1 can inhibit CCL21 activity in vivo.An- Research Foundation (M.S. Emmett), Michael Brough Healing Foundation other chemokine-binding protein, the M3 protein encoded (D.B. Dunn), and British Heart Foundation grant BS06/009 (D.O. Bates). by murine gammaherpesvirus-68, has previously been 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 shown to bind CCL21 with high affinity and to inhibit accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 01/19/2010; revised 06/13/2010; accepted 07/16/2010; published 5 J-P. Girard, unpublished data. OnlineFirst 08/24/2010.

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8148 Cancer Res; 70(20) October 15, 2010 Cancer Research

Chemotrap-1: An Engineered Soluble Receptor That Blocks Chemokine-Induced Migration of Metastatic Cancer Cells In vivo

Silvia Lanati, Darryl B. Dunn, Myriam Roussigné, et al.

Cancer Res 2010;70:8138-8148. Published OnlineFirst August 24, 2010.

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