Many human diseases are caused by mutant or abnormal protein functions that are largely confined to the inside of cells, rather than being displayed on the abnormal cell surface. Furthermore, many of the functional consequences of aberrant proteins, such as in cancer cells, are due to protein–protein interactions (PPIs). Developing reagents that can specifically interfere with PPI is an important goal for both therapeutic use and as reagents to interrogate the functional importance of PPI. Antibody fragments can be used for inhibiting PPI. Our recent technology development has provided a set of simple protocols that allow development of single antibody variable (V) region domains that can function inside the reducing environment of the cell. The heavy chain variable region (VH) segments mainly used in this technology are based on a designer framework that folds inside cells without the need for the intra-chain disulphide bond and can be used as drug surrogates to determine on-target effects (target validation) and as templates for small molecule drug development. In this review, we discuss our work on single domains as intracellular antibodies and where this work might in the future.
References
[1]
Mulcahy, L.S.; Smith, M.R.; Stacey, D.W. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature 1985, 313, 241–243, doi:10.1038/313241a0.
[2]
Lobato, M.N.; Rabbitts, T.H. Intracellular antibodies and challenges facing their use as therapeutic agents. Trends Mol. Med. 2003, 9, 390–396, doi:10.1016/S1471-4914(03)00163-1.
[3]
Perez-Martinez, D.; Tanaka, T.; Rabbitts, T.H. Intracellular antibodies and cancer: New technologies offer therapeutic opportunities. Bioessays 2010, 32, 589–598, doi:10.1002/bies.201000009.
[4]
Ward, E.S.; Gussow, D.; Griffiths, A.D.; Jones, P.T.; Winter, G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 1989, 341, 544–546, doi:10.1038/341544a0.
[5]
Tanaka, T.; Williams, R.L.; Rabbitts, T.H. Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS. EMBO J. 2007, 26, 3250–3259, doi:10.1038/sj.emboj.7601744.
[6]
Tanaka, T.; Sewell, H.; Waters, S.; Phillips, S.E.; Rabbitts, T.H. Single domain intracellular antibodies from diverse libraries: Emphasizing dual functions of LMO2 protein interactions using a single VH domain. J. Biol. Chem. 2011, 286, 3707–3716.
[7]
Colby, D.W.; Garg, P.; Holden, T.; Chao, G.; Webster, J.M.; Messer, A.; Ingram, V.M.; Wittrup, K.D. Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display. J. Mol. Biol. 2004, 342, 901–912, doi:10.1016/j.jmb.2004.07.054.
[8]
Rabbitts, T.H. Commonality but diversity in cancer gene fusions. Cell 2009, 137, 391–395, doi:10.1016/j.cell.2009.04.034.
[9]
Wells, J.A.; McClendon, C.L. Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature 2007, 450, 1001–1009, doi:10.1038/nature06526.
[10]
Forster, A.; Pannell, R.; Drynan, L.; Cano, F.; Chan, N.; Codrington, R.; Daser, A.; Lobato, N.; Metzler, M.; Nam, C.H.; et al. Chromosomal translocation engineering to recapitulate primary events of human cancer. Cold Spring Harb. Symp. Quant Biol. 2005, 70, 275–282, doi:10.1101/sqb.2005.70.008.
[11]
Marasco, W.A.; Haseltine, W.A.; Chen, S.Y. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc. Natl. Acad. Sci. USA 1993, 90, 7889–7893, doi:10.1073/pnas.90.16.7889.
[12]
Tse, E.; Lobato, M.N.; Forster, A.; Tanaka, T.; Chung, G.T.; Rabbitts, T.H. Intracellular antibody capture technology: Application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein. J. Mol. Biol. 2002, 317, 85–94, doi:10.1006/jmbi.2002.5403.
[13]
Visintin, M.; Settanni, G.; Maritan, A.; Graziosi, S.; Marks, J.D.; Cattaneo, A. The intracellular antibody capture technology (IACT): Towards a consensus sequence for intracellular antibodies. J. Mol. Biol. 2002, 317, 73–83, doi:10.1006/jmbi.2002.5392.
[14]
Visintin, M.; Tse, E.; Axelson, H.; Rabbitts, T.H.; Cattaneo, A. Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc. Natl. Acad. Sci. USA 1999, 96, 11723–11728, doi:10.1073/pnas.96.21.11723.
[15]
Vascotto, F.; Visintin, M.; Cattaneo, A.; Burrone, O.R. Design and selection of an intrabody library produced de-novo for the non-structural protein NSP5 of rotavirus. J. Immunol. Methods 2005, 301, 31–40, doi:10.1016/j.jim.2005.03.011.
[16]
Mukhtar, M.M.; Li, S.; Li, W.; Wan, T.; Mu, Y.; Wei, W.; Kang, L.; Rasool, S.T.; Xiao, Y.; Zhu, Y.; et al. Single-chain intracellular antibodies inhibit influenza virus replication by disrupting interaction of proteins involved in viral replication and transcription. Int. J. Biochem. Cell. Biol. 2009, 41, 554–560, doi:10.1016/j.biocel.2008.07.001.
[17]
Dixon, A.S.; Constance, J.E.; Tanaka, T.; Rabbitts, T.H.; Lim, C.S. Changing the subcellular location of the oncoprotein Bcr-Abl using rationally designed capture motifs. Pharm. Res. 2012, 29, 1098–1109, doi:10.1007/s11095-011-0654-8.
[18]
Tanaka, T.; Rabbitts, T.H. Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation. EMBO J. 2003, 22, 1025–1035, doi:10.1093/emboj/cdg106.
[19]
Tanaka, T.; Lobato, M.N.; Rabbitts, T.H. Single domain intracellular antibodies: A minimal fragment for direct in vivo selection of antigen-specific intrabodies. J. Mol. Biol. 2003, 331, 1109–1120, doi:10.1016/S0022-2836(03)00836-2.
[20]
Tanaka, T.; Rabbitts, T.H. Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds. J. Mol. Biol. 2008, 376, 749–757, doi:10.1016/j.jmb.2007.11.085.
[21]
Tanaka, T.; Rabbitts, T.H. Protocol for the selection of single-domain antibody fragments by third generation intracellular antibody capture. Nat. Protoc. 2010, 5, 67–92, doi:10.1038/nprot.2009.199.
[22]
Schwimmer, L.J.; Huang, B.; Giang, H.; Cotter, R.L.; Chemla-Vogel, D.S.; Dy, F.V.; Tam, E.M.; Zhang, F.; Toy, P.; Bohmann, D.J.; et al. Discovery of diverse and functional antibodies from large human repertoire antibody libraries. J. Immunol. Methods 2013, 391, 60–71, doi:10.1016/j.jim.2013.02.010.
[23]
Tanaka, T.; Rabbitts, T.H. Selection of functional single domain antibody fragments for interfering with protein-protein interactions inside cells: A "one plasmid" mammalian two-hybrid system. Methods Mol. Biol. 2012, 911, 175–182.
[24]
Downward, J. Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 2003, 3, 11–22, doi:10.1038/nrc969.
[25]
Downward, J. PI 3-kinase, Akt and cell survival. Semin. Cell Dev. Biol. 2004, 15, 177–182, doi:10.1016/j.semcdb.2004.01.002.
Tanaka, T.; Rabbitts, T.H. Interfering with RAS-effector protein interactions prevent RAS-dependent tumour initiation and causes stop-start control of cancer growth. Oncogene 2010, 29, 6064–6070, doi:10.1038/onc.2010.346.
[28]
Torchilin, V.P.; Lukyanov, A.N. Peptide and protein drug delivery to and into tumors: Challenges and solutions. Drug Discov. Today 2003, 8, 259–266, doi:10.1016/S1359-6446(03)02623-0.
[29]
Li, X.; Stuckert, P.; Bosch, I.; Marks, J.D.; Marasco, W.A. Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells. Cancer Gene Ther. 2001, 8, 555–565, doi:10.1038/sj.cgt.7700337.
[30]
Song, E.; Zhu, P.; Lee, S.K.; Chowdhury, D.; Kussman, S.; Dykxhoorn, D.M.; Feng, Y.; Palliser, D.; Weiner, D.B.; Shankar, P.; et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 2005, 23, 709–717.
[31]
Thompson, D.B.; Cronican, J.J.; Liu, D.R. Engineering and identifying supercharged proteins for macromolecule delivery into mammalian cells. Methods Enzymol. 2012, 503, 293–319, doi:10.1016/B978-0-12-396962-0.00012-4.
[32]
Cronican, J.J.; Thompson, D.B.; Beier, K.T.; McNaughton, B.R.; Cepko, C.L.; Liu, D.R. Potent delivery of functional proteins into Mammalian cells in vitro and in vivo using a supercharged protein. ACS Chem. Biol. 2010, 5, 747–752, doi:10.1021/cb1001153.
[33]
McNaughton, B.R.; Cronican, J.J.; Thompson, D.B.; Liu, D.R. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proc. Natl. Acad. Sci. USA 2009, 106, 6111–6116.
[34]
Torchilin, V.P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discov. 2005, 4, 145–160, doi:10.1038/nrd1632.
[35]
Thompson, D.B.; Villasenor, R.; Dorr, B.M.; Zerial, M.; Liu, D.R. Cellular uptake mechanisms and endosomal trafficking of supercharged proteins. Chem. Biol. 2012, 19, 831–843, doi:10.1016/j.chembiol.2012.06.014.
[36]
Kreuter, J. Nanoparticles—A Historical Perspective. Int. J. Pharm. 2007, 331, 1–10, doi:10.1016/j.ijpharm.2006.10.021.
[37]
Bangham, A.D.; Standish, M.M.; Watkins, J.C. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 1965, 13, 238–252, doi:10.1016/S0022-2836(65)80093-6.
[38]
Adler-Moore, J.; Proffitt, R.T. AmBisome: Liposomal formulation, structure, mechanism of action and pre-clinical experience. J. Antimicrob. Chemother. 2002, 49 (Suppl. 1), 21–30.
[39]
Slingerland, M.; Guchelaar, H.J.; Gelderblom, H. Liposomal drug formulations in cancer therapy: 15 years along the road. Drug Discov. Today 2012, 17, 160–166, doi:10.1016/j.drudis.2011.09.015.
[40]
Wang, A.Z.; Langer, R.; Farokhzad, O.C. Nanoparticle delivery of cancer drugs. Annu. Rev. Med. 2012, 63, 185–198, doi:10.1146/annurev-med-040210-162544.
[41]
Torchilin, V.; Klibanov, A. Immobilization of proteins on liposome surface. Enzym. Microb. Technol. 1981, 3, 297–304, doi:10.1016/0141-0229(81)90003-X.
Assi, S.A.; Tanaka, T.; Rabbitts, T.H.; Fernandez-Fuentes, N. PCRPi: Presaging Critical Residues in Protein interfaces, a new computational tool to chart hot spots in protein interfaces. Nucleic Acids Res. 2010, 38, e86, doi:10.1093/nar/gkp1158.