A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom

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A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom Q8

J.M.A. Castro a, T.S. Oliveira a, C.R.F. Silveira a, M.C. Caporrino a, D. Rodriguez b,
rrez d, A.M. Moura-da-Silva a, O.H.P. Ramos c, A. Rucavado d, J.M. Gutie a a a , * ~es , E.L. Faquim-Mauro , I. Fernandes G.S. Magalha ~, CEP 05503-900 Sa ~o Paulo, SP, Brazil
rio de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, Butanta Laborato ~o Paulo, Brazil
rio de Biotecnologia IV, Instituto Butantan, Sa Laborato CEA, iBiTecS, Service d'Ing
enierie Mol
eculaire des Prot
eines, Gif sur Yvette, France d Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jos
e, Costa Rica a

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 January 2014 Received in revised form 20 May 2014 Accepted 21 May 2014 Available online xxxx

BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of recombinant antibody fragments for improving the neutralization of local tissue damage in snakebite envenoming. © 2014 Published by Elsevier Ltd.

Keywords: Neutralizing antibody scFv Metalloproteinase BaP1 Hemorrhage Snake venom

Q1

* Corresponding author. Tel.: þ55 11 2627 9783. E-mail addresses: [email protected] (J.M.A. Castro), [email protected] (T.S. Oliveira), [email protected] (C.R.F. Silveira), [email protected] (M.C. Caporrino), [email protected] (D. Rodriguez), [email protected] (A.M. Moura-da-Silva), oscarhpr@
rrez), [email protected] (G.S. gmail.com (O.H.P. Ramos), [email protected] (A. Rucavado), [email protected] (J.M. Gutie Magalh~ aes), [email protected] (E.L. Faquim-Mauro), [email protected], [email protected] (I. Fernandes). http://dx.doi.org/10.1016/j.toxicon.2014.05.017 0041-0101/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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1. Introduction Envenoming by snakebites constitutes a significant public health problem on a worldwide basis, particularly in tropical regions of Africa, Asia and Latin America
rrez et al., 2006; WHO, 2013). According to WHO, (Gutie 2013, the global total of snakebite envenoming cases is estimated to 2, 4 million with deaths ranging from 94,000 to 125,000 per year. At present, passive immunotherapy is the only specific treatment for snakebite envenoming used worldwide, which is based on the administration of antibodies produced by the hyperimmunization of animals, generally horses, with snake venoms. These antivenoms are preparations of immunoglobulins (Igs), or Ig fragments such as F(ab0 )2 or Fab, obtained by fractionating hyperimmune plasma either by treatment with caprylic acid to obtain
rrez et al., whole IgG preparations (Rojas et al., 1994; Gutie 2005) or by enzymatic digestion followed by ammonium sulfate precipitation and chromatographic steps to obtain IgG fragments (Cardoso, 2000; WHO, 2010). Antivenoms are generally very efficient for neutralizing the most relevant systemic effects of snakebite envenom
rrez Q2 ing (Warrell, 1992; Lalloo and Theakston, 2003; Gutie
n, 2009); however, they poorly neutralize toxins and Leo involved in local pathological effects (edema, dermonecrosis, local hemorrhage and myonecrosis). This is due to the early onset of these effects upon envenoming and, to some degree, to the poor distribution of the Igs and their fragments to the local tissues where venom is injected
rrez et al., 1998). Furthermore, administration of (Gutie antivenom may be associated, in a variable percentage of cases, with early and late adverse reactions to the heter
n et al., 2013). ologous proteins (Warrell, 1995; Leo A new perspective for the treatment of snakebite envenoming has emerged with the use of recombinant antibodies, particularly the Fv fragment, named the single-chain variable fragment (scFv). The scFv antibody format presents several distinctive features as compared to the whole antibody, such as higher diffusion to the affected tissues, low immunogenicity and faster elimination (Azzazy and Highsmith, 2002; Zhang et al., 2014; Yu et al., 2014). ScFv antibody is composed exclusively of Ig VL and VH regions joined by a flexible peptide linker, usually composed of 15 amino acid residues with the sequence (Gly4Ser)3, and is expressed as a single polypeptide chain. The linker allows the association of the VH and VL to form the antigen-binding site (Azzazy and Highsmith, 2002). Recombinant scFv antibodies may be obtained by phage display technology or from hybridoma cells secreting monoclonal antibodies. Our group has obtained and characterized six monoclonal antibodies (MoAb) against BaP1 (MABaP1) from the venom of the pit viper Bothrops asper. BaP1 is an abundant P-I snake venom metalloproteinase (SVMP) in the venom of this species, and plays a relevant role in the local tissue damage associated with envenomings by B. asper, a medically important species in Central America and parts of South and North America. We previously showed that three monoclonal antibodies were able to neutralize BaP1-

induced hemorrhagic and proteolytic activities (Fernandes et al., 2010). Herein we describe the generation of a recombinant single chain antibody fragment (scFv) produced from the mRNA isolated from MABaP1-8, expressed in Escherichia coli cytoplasm, and possessing neutralizing activities similar to those of the original monoclonal antibody. In addition to its value as a molecular tool to assess the structureefunction relationships of this SVMP, this scFv will allow the evaluation of small recombinant antibody fragments in the neutralization of venom-induced local tissue damage. 2. Materials and methods 2.1. Animals, venoms and enzymes BALB/c female mice (18e20 g) were used throughout. All procedures were approved by the Ethical Committee for Animal Research of Instituto Butantan (661/09) while CGEN (Board of Genetic Heritage Management) provided the license for genetic patrimony access (02001005148/ 2008e11). The Herpetology Laboratory of Instituto Butantan provided Bothrops neuwiedi venom, while Instituto Clodomiro Picado, Costa Rica, provided B. asper venom. The venoms corresponded to pools obtained from many specimens and were lyophilized and stored at 20  C. The SVMPs BaP1 and BnP1 were purified as previously described by
rrez et al. (1995) and by Baldo et al. (2008), respectively. Gutie 2.2. Cloning of single-chain variable fragment (scFv) derived from the monoclonal anti-BaP1 antibody Protocols for DNA manipulation were used as described in Sambrook and Russell (2001). Briefly, MABaP1-8 hybridoma cells secreting anti-BaP1 monoclonal antibody (Fernandes et al., 2010) were cultivated in RPMI medium (Invitrogen, Brazil) plus 10% fetal bovine serum at 37  C in 5% CO2. Hybridoma mRNA was extracted (IlustraQuickprep mRNA Purification Kit e GE Healthcare, UK) from the cells and reversely transcribed into cDNA (First Strand cDNA Synthesis Kit e GE Healthcare, UK). Commercially available primers [Light primer mix, Heavy primer 1, Heavy primer 2 (GE Healthcare, UK)] were employed to amplify the variable domain of heavy (VH) and light (VL) chains of the antibody, following the manufacturer's instructions. The cDNA inserts corresponding to VL and VH were cloned into the pGEM-T Easy vector and submitted to sequencing. The synthetic gene ScFvBaP1 was built by GeneArt containing VH sequence joined to VL by a flexible linker sequence that encodes (Gly4Ser)3 and codon-optimized for E. coli with BamHI and HindIII sequence flanking the construction. GeneArt vector holding scFvBaP1 sequence was digested with BamHI and HindIII endonucleases to release the construct and cloned into pMST3 vector, a modified pET28b vector with the small ubiquitin-related modifier (SUMO) sequence cloned 30 to the His6 tag (Yunus and Lima, 2009), resulting in the expression of SUMO-scFvBaP1. All microorganisms manipulation was approved by CTNBio (National Technical Commission on Biosecurity) (CQB-0039/98 de 31/07/1998).

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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2.3. scFvBaP1 expression and purification Chemically competent E. coli C43 (DE3) (Miroux and Walker, 1996) was transformed with pMST3-scFvBaP1 construction and plated on LB agar plates containing 100 mg/mL ampicillin and grown overnight at 37  C, shaking at 150 rpm. Next day, a single colony was inoculated into LB broth (100 mg/mL ampicillin) and grown overnight at 37  C. The overnight culture was then used to inoculate 250 mL of fresh LB (100 mg/mL ampicillin) medium at 1:100 dilution. At an optical density (600 nm) of 0.6, isopropyl b-D-1 thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce scFvBaP1 expression. The culture was allowed to express for 4 h before the cells were harvested by centrifugation at 5000 g for 10 min at 4  C. Cells were resuspended in binding buffer (50 mM sodium phosphate pH 7.0, 300 mM NaCl and 10 mM of imidazole) and intermittently sonicated on ice for 60 s with intervals of 5 min for sample cooling (4 cycles). Cells were then harvested by centrifugation (7000 g, 10 min, 4  C) and the recombinant protein was purified from the supernatant by immobilized metal affinity chromatography (IMAC) using Ni-Sepharose resin (GE Healthcare, UK), following the manufacturer's recommendations. After purification, the recombinant protein was dialyzed against phosphate-buffered saline solution (PBS) and analyzed in a 12.5% SDS-PAGE under reducing conditions. 2.4. Ability of scFvBaP1 to recognize SVMPs BaP1 and BnP1, and whole B. asper venom, by ELISA ELISA was carried out according to Theakston et al. (1977). Briefly, plates were coated with either P-I class SVMPs (BaP1 or BnP1) or venoms (2 mg/well) and, after blocking with 3% bovine serum albumin, scFv antibody was added, followed by incubation with mouse anti-HIS antibody. Antigeneantibody reaction was detected by addition of anti-mouse IgG-peroxidase conjugate and revealed by incubation in presence of ortho-phenylenediamine (1 mg/ mL, Sigma) plus H2O2 as substrates. As controls, the reactivity of scFv anti-LDL() and its MoAb partner were tested against BaP1. These antibodies were kindly provided by D. Abdalla (Kazuma et al., 2013). 2.5. Neutralization of BaP1-induced hemorrhage by scFvBaP1 The ability of the scFvBaP1 to neutralize BaP1-induced hemorrhage was studied by incubating scFv (10:1 scFv:BaP1 molar ratio) or IgG purified from MABaP1 (1.5:1 MABaP1:BaP1 molar ratio) with BaP1 for 1 h at 37  C. After incubation, the mixture was centrifuged, and aliquots of 100 mL of the supernatant, containing one Minimum Hemorrhagic Dose (MHD) of BaP1 (35 mg), were injected intradermally (i.d.) into the dorsal skin of mice (Fernandes et al., 2010). Animals were killed 3 h after injection, the skin was removed, and the area of the hemorrhagic spots (A) calculated according to the formula A ¼ p. R2, where R is the radius of the hemorrhagic lesion. The results were expressed in mm2. Normal mouse serum (NMS) and SUMO were used as negative controls for all experiments.

3

2.6. Neutralization of fibrinolytic activity of BaP1 and BnP1 by scFvBaP1 antibodies Fibrinolytic activity was assayed by the fibrin-plate method, as previously described (Jespersen and Astrup, 1983). Briefly, a fibrin-agarose gel was prepared by mixing 1 mg/mL solution of human fibrinogen (Calbiochem) with a pre-heated solution of 2% agarose in 50 mM TriseHCl, pH 7.3, buffer containing 200 mM NaCl, 50 mM CaCl2 and 2 U/mL thrombin. Samples of SVMPs (5 mg) or samples of SVMPs previously incubated (30 min/37  C) with scFvBaP1 (20:1 or 10:1 M ratios) or MABaP1 (5:1 or 2.5:1 M ratios) were applied to wells pierced in the solidified gel. Plates were incubated at 37  C overnight, and then the area of fibrin hydrolysis was measured. The results were expressed in cm2 of fibrinolytic area. 2.7. Neutralization of BaP1-induced myotoxic activity by scFvBaP1 The myotoxic activity of BaP1 and the neutralizing potential of scFvBaP1 on this activity were evaluated in mice injected i.m. with 20 mg BaP1 either alone or previously incubated with scFvBaP1 (10:1 M ratio). After 4 h, the animals were bled and the sera were assayed for creatine kinase (CK) activity (U/L) with a commercial kit (Bioclin, K069). 2.8. Neutralization of BaP1 and BnP1-induced inflammation by scFvBaP1 Inflammatory reaction induced by BaP1 or BnP1, and their neutralization by scFvBaP1, were evaluated in the peritoneal cavity of mice. The enzymes (5 mg), or samples of enzymes previously incubated with scFvBaP1 (20:1 or 10:1 M ratios) or MABaP1 (5:1 or 2.5:1 M ratios), were injected in the peritoneal cavity and, after 6 h, the cellular infiltrate was collected. Mice injected with PBS or SUMO were used as controls. For peritoneal cell harvesting, mice were killed by CO2 inhalation, and the peritoneal cells were aseptically collected by washing the peritoneal cavity with 5 mL of sterile ice-cold PBS devoid of calcium and magnesium ions. For total cell determination, nine volumes of peritoneal cells were added to one volume of 0.05% crystal violet dissolved in 30% acetic acid, and counts were performed using a bright-line hemocytometer (Sigma, St. Louis, MO, USA). Differential cell counts were determined by cytospin preparations stained with Instant-Prov (Newprov, Pinhais, Brazil). To determine H2O2-release, a horseradish peroxidasedependent phenol red oxidation microassay was used (Pick and Mizel, 1980). Briefly, two million peritoneal cells Q3 were suspended in one mL of freshly prepared phenol red solution consisting of ice-cold Dulbecco's PBS containing 5.5 mM dextrose, 0.56 mM phenol red (Sigma) and 8.5 U/mL horseradish peroxidase type II (Sigma). One hundred microliters of the cell suspension were added to each well and incubated for 1 h at 37  C in a humid atmosphere containing 5% CO2 with 10 mL phorbol myristate acetate (PMA) 1 mg/mL (SigmaeAldrich). The reaction was stopped with 10 mL 1 N NaOH. The absorbance was measured at 620 nm with a

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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micro-plate reader (MR 5000, Dynatech Laboratories Inc., Gainesville, VA, USA). Conversion of absorbance to mM H2O2 was done by comparison to a standard curve obtained with known concentrations of H2O2 (5e40 mM), as described previously (Pick and Keisari, 1980). 2.9. Statistical analysis The significance of the differences of two mean values was analyzed by the Student's t-test. When more than two experimental groups were compared, the significance of the differences was determined by ANOVA, followed by Tukey test (p values < 0.05 were considered significant). 2.10. Tertiary structure prediction The tertiary structure of scFvBaP1 [GenBank: KF724934] was predicted using the method detailed elsewhere (Ramos, 2012). Briefly, models of heavy- and light-chain variable domains were independently built by grafting segments (framework regions and CDRs) from homologous templates. Next, hybrid models of heavy-chain (HV) and light-chain (LV) were mutated to comply with target's sequence and associated by fitting into a template structure that closely matches their predicted packing angle. The associated variable domains, corresponding to the scFv, were submitted to energy minimization (1 step of steepest descent each 100 steps of conjugate gradient). Next, CDRH3 was optimized by a simulated annealing protocol (500 ps) using an implicit solvent model (GBSA) implemented GROMACS (Hess et al., 2008). The resulting model was employed to calculate its quaternary structure with BaP1 (PDB: 1ND1) using ZDOCK 3.0 restrained search (Pierce et al., 2011) by excluding any possible interaction with structural regions that are not expected to be involved in the formation of the complex interface (i.e., residues opposed to the catalytic site of BaP1 or opposed to the predicted antigen binding site of scFvBaP1). The top ranked model was used as starting conformation for refinement using Rosetta Dock (Chaudhury et al., 2007) for the production of 10 complex models that were assumed as possible binding modes. 3. Results 3.1. Cloning strategy of scFv anti-BaP1 recombinant antibody The amplified fragments corresponding to VL and VH were cloned into pGEM-T Easy vector and sequenced using the M13 primers (forward and reverse). These sequences were used to build the synthetic gene scFvBaP1 that included the linker between VH and VL, as described in Fig. 1. The construct was subsequently cloned into pMST3 expression vector in frame with SUMO sequence that contains a N-terminal 6xHis tag, which allows solubilization and purification through immobilized metal affinity chromatography (IMAC), respectively. One positive clone was selected by PCR and the insert was fully sequenced (data not shown). The predicted amino acid sequences of VL and VH were analyzed in the NCBI database (blast.ncbi. nlm.nih.gov) using the BLAST tool, and were classified as

mouse immunoglobulin variable domains. The complementarity determining regions (CDRs) were identified according to Chothia scheme, following pre-stipulated rules (Ramos, 2012, Fig. 1). 3.2. Purification of the recombinant scFvBaP1 The scFvBaP1 protein fused to SUMO was expressed in E. coli C43 (DE3) and purified using Ni-Sepharose resin. The purified protein was then quantified and subjected to analysis by 12.5% SDS/PAGE (Fig. 2), revealing a protein of about 40 kDa, as predicted for SUMO-scFvBaP1 (38.9 kDa). The final process yield was approximately 280 mg of scFv per liter of bacterial culture. SUMO was also purified and used as control in neutralization experiments. 3.3. Ability of scFvBaP1 to recognize BaP1 toxin or whole venom by ELISA The ability of scFvBaP1 to recognize purified BaP1 or B. asper whole venom was analyzed by ELISA. In this assay the MABaP1 was used as positive control and SUMO as negative control. Fig. 3 shows that MABaP1 and scFvBaP1 recognized both BaP1 and B. asper venom. No binding was observed for SUMO protein alone. The irrelevant scFv antiLDL() or its MoAb partner did not recognize BaP1 (data not shown). 3.4. Neutralization of enzymatic and toxic activities of BaP1 by anti-BaP1 antibodies ScFvBaP1 was able to neutralize fibrin degradation induced by BaP1 in a dose-dependent manner (molar ratios 20:1 and 10:1) (Fig. 4). On the other hand, MABaP1 completely abrogated this activity at molar ratios of 5:1 and 2.5:1. Both scFvBaP1 (at molar ratio of 10:1) and MABaP1 (at molar ratio of 1.5: 1) completely neutralized hemorrhagic activity of BaP1 (Fig. 5). In addition, scFvBaP1 was able to completely neutralize myotoxicity induced by BaP1 at a molar ratio of 10:1 (Fig. 6). 3.5. Neutralization of BaP1-induced inflammation by scFvBaP1 BaP1 induced an intense inflammatory response in the peritoneal cavity of mice, when compared to that observed in mice injected with PBS (Fig. 7A). The differential cell counts evidenced the predominant presence of neutrophils in peritoneal lavage of mice injected with BaP1 (data not shown). The neutralizing activity of scFvBaP1 on this leukocyte influx induced by BaP1 was evident, as shown in Fig. 7A. Hydrogen peroxide (H2O2) production was also analyzed as an indicator of the cellular activation of peritoneal cells by BaP1. Fig. 7B shows that production of H2O2 by peritoneal cells stimulated in vitro with PMA was significantly higher in mice that were injected with BaP1 than in mice injected with PBS. As shown in Fig. 7B, the previous incubation of the toxin with scFvBaP1 prevented H2O2 production.

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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Fig. 1. DNA and predicted amino acid sequences of scFvBaP1. The complementarity-determining regions (CDR1eCDR3 in bold) of heavy (VH) and light (VL) chain variable domains were identified according to Chothia scheme. The synthetic flexible linker between the VH and VL domains is underlined.

3.6. ScFvBaP1 recognizes and neutralizes the biological activities of BnP1, a P-I SVMP isolated from Bothrops neuwiedi venom The ability of scFvBaP1 to recognize BnP1, a P-I SVMP isolated from B. neuwiedi venom, was evaluated. ScFvBaP1 was able to recognize the purified BnP1 (Fig. 8A), and to neutralize fibrinolytic (Fig. 8B), inflammatory (Fig. 8C) and H2O2-generating activities of this enzyme (Fig. 8D).

surface is composed of positively (present in light- and heavy-chain) and negatively (mainly present in the heavychain variable domain) charged regions. This surface area is adjacent to S10 substrate binding pocket usually found in SVMPs. One of the docking solutions completely blocks the access to the catalytic zinc (Fig 9B). Alternative graphical representations of the docking solutions are presented in Supplementary Figs. 1S and 2S. The coordinates of scFVBaP1 model and docking solutions are available as Supplementary data.

3.7. Tertiary and quaternary structure predictions 4. Discussion The scFVBaP1 model is represented in Fig. 9A. It comprises typical features of antibody variable domains and a relatively short CDR-H3. The docking between scFvBaP1 model and BaP1 crystallographic structure resulted in 10 possible binding modes. The main cluster of solutions interacts with BaP1 surface delineated by residues positioned at the N-terminal region of the helix harboring part of the zinc binding motif and the subsequent loop that links this helix with the most C-terminal one. The antigen binding

SVMPs are key components of viperid snake venoms, as
n et al., 2008; revealed by proteomic analysis (Alape-Giro Calvete, 2011; Lomonte et al., 2012; Sousa et al., 2013). Venoms present a variable balance between P-I and P-III SVMPs, a finding that has implications for the pathophysiology of envenomings. It has been demonstrated that P-III SVMPs have higher toxicity, i.e. hemorrhagic and procoagulant activities, than P-I SVMPs, and that the latter are

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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Fig. 2. SDS-PAGE analysis of scFvBaP1 during production steps. Separation of proteins was performed in 12.5% acrylamide gel, and revealed by Coomassie blue staining. MW: molecular-weight marker (Page Ruler TM, Thermo Scientific); lysate samples of bacteria transformed by SUMOscFvBaP1 expression vector without (1) and with IPTG induction (2); purified SUMO-scFvBaP1 (3); lysate samples of bacteria transformed by SUMO expression vector without (4) and with IPTG induction (5); purified recombinant SUMO protein (6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

likely to play a predominantly digestive role owing to their high enzymatic activity, also contributing to the prominent local tissue damage characteristic of these envenomings
rrez et al., 2010a). This is illustrated by the case of (Gutie BaP1, an enzyme that comprises approximately 10% of B. asper venom from the Pacific region of Costa Rica (Alape
n et al., 2009). BaP1 has low systemic toxicity Giro
rrez et al., 1995; Escalante et al., 2004), but exerts (Gutie significant local tissue damage associated with hemorrhage, myonecrosis (Rucavado et al., 1995), dermonecrosis
nez et al., 2008), pain (Fernandes et al., and blistering (Jime

Fig. 3. scFvBaP1 recognition of BaP1 toxin or whole venom by ELISA. Samples of purified BaP1 or crude B. asper venom were coated onto 96 well plates and incubated with scFvBaP1, MABaP1 or SUMO. After washing and incubation with anti-HIS antibody, the antigeneantibody complex formation was detected using anti-mouse IgG-HRP and revealed under suitable enzymatic condition.

Fig. 4. scFvBaP1 neutralization of the fibrinolytic activity induced by BaP1. A sample of BaP1 toxin (5 mg) was incubated (30 min/37  C) with MABaP1 (at 2.5:1 or 5:1, antibody: toxin molar ratio) or scFvBaP1 (20:1 or 10:1 M ratio). Samples were applied to agarose plates containing human fibrinogen clotted by bovine thrombin, incubated (37  C, overnight), subsequently, the hydrolyzed area was measured and expressed in mm2. *p < 0.05 compared to PBS group values, #p < 0.05 compared to BaP1.

2007), and inflammation (Farsky et al., 2000; Fernandes et al., 2006). Thus, BaP1 is a typical example of a P-I SVMP provoking complex local pathology. Antivenoms are highly effective at abrogating the sys
rrez temic effects induced by Bothrops sp venoms (Gutie
n, 2009). As a consequence, antivenom adminisand Leo tration readily controls systemic bleeding and coagulopathy in patients suffering envenomings by Bothrops sp ~ o et al., 1998, 2006). bites (Pardal et al., 2004; Otero-Patin In contrast, antivenoms are relatively inefficient for controlling the extent of local tissue damage, including local

Fig. 5. Ability of MABaP1 and scFvBaP1 to neutralize BaP1-induced hemorrhage. Hemorrhage was induced by BaP1 (35 mg) intradermally injected into the dorsal skin of Swiss mice. To neutralize BaP1-induced hemorrhage, MABaP1 or scFvBaP1 was incubated with BaP1 (1.5:1 or 10:1 M ratio, respectively). Mice were killed 3 h after injection, the skin was removed, the area of the hemorrhagic spots was assessed and expressed in mm2. *p < 0.05 compared to saline values, #p < 0.05 compared to BaP1.

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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Fig. 6. Ability of scFvBaP1 to neutralize BaP1-induced myonecrosis. To evaluate the myotoxic activity and the neutralizing potential of scFvBaP1 on this activity, mice were injected (i.m.) with either 20 mg BaP1 alone or previously incubated with scFvBaP1 (10:1 M ratio). After 4 h, the animals were bled and the sera were assayed for creatine kinase activity. *p < 0.05 compared to PBS values, #p < 0.05 compared to BaP1.


rrez et al., 1998; hemorrhage and myonecrosis (Gutie ~ o, 2009). This is likely to depend on several Otero-Patin factors, such as rapid development of local pathology after the bite, poor access of antivenom antibodies to the affected tissues, and low immunogenicity of toxins responsible for local tissue damage, i.e. phospholipases A2 (PLA2s) and P-I SVMPs. This agrees with antivenomics studies, which have shown that antivenoms have high capacity to recognize P-III SVMPs, but are much less effi
rrez et al., cient at recognizing P-I SVMPs and PLA2s (Gutie 2010b; Sousa et al., 2013). Thus neutralization of venom induced local tissue damage remains one of the most difficult challenges to improve the management of viperid snakebite envenomings.

7

To this end, the use of recombinant scFv fragments offers a promising alternative to improve treatment, mainly owing to the pharmacokinetic properties of such small molecular mass antibody fragments, which enable them to reach extravascular spaces in the affected tissues much more readily than whole IgG or its fragments. In this work, a scFv version of MABaP1, a neutralizing monoclonal antibody, was obtained. To construct scFv, the variable regions can be connected in either the VH- linker- VL or VL-linker-VH orientation. These orientations can affect expression efficiency (Merk et al., 1999), stability, and antigen binding activity (Desplancq et al., 1994). We chose the former because it is the one preferred by most researchers. Our scFv was constructed using the synthetic gene containing VH and VL joined by the linker (G4S)3, and was cloned in pMST3 vector that contains the sequence of Small Ubiquitin-like Modifier (or SUMO) protein. It was verified that scFvBaP1 and MABaP1 presented similar results regarding the recognition of its target, i.e. BaP1. The scFvBaP1 was expressed in fusion with SUMO that could have been removed using the proteolytic enzyme Ulp1. However, we decided to maintain SUMO in fusion with the scFv because removal would decrease the yield of the recombinant antibody and mainly because we did not observe any interaction between SUMO and BaP1 or other venom components in our assays. This result was expected since SUMO is a recombinant protein related only to solubility of scFv and it is not expected to be involved in antigen specific recognition (data not shown) (Lee et al., 2008). SUMO is a small protein of around 100 amino acids and 12 kDa. Our observations clearly show that scFvBaP1 was able to neutralize proteolytic, i.e. fibrinolytic, as well as hemorrhagic, myotoxic, and pro-inflammatory effects of BaP1. Moreover, it also neutralized enzymatic and proinflammatory activities of BnP1 from the venom of B. neuwiedi. In terms of molar ratio, the scFv fragment was less effective than the monoclonal antibody in the

Fig. 7. Ability of scFvBaP1 to neutralize BaP1-induced inflammatory cell migration and H2O2 production in the peritoneal cavity of mice. Total peritoneal exudate cell (PEC) (A) and production of H2O2 by exudate cells (B) were evaluated. BALB/c mice were injected (i.p.) with either PBS, BaP1 (5 mg) or BaP1 previously incubated with scFvBaP1 (10:1 M ratio). Mice were killed 6 h after treatment and total cells were counted in a Neubauer chamber. For detection of H2O2, 2.5  106 cells/mL were incubated in the presence or absence of PMA (10 ng/well) followed by horseradish peroxidase (HRPO)-mediated oxidation of phenol red, consequently, resulting in increased absorbance at 620 nm. The data are expressed as mean ± SD (4e5 animals/group). *p < 0.05 compared to PBS values, # p < 0.05 compared to BaP1.

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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Fig. 8. Ability of scFvBaP1 to recognize and neutralize the biological activities of BnP1 (a P-I SVMP isolated from Bothrops neuwiedi venom). To evaluate the scFvBaP1 recognition of BnP1, samples of this SVMP were used to coat plates that were incubated with scFvBaP1. After addition of anti-HIS antibody, antigeneantibody complex formation was detected using anti-mouse IgG-HRP followed by incubation with suitable substrate (A). To evaluate the ability of scFvBaP1 to neutralize fibrinolysis (B) induced by BnP1, 5 mg of BnP1 was incubated (30 min/37  C) with scFvBaP1 (20:1 or 10:1 M ratio). Samples were applied to agarose plates containing human fibrinogen clotted by bovine thrombin. After incubation (37  C, overnight), the hydrolyzed area was measured and expressed in mm2. The inhibition of BnP1-proinflammatory activities by scFvBaP1 was evaluated in mice. Total peritoneal exudate cell (PEC) (C) and production of H2O2 by exudate cells (D) was determined as described in the legend of Fig. 7 and in materials and methods section. The data are expressed as mean ± SD (4e5 animals/group). *p < 0.05 compared to PBS values, #p < 0.05 compared to BnP1.

neutralization of fibrinolytic and hemorrhagic activities. This is likely to depend on differences in stability and affinity owing to the different nature of these molecules. Also, steric hindrance effects imposed by the larger form (MABaP1), its bivalence and probable post-translational modification can not be discarded at this point as plausible explanations for its increased efficacy. Thus, when preincubated with BaP1 before injection in mice, the scFv fragment was effective concerning the neutralization of the most important local effects induced by this P-I SVMP. Moreover, our findings provide clues from the structureefunction perspective, since they strongly suggest that pathological and pro-inflammatory activities of BaP1 depend on its proteolytic activity, i.e. on the accessibility of the catalytic site, suggested by the fact that the inhibition of enzymatic and pharmacological effects by the scFv antibody could not be dissociated. This is in agreement with previous studies in which metalloproteinase inhibitors were able to blockade hemorrhagic and pro-inflammatory actions of BaP1 (Escalante et al., 2000; Fernandes et al., 2006). Docking analysis suggests that such inhibition of

catalytic activity might be due to either a direct recognition of scFv antibody to the catalytic cleft or, alternatively, to steric hindrance after binding to an epitope located in a nearby molecular region. The use of scFv fragments to neutralize venom components at the experimental level has been explored by various groups. Significant advances have been made in the generation of scFv against scorpion venoms (Mousli et al.,
ndez et al., 2012; Rian ~ o-Umbarila 1999; Quintero-Herna et al., 2013; Pucca et al., 2013). Regarding snake venom toxins, Cardoso et al. (2000) and Oliveira et al. (2009) described the ability of scFvs produced by phage display methodology to neutralize myonecrosis induced in mice by crotoxin, a PLA2 complex from Crotalus durissus terrificus venom. Similar results were obtained using a pool of scFv produced against Bothrops jararacussu venom, which was able to reduce in vivo the myotoxic activity of bothropstoxin-I, a PLA2 homologue, and bothropstoxin-II, a PLA2 (Tamarozzi et al., 2006), as well as other Bothrops sp venoms (Roncolato et al., 2013). Most scFvs with neutralizing activity produced against toxins from snake venoms

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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9

Fig. 9. Structure prediction of scFVBaP1 (A) and its complex with BaP1 (B). (A) scFvBaP1 model represented as cartoon. Conserved framework regions of light (VL) and heavy (VH) chain variable domains are represented in gray. CDRs are represented in white (L1 and H1), dark gray (L2 and H2) or black (L3 and H3). C-alpha carbons corresponding to the first and last residues of each CDR are represented as spheres. The image as created using Pymol (The PyMOL Molecular Graphics €dinger, LLC). The web version of this article provides a color image. The atomic positions corresponding to scFvBaP1 molecular model System, Version 1.6 Schro are available as Supplemental data (scfvBaP1.pdb). (B) Ten possible binding modes for BaP1/scFvBaP1 complex. The complex was calculated using scFvBaP1 model and BaP1 crystallographic structure (1ND1). The histidine triad that coordinates the Zn atom (dark gray) are represented as sticks. The catalytic Zn atom is represented as a gray sphere. The black spheres indicate the unweighted center of mass of the scFvBaP1 corresponding to each solution in order to represent different binding positions. The molecular graphic was created using UCSF Chimera 1.9. The web version of this article provides a color figure and alternative graphical representations of the docking solutions (Supplementary Figs. 1S and 2S). The atomic positions of docking solutions are available as Supplemental data (Docking_solutions.pdb). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

were obtained by phage display, whereas scFvBaP1 was generated from hybridoma technology, a platform that has been used in the generation of therapeutics against various diseases (Chester et al., 2004). Low molecular mass neutralizing molecules, such as scFvs, represent an alternative to improve the immunotherapy of envenomings by animal bites and stings. In the case of viperid snakebite envenomings, it is necessary to explore the potential of these recombinant antibody fragments to neutralize toxins provoking local tissue damage. These toxins present two features that might be confronted with the aid of scFvs: (a) SVMPs and PLA2s causing local hemorrhage, myonecrosis, and blistering are poor immunogens and, hence, antivenoms have relatively low titers against these toxins; (b) owing to their pharmacokinetic properties, IgGs and their fragments in antivenoms have limitations for reaching the affected tissues in viperid envenomings. These two aspects can be improved by the use of scFv fragments, since the pharmacokinetic properties of such small antibody fragments allow them to reach extravascular areas where locally acting toxins are present. One disadvantage of scFv fragments has to do with their reduced half-life, owing to a
rrez et al., rapid renal clearance of these fragments (Gutie 2003). This drawback can be confronted by preparing antivenoms containing a combination of large neutralizing molecules, i.e. IgGs or F(ab0 )2 fragments, and scFv frag
rrez et al., 2003, 2007). The former would ments (Gutie remain in the bloodstream for prolonged time periods whereas the latter would rapidly diffuse to extravascular spaces where toxins are distributed. This hypothesis can now be tested in the case of B. asper venom by using whole IgG antivenom enriched with the scFv fragment described in this work. Another option would be the modification of the scFv by PEGylation or its fusion to Fc antibody domain.

In conclusion, our data show that scFv specifically recognizes BaP1 and whole B. asper venom, and neutralizes the main biological effects of BaP1, an abundant SVMP in this venom. The docking analyses performed revealed possible modes of interaction of the recombinant antibody and BaP1, thus providing clues for understanding its mode of neutralization. In addition, this scFv antibody may become a useful tool for exploring the development of novel therapeutic alternatives for the neutralization of the local tissue damage induced by this SVMP. Future studies are necessary to test the therapeutic potential of this scFv antibody in preclinical experimental models. Uncited reference Pick and Mizel, 1981.

Q7

Acknowledgments ~o Paulo Research Foundation (FAPESP), 2012/01028-3 Sa PIPE/FAPESP 2004/08297-3, INCT-TOX program of Conselho
gico Nacional de Desenvolvimento Científico e Tecnolo ~o de Amparo a Pesquisa do Estado de (CNPq) and Fundaça ~o Paulo (Fapesp), Brazil, CAPES (fellowship to JA Castro), Sa
n (Universidad de CNPq and Vicerrectoría de Investigacio Costa Rica). Conflict of interest The authors declare that there are no conflicts of interest. Q4 Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.toxicon.2014.05.017.

Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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~ eda, N., Silva-Díaz, J.F., Murcia, M., C
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Please cite this article in press as: Castro, J.M.A., et al., A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom, Toxicon (2014), http://dx.doi.org/10.1016/ j.toxicon.2014.05.017

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