Bivalent single chain (sc)Fv-Fc antibodies have been used for years as recombinant alternatives of natural immunoglobulins. We have extended this approach to the scFv-Fc-scFv antibody format to obtain tetravalent antigen binding and the possibility to generate bispecific antibodies. We developed a mammalian expression vector system to construct tetravalent scFv-Fc-scFv antibodies with two NcoI+ NotI compatible cloning sites flanking the Fc gene fragment. We demonstrated direct cloning from single chain antibody gene libraries and tested various scFv combinations. Transient production of scFv-Fc-scFv antibodies in human embryonic kidney (HEK) 293T cells achieved volumetric yields of up to 10 mg/L. However, expression levels were strongly dependent on the carboxyterminal scFv and the scFv combination. All scFv-Fc-scFv antibodies exclusively formed disulfide-linked homodimers. Antigen binding studies revealed dual specificity for all scFv-Fc-scFv employing different scFv fragments. Comparison of C-reactive protein (CRP) specific monovalent scFv LA13-IIE3, bivalent scFv-Fc and Fc-scFv LA13-IIE3, and tetravalent scFv-Fc-scFv (scFv LA13-IIE3 in combination with scFvs LA13-IIE3, TOB4-B11, or TOB5-D4) revealed an up to 500-fold increased antigen binding. This novel scFv-Fc-scFv antibody expression system allows simple and fast testing of various scFv combinations.
References
[1]
Hust, M.; Meyer, T.; Voedisch, B.; Rülker, T.; Thie, H.; El-Ghezal, A.; Kirsch, M.I.; Schütte, M.; Helmsing, S.; Meier, D.; et al. A human scFv antibody generation pipeline for proteome research. J. Biotechnol. 2011, 152, 159–170, doi:10.1016/j.jbiotec.2010.09.945. 20883731
[2]
Thie, H.; Voedisch, B.; Dübel, S.; Hust, M.; Schirrmann, T. Affinity maturation by phage display. Methods Mol. Biol 2009, 525, 309–322, xv, doi:10.1007/978-1-59745-554-1_16.
[3]
Thie, H.; Toleikis, L.; Li, J.; von Wasielewski, R.; Bastert, G.; Schirrmann, T.; Esteves, I.T.; Behrens, C.K.; Fournes, B.; Fournier, N.; et al. Rise and fall of an anti-MUC1 specific antibody. PLoS One 2011, 6, e15921, doi:10.1371/journal.pone.0015921. 21264246
[4]
Kipriyanov, S.M.; Little, M.; Kropshofer, H.; Breitling, F.; Gotter, S.; Dübel, S. Affinity enhancement of a recombinant antibody: Formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion. Protein Eng. 1996, 9, 203–211, doi:10.1093/protein/9.2.203. 9005442
[5]
Thie, H.; Binius, S.; Schirrmann, T.; Hust, M.; Dübel, S. Multimerization domains for antibody phage display and antibody production. N. Biotechnol. 2009, 26, 314–321, doi:10.1016/j.nbt.2009.07.005.
[6]
Schirrmann, T.; Büssow, K. Transient production of scFv-Fc fusion proteins in mammalian cells. In Antibody Engineering; Kontermann, R., Dübel, S., Eds.; Springer Berlin Heidelberg: Berlin, Germany, 2010; pp. 387–398.
[7]
Schirrmann, T.; Menzel, C.; Hust, M.; Prilop, J.; Jostock, T.; Dübel, S. Oligomeric forms of single chain immunoglobulin (scIgG). MAbs 2010, 2, 73–76, doi:10.4161/mabs.2.1.10784.
[8]
McCall, A.M.; Adams, G.P.; Amoroso, A.R.; Nielsen, U.B.; Zhang, L.; Horak, E.; Simmons, H.; Schier, R.; Marks, J.D.; Weiner, L.M. Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis. Mol. Immunol. 1999, 36, 433–445, doi:10.1016/S0161-5890(99)00057-7.
[9]
Hammond, S.A.; Lutterbuese, R.; Roff, S.; Lutterbuese, P.; Schlereth, B.; Bruckheimer, E.; Kinch, M.S.; Coats, S.; Baeuerle, P.A.; Kufer, P.; et al. Selective targeting and potent control of tumor growth using an EphA2/CD3-bispecific single-chain antibody construct. Cancer Res. 2007, 67, 3927–3935, doi:10.1158/0008-5472.CAN-06-2760. 17440108
[10]
M？lh？j, M.; Crommer, S.; Brischwein, K.; Rau, D.; Sriskandarajah, M.; Hoffmann, P.; Kufer, P.; Hofmeister, R.; Baeuerle, P.A. CD19-/CD3-bispecific antibody of the BiTE class is far superior to tandem diabody with respect to redirected tumor cell lysis. Mol. Immunol. 2007, 44, 1935–1943, doi:10.1016/j.molimm.2006.09.032. 17083975
[11]
Schanzer, J.; Jekle, A.; Nezu, J.; Lochner, A.; Croasdale, R.; Dioszegi, M.; Zhang, J.; Hoffmann, E.; Dormeyer, W.; Stracke, J.; et al. Development of tetravalent, bispecific CCR5 antibodies with antiviral activity against CCR5 monoclonal antibody-resistant HIV-1 strains. Antimicrob. Agents Chemother. 2011, 55, 2369–2378, doi:10.1128/AAC.00215-10.
[12]
Mabry, R.; Gilbertson, D.G.; Frank, A.; Vu, T.; Ardourel, D.; Ostrander, C.; Stevens, B.; Julien, S.; Franke, S.; Meengs, B.; et al. A dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo. MAbs 2010, 2, 20–34, doi:10.4161/mabs.2.1.10498.
[13]
Cao, Y.; Lam, L. Bispecific antibody conjugates in therapeutics. Adv. Drug Deliv. Rev. 2003, 55, 171–197, doi:10.1016/S0169-409X(02)00178-3.
[14]
Müller, D.; Kontermann, R.E. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010, 24, 89–98, doi:10.2165/11530960-000000000-00000.
Bargou, R.; Leo, E.; Zugmaier, G.; Klinger, M.; Goebeler, M.; Knop, S.; Noppeney, R.; Viardot, A.; Hess, G.; Schuler, M.; et al. Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science 2008, 321, 974–977, doi:10.1126/science.1158545. 18703743
[17]
Schaefer, W.; Regula, J.T.; B？hner, M.; Schanzer, J.; Croasdale, R.; Dürr, H.; Gassner, C.; Georges, G.; Kettenberger, H.; Imhof-Jung, S.; et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc. Natl. Acad. Sci. USA 2011, 108, 11187–11192, doi:10.1073/pnas.1019002108. 21690412
[18]
Muda, M.; Gross, A.W.; Dawson, J.P.; He, C.; Kurosawa, E.; Schweickhardt, R.; Dugas, M.; Soloviev, M.; Bernhardt, A.; Fischer, D.; et al. Therapeutic assessment of SEED: A new engineered antibody platform designed to generate mono- and bispecific antibodies. Protein Eng. Des. Sel. 2011, 24, 447–454, doi:10.1093/protein/gzq123.
[19]
Asano, R.; Watanabe, Y.; Kawaguchi, H.; Fukazawa, H.; Nakanishi, T.; Umetsu, M.; Hayashi, H.; Katayose, Y.; Unno, M.; Kudo, T.; et al. Highly effective recombinant format of a humanized IgG-like bispecific antibody for cancer immunotherapy with retargeting of lymphocytes to tumor cells. J. Biol. Chem. 2007, 282, 27659–27665, doi:10.1074/jbc.M704719200. 17644522
[20]
Wu, C.; Ying, H.; Grinnell, C.; Bryant, S.; Miller, R.; Clabbers, A.; Bose, S.; McCarthy, D.; Zhu, R.-R.; Santora, L.; et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat. Biotechnol. 2007, 25, 1290–1297, doi:10.1038/nbt1345.
Dong, J.; Sereno, A.; Aivazian, D.; Langley, E.; Miller, B.R.; Snyder, W.B.; Chan, E.; Cantele, M.; Morena, R.; Joseph, I.B.J.K.; et al. A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity. MAbs 2011, 3, 273–288, doi:10.4161/mabs.3.3.15188.
[23]
Alt, M.; Müller, R.; Kontermann, R.E. Novel tetravalent and bispecific IgG-like antibody molecules combining single-chain diabodies with the immunoglobulin gamma1 Fc or CH3 region. FEBS Lett. 1999, 454, 90–94, doi:10.1016/S0014-5793(99)00782-6.
[24]
Connelly, R.J.; Hayden, M.S.; Scholler, J.K.; Tsu, T.T.; Dupont, B.; Ledbetter, J.A.; Kanner, S.B. Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes. Int. Immunol. 1998, 10, 1863–1872, doi:10.1093/intimm/10.12.1863.
[25]
Mabry, R.; Lewis, K.E.; Moore, M.; McKernan, P.A.; Bukowski, T.R.; Bontadelli, K.; Brender, T.; Okada, S.; Lum, K.; West, J.; et al. Engineering of stable bispecific antibodies targeting IL-17A and IL-23. Protein Eng. Des. Sel. 2010, 23, 115–127, doi:10.1093/protein/gzp073.
[26]
Jendreyko, N.; Popkov, M.; Beerli, R.R.; Chung, J.; McGavern, D.B.; Rader, C.; Barbas, C.F., 3rd. Intradiabodies, bispecific, tetravalent antibodies for the simultaneous functional knockout of two cell surface receptors. J. Biol. Chem. 2003, 278, 47812–47819, doi:10.1074/jbc.M307002200. 12947084
[27]
Schirrmann, T.; Pecher, G. Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo. Cancer Gene Ther. 2002, 9, 390–398, doi:10.1038/sj.cgt.7700453.
[28]
Schirrmann, T.; Pecher, G. Specific targeting of CD33(+) leukemia cells by a natural killer cell line modified with a chimeric receptor. Leuk. Res. 2005, 29, 301–306, doi:10.1016/j.leukres.2004.07.005.
[29]
Jostock, T.; Vanhove, M.; Brepoels, E.; Van Gool, R.; Daukandt, M.; Wehnert, A.; Van Hegelsom, R.; Dransfield, D.; Sexton, D.; Devlin, M.; et al. Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries. J. Immunol. Methods 2004, 289, 65–80, doi:10.1016/j.jim.2004.03.014.
[30]
Li, J.; Menzel, C.; Meier, D.; Zhang, C.; Dübel, S.; Jostock, T. A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J. Immunol. Methods 2007, 318, 113–124, doi:10.1016/j.jim.2006.10.010.
[31]
Goldsmith, M.E.; Konigsberg, W.H. Adsorption protein of the bacteriophage fd: Isolation, molecular properties, and location in the virus. Biochemistry 1977, 16, 2686–2694, doi:10.1021/bi00631a016.
[32]
Al-Halabi, L.; Balck, A.; Michalzik, M.; Fr？de, D.; Büttgenbach, S.; Hust, M.; Schirrmann, T.; Dübel, S. Recombinant antibody fragments allow repeated measurements of C-reactive protein with a quartz crystal microbalance immunosensor. J. Biotechnol. 2012. submitted.
[33]
Marks, J.D.; Griffiths, A.D.; Malmqvist, M.; Clackson, T.P.; Bye, J.M.; Winter, G. By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (N.Y.) 1992, 10, 779–783, doi:10.1038/nbt0792-779.
[34]
Hust, M.; Toleikis, L.; Dübel, S. Antibody phage display. Handbook of therapeutic antibodies. In Handbook of Therapeutic Antibodies; Wiley-VCH: Weinheim, Germany, 2007; Volume 1, pp. 45–68.
[35]
Hust, M.; Dübel, S.; Schirrmann, T. Selection of recombinant antibodies from antibody gene libraries. Methods Mol. Biol 2007, 408, 243–255, doi:10.1007/978-1-59745-547-3_14.
[36]
Hust, M.; Steinwand, M.; Al-Halabi, L.; Helmsing, S.; Schirrmann, T.; Dübel, S. Improved microtitre plate production of single chain Fv fragments in Escherichia coli. N. Biotechnol. 2009, 25, 424–428, doi:10.1016/j.nbt.2009.03.004.
[37]
Schmiedl, A.; Breitling, F.; Winter, C.H.; Queitsch, I.; Dübel, S. Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli.. J. Immunol. Methods 2000, 242, 101–114, doi:10.1016/S0022-1759(00)00243-X. 10986393
[38]
Jordan, E.; Hust, M.; Roth, A.; Biedendieck, R.; Schirrmann, T.; Jahn, D.; Dübel, S. Production of recombinant antibody fragments in Bacillus megaterium. Microb. Cell Fact. 2007, 6, 2, doi:10.1186/1475-2859-6-2.
[39]
Rülker, T.; Meier, D.; Schirrmann, T. Quantification of human IgG and related Fc fusion proteins by a human IgG/Fc capture ELISA. In Antibody Engineering; Kontermann, R., Dübel, S., Eds.; Springer Berlin Heidelberg: Berlin, Germany, 2010; pp. 743–748.
[40]
Sambrook, J.; Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 2001.
[41]
Blum, H.; Beier, H.; Gross, H.J. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 1987, 8, 93–99, doi:10.1002/elps.1150080203.
