Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks. SdAb are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. Starting with our previously isolated ricin binding sdAb determined to bind to four non-overlapping epitopes, we constructed a series of sdAb pairs, which were genetically linked through peptides of different length. We designed the series so that the sdAb are linked in both orientations with respect to the joining peptide. We confirmed that each of the sdAb in the constructs was able to bind to the ricin target, and have evidence that they are both binding ricin simultaneously. Through this work we determined that the order of genetically linked sdAb seems more important than the linker length. The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb.
References
[1]
Ghahroudi, M.A.; Desmyter, A.; Wyns, L.; Hamers, R.; Muyldermans, S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 1997, 414, 521–526.
[2]
Greenberg, A.S.; Avila, D.; Hughes, M.; Hughes, A.; McKinney, E.C.; Flajnik, M.F. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 1995, 374, 168–173.
Nuttall, S.D.; Krishnan, U.V.; Hattarki, M.; de Gori, R.; Irving, R.A.; Hudson, P.J. Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries. Mol. Immunol. 2001, 38, 313–326, doi:10.1016/S0161-5890(01)00057-8.
[5]
Wesolowski, J.; Alzogaray, V.; Reyelt, J.; Unger, M.; Juarez, K.; Urrutia, M.; Cauerhff, A.; Danquah, W.; Rissiek, B.; Scheuplein, F.; Schwarz, N.; Adriouch, S.; Boyer, O.; Seman, M.; Licea, A.; Serreze, D.V.; Goldbaum, F.A.; Haag, F.; Koch-Nolte, F. Single domain antibodies: Promising experimental and therapeutic tools in infection and immunity. Med. Microbiol. Immunol. 2009, 198, 157–174.
[6]
De Marco, A. Biotechnological applications of recombinant single-domain antibody fragments. Microb. Cell Factories 2011.
[7]
Van der Linden, R.H.J.; Frenken, L.G.J.; de Geus, B.; Harmsen, M.M.; Ruuls, R.C.; Stok, W.; de Ron, L.; Wilson, S.; Davis, P.; Verrips, C.T. Comparison of physical chemical properties of llama V-HH antibody fragments and mouse monoclonal antibodies. Biochim. Biophys. Acta 1999, 1431, 37–46.
[8]
Perez, J.M.J.; Renisio, J.G.; Prompers, J.J.; van Platerink, C.J.; Cambillau, C.; Darbon, H.; Frenken, L.G.J. Thermal unfolding of a llama antibody fragment: A two-state reversible process. Biochemistry 2001, 40, 74–83.
[9]
Ewert, S.; Cambillau, C.; Conrath, K.; Pluckthun, A. Biophysical properties of camelid V-HH domains compared to those of human VH3 domains. Biochemistry 2002, 41, 3628–3636.
[10]
Pluckthun, A.; Pack, P. New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology 1997, 3, 83–105.
[11]
Crothers, D.M.; Metzger, H. Influence of polyvalency on binding properties of antibodies. Immunochemistry 1972, 9, 341–357, doi:10.1016/0019-2791(72)90097-3.
[12]
Zhou, H.X. Quantitative account of the enhanced affinity of two linked scFvs specific for different epitopes on the same antigen. J. Mol. Biol. 2003, 329, 1–8.
[13]
Neri, D.; Momo, M.; Prospero, T.; Winter, G. High affinity antigen-ginding by chelating-recombinant-antibodies (CRABS). J. Mol. Biol. 1995, 246, 367–373.
[14]
Korn, T.; Nettelbeck, D.M.; Volkel, T.; Muller, R.; Kontermann, R.E. Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: A comparative analysis of bacterially expressed single-chain diabody and tandem scFv. J. Gene Med. 2004, 6, 642–651, doi:10.1002/jgm.555.
Conrath, K.E.; Lauwereys, M.; Wyns, L.; Muyldermans, S. Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J. Biol. Chem. 2001, 276, 7346–7350.
[17]
Coppieters, K.; Dreier, T.; Silence, K.; de Haard, H.; Lauwereys, M.; Casteels, P.; Beirnaert, E.; Jonckheere, H.; de Wiele, C.V.; Staelens, L.; Hostens, J.; Revets, H.; Remaut, E.; Elewaut, D.; Rottiers, P. Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. Arthritis Rheum. 2006, 54, 1856–1866.
[18]
Hmila, I.; Abdallah, B.A.B.; Saerens, D.; Benlasfar, Z.; Conrath, K.; El Ayeb, M.; Muyldermans, S.; Bouhaouala-Zahar, B. VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI’. Mol. Immunol. 2008, 45, 3847–3856.
Hultberg, A.; Temperton, N.J.; Rosseels, V.; Koenders, M.; Gonzalez-Pajuelo, M.; Schepens, B.; Ibanez, L. I.; Vanlandschoot, P.; Schillemans, J.; Saunders, M.; Weiss, R.A.; Saelens, X.; Melero, J.A.; Verrips, C.T.; Van Gucht, S.; de Haard, H.J. Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules. PLoS One 2011.
[21]
Anderson, G.P.; Liu, J.L.; Hale, M.L.; Bernstein, R.D.; Moore, M.; Swain, M.D.; Goldman, E.R. Development of antiricin single domain antibodies toward detection and therapeutic reagents. Anal. Chem. 2008, 80, 9604–9611.
[22]
Anderson, G.P.; Bernstein, R.D.; Swain, M.D.; Zabetakis, D.; Goldman, E.R. Binding kinetics of antiricin single domain antibodies and improved detection using a B chain specific binder. Anal. Chem. 2010, 82, 7202–7207.
[23]
Goldman, E.R.; Anderson, G.P.; Liu, J.L.; Delehanty, J.B.; Sherwood, L.J.; Osborn, L.E.; Cummins, L.B.; Hayhurst, A. Facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisynthetic llama library. Anal. Chem. 2006, 78, 8245–8255.
[24]
Conway, J.O.; Sherwood, L.J.; Collazo, M.T.; Garza, J.A.; Hayhurst, A. Llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagents. PLoS One 2010.
[25]
Anderson, G.P.; Zabetakis, D.; Bernstein, R.D.; Cai, S.W.; Singh, B.R.; Goldman, E.R. Evaluation of anti-hemagglutinin Hn-33 single domain antibodies: Kinetics, binding epitopes, and thermal stability. Botulinum J. 2011, 2, 59–71, doi:10.1504/TBJ.2011.041816.
[26]
Roberts, L.M.; Lamb, F.I.; Pappin, D.J.C.; Lord, J.M. The primary sequence of ricin-communis agglutinin—Comparison with ricin. J. Biol. Chem. 1985, 260, 5682–5686.
[27]
Kimura, M.; Sumizawa, T.; Funatsu, G. The complete amino-acid sequences of the B-chains of abrin-A and abrin-G, toxic proteins from the seeds of Abrus-precatorius. Biosci. Biotechnol. Biochem. 1993, 57, 166–169, doi:10.1271/bbb.57.166.
[28]
Wood, K.A.; Lord, J.M.; Wawrzynczak, E.J.; Piatak, M. Preproabrin—Genomic cloning, characterization and the expressin of the A-chain in Escherichia-coli. Eur. J. Biochem. 1991, 198, 723–732, doi:10.1111/j.1432-1033.1991.tb16072.x.
[29]
Robertus, J.D.; Monzingo, A.F. The structure of ribosome inactivating proteins. Mini Rev. Med. Chem. 2004, 4, 477–486.
[30]
Zhang, J.B.; Li, Q.G.; Nguyen, T.D.; Tremblay, T.L.; Stone, E.; To, R.; Kelly, J.; MacKenzie, C.R. A pentavalent single-domain antibody approach to tumor antigen discovery and the development of novel proteomics reagents. J. Mol. Biol. 2004, 341, 161–169.
[31]
Sherwood, L.J.; Osborn, L.E.; Carrion, R., Jr.; Patterson, J.L.; Hayhurst, A. Rapid assembly of sensitive antigen-capture assays for Marburg virus, using in vitro selection of llama single-domain antibodies, at biosafety level 4. J. Infect. Dis. 2007, 196, S213–S219, doi:10.1086/520586.
