Andersen, Mikael Rørdam

B. Hallgren, Malte

H. Dam, Søren

Kilstrup, Mogens

Laustsen, Andreas Hougaard

M. Haack, Aleksander

Martos Esteban, Andrea

U. W. Friis, Rasmus

Snakebite is one of the world’s most neglected tropical diseases, with an estimated 5 million bites per year, resulting in about 125.000 deaths. The only current treatment for snakebite envenoming is antiserum derived from the blood of immunized mammals(typically horses). These antisera are expensive to produce and carry a high risk of causing hyper-allergic reactions in human recipients due to their heterologous origin. Here we report the discovery of chimeric scFvs against Bothrops asper toxins

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Dam, Søren; Martos Esteban, Andrea;Andersen, Mikael Rørdam; Kilstrup, Mogens; Laustsen, Andreas HougaardPublication date:2016Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):M. Haack, A., B. Hallgren, M., U. W. Friis, R., H. Dam, S., Martos Esteban, A., Andersen, M. R., ... Laustsen, A.H. (2016). Design of Fab-based chimeric antibodies against Bothrops asper toxins. Poster session presented atSymposium for Biological and Life Science Students (SymBLS) 2016, Cambridge, United Kingdom.D E P A R T M E N T   O F   B I O T E C H N O L O G Y   A N D   B I O M E D I C I N E   T E C H N I C A L   U N I V E R S I T Y    O F   D E N M A R K   Design of Fab-based chimeric antibodies against Bothrops asper  toxins Aleksander M. Haack1,2, Malte B. Hallgren1,2, Rasmus U. W. Friis1,2,  Søren H. Dam1,2, Andrea Martos Esteban1, Mikael Rørdam Andersen1, Mogen Kilstrup1, Andreas  H. Laustsen1  1Department of Biotechnology and Biomedicine, Technical University of Denmark  2Department of Bio and Health Informatics, Technical University of Denmark Composition of B. asper  venom References  [1] Richard G., Meyers A.J., McLean M.D., Arbabi-Ghahroudi M., MacKenzie R., et al. (2013) In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody. PLoS ONE 8.7:e69495 [2] Gutiérrez, J.M., León, G., Lomonte, B., Angulo, Y. (2011) Antivenoms for snakebite envenomings. Inflammation and Allergy - Drug Targets 10.5:369-380 [3] Harrison R.A., Cook, D.A., Renjifo, C., Casewell N.R., Currier R.B., Wagstaff, S.C. (2011) Research strategies to improve snakebite treatment: Challenges and progress. Journal of Proteomics 74.9:1768-1780 [4] Alape-Giron A., Sanz L., Escolano J., Flores-Díaz M., Madrigal M., Sasa M., Calvete J.J. et al. (2008) Snake Venomics of the Lancehead Pitviper Bothrops Asper Geographic, Individual, and Ontogenetic Variations. Journal of Proteome Research 7.8:3556–3571 [5] Castro J.M.A., Oliveira T.S., Silveira C.R.F., Caporrino M.C., Rodriguez D. et al. (2014) A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom. Toxicon 87:81-91 [6] I-TASSER, protein modeling tool, http://zhanglab.ccmb.med.umich.edu/I-TASSER// [Accessed 9th of November]    Contact information  Aleksander M. Haack:  s153292@student.dtu.dk Søren H. Dam:   s153398@student.dtu.dk Malte Hallgren:   s153002@student.dtu.dk Rasmus U.W. Friis:   s154269@student.dtu.dk  Acknowledgements We thank the Novo Nordisk Foundation (NNF16OC0019248) and Symphogen A/S for financial support. Next steps: Determining the best antibody format Addressing the problem of immunogenic antivenoms Composition of B. asper venom[4] according to protein families, as percentages of the total protein content:  SVMPs: Snake Venom MetalloProteinase PLA2:  Phospholipases A2 CRISP:  Cysteine-Rich Secretory Proteins LAO:   L-amino Acid Oxidases SP:   Serine Proteinases DC:   Dendritic Cell Fragments 2 1 7 4 Construction of scFvBaP1 from a monoclonal antibody Results from the recombinant scFvBaP1 5 3 Transformation of antibody formats into E. coli Hybridoma cells secreting the monoclonal antibody against BaP1 (MABaP1) were made, and from these a recombinant single chain variable fragment (scFv) was constructed.  6 BaP1 Hybridoma cells B cells are harvested  Myeloma cells MABaP1 (monoclonal antibody) scFvBaP1 Binding strength testing of chimeric antibodies by ELISA Binding strengths of the antibodies to BaP1 will be determined by ELISA. This will allow us to compare the different antibody formats in regards to how likely they are to neutralize BaP1. The scFvBaP1 was cloned into the pMST3 expression vector in frame with a SUMO (Small Ubiquitin-like Modifier) sequence. When subjected to SDS-PAGE, the sequenced SUMO-scFvBaP1 showed a band at 40 kDa as was predicted and SUMO protein around 13 kDa[5] (well 3 and 6 respectively). In vivo tests showed that scFvBaP1 specifically recognizes BaP1 and whole B. asper venom, and to a large extend neutralizes the main toxic effects of BaP1.  Recombinant FabBaP1 and scFabBaP1 will be made from scFvBaP1. These are going to be expressed in Escherichia coli using a pUHE23-2 expression vector. The final protein model shown here is of the recombinant scFab.[6] pUHE23-2 E. coli If good chimeric antibodies are obtained in good yield, the next steps will be to determine their KD and test the antibodies in a preclinical model. This will allow an in vivo  comparison between the different formats. Snakebite is one of the world’s most neglected tropical diseases, with an estimated 5 million bites per year, resulting in about 125.000 deaths.[1] The only current treatment for snakebite envenoming is antiserum derived from the blood of immunized mammals (typically horses).[2] These antisera are expensive to produce and carry a high risk of causing hyper-allergic reactions in human recipients due to their heterologous origin.[3] Here we report the discovery of chimeric scFvs against Bothrops asper  toxins.  scFv scFab Fab kDa   50- 40-  30- 25- 15- MW  1     2      3     4    5     6 Results presented here were obtained prior to the project[5] Results presented here were obtained prior to the project[5] Results presented here were obtained prior to the project[4] E Substrate BaP1 Chimeric Fab Indirect ELISA Secondary antibody conjugate Bothrops  asper 

Design of Fab-based chimeric antibodies against <i>Bothrops asper</i> toxins

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Design of Fab-based chimeric antibodies against <i>Bothrops asper</i> toxins

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