Abstract
The intracellular expression of recombinant single-chain Fv (scFv) molecules—termed intrabodies—has potential advantages for therapeutic use (1,2). These single polypeptide chains are made of one heavy-chain variable region (VH) linked through a flexible spacer, usually a repeated motif of 3× GGGGS (although some others have also been described) (3), to one light chain variable region (VL). The VH-linker-VL sequence can be inverted with no loss of the binding. Shortened linkers have been used to produce scFvs of divalent or bispecific forms (3,4).
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References
Marasco W.A. (1997) Intrabodies: turning the humoral immune system outside in for intracellular immunization. Gene Ther. 4, 11–15.
Deshane J., Grim J., Loechel S., Siegal G.P., Alvarez R.D. and Curiel D.T. (1996) Intracellular antibody against erbB-2 mediates targeted tumor cell eradication by apoptosis. Cancer Gene Ther. 3, 89–98.
Huston J.S., Mudgett-Hunter M., Tai M.S., McCartney J., Warren F., Haber E., et al. (1991) Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol. 203, 46–88.
Kontermann R.E., and Muller R. (1999) Intracellular and cell surface displayed single-chain diabodies. J. Immunol. Methods 226, 179–88.
Richardson J.H., and Marasco W.A. (1995) Intracellular antibodies: development and therapeutic potential. Trends Biotechnol. 13, 306–310.
Cochet O., Kenigsberg M., Delumeau I., Virone-Oddos A., Multon M.-C., Fridman W.H., et al. (1998) Intracellular expression of an antibody fragment neutralizing p21ras promotes tumor regression. Cancer Res. 58, 1170–1176.
Caron de Fromentel C., Gruel N., Venot C., Debuscche L., Conseiller E., Dureuil C., et al. (1999) Restoration of transcriptional activity of p53 mutants in human tumor cells by intracellular expression of anti-p53 single-chain Fv fragments. Oncogene 18, 551–557.
Chester K.A., Begent R.H., Robson L., Keep P., Pedley R.B., Boden J.A., et al. (1994) Phage libraries for generation of clinically useful antibodies. Lancet 343, 455–456.
Griffiths A.D., Williams S.C., Hartley O., Tomlinson I.M., Waterhouse P., Crosby W.L., et al. (1994) Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13, 3245–3260.
Vaughan T.J., Williams A.J., Pritchard K., Osbourn J.K., Pope A.R., Earnshaw J.C., et al. (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nature Biotech. 14, 309–314.
Marasco W.A., Haseltine W.A., and Chen S. (1993) Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc. Natl. Acad. Sci. USA 90, 7889–7893.
Hancock J.F., Paterson H. and Marshall J. (1990) A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell 63, 133–139.
Cochet O., Delumeau I., Kenigsberg M., Gruel N., Schweighoffer F., Bracco L., et al. (1998) Intracellular targeting of oncogenes: a novel approach for cancer therapy. in: Intrabodies, Basic Research and Clinical Gene Therapy Applications Marasco W.A. ed., R. G. Landes Company, Springer-Verlag, New York, 129–146.
Lei S.P., Lin H.C., Wang S.S., Callaway J. and Wilcox G. (1987) Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. J. Bacteriol. 169, 4379–4383.
Evan G.I., Lewis G.K., Ramsay G., and Bishop J.M. (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5, 3610–3616.
Kabat E.A., Wu T.T., Perry H.M., Gottesman K.S. and Foeller C. (1991) Sequences of proteins of immunological interest, 5th ed. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD.
Orlandi R., Güssow D.H., Jones P.T., and Winter G. (1989) Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 3833–3837.
Ward E.S., Güssow D., Griffiths A.D., Jones P.T., and Winter G. (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 314, 544–546.
Cochet O., Kenigsberg M., Delumeau I., Duchesne M., Schweighoffer F., Tocqué B., et al. (1998) Intracellular expression and functional properties of an anti-p21ras scFv derived from a rat hybridoma containing specific γ and irrelevant κ light chains. Mol. Immunol. 35, 1097–1110.
Cuisinier A.M., Fumoux F., Moinier D., Boubli L., Guigou V., Milili M., et al. (1990) Rapid expansion of human immunoglobulin repertoire (VH, Vκ, V) expressed in early fetal bone marrow. New Biol. 2, 689–699.
Jones S.T., and Bending M.M. (1991) Rapid PCR-cloning of full-length mouse immunoglobulin variable regions. BioTechnol. 9, 579.
Cochet O., Martin E., Fridman W.H., and Teillaud J-L. (1999) Selective PCR amplification of functional immunoglobulin light chain from hybridoma containing the aberrant MOPC 21-derived Vκ by PNA-mediated PCR clamping. BioTechniques 26, 818–822.
Egholm M., Buchardt O., Christensen L., Behrens C., Freier S.M., Driver D.A., et al. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566–568.
Orum H., Nielsen P.E., Egholm M., Berg R.H., Buchardt O., and Stanley C. (1993) Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res. 21, 5332–5336.
Pelegrin M., Marin M., Noel D., and Piechaczyk M. (1998) Genetically engineered antibodies in gene transfer and gene therapy. Human Gene Ther. 9, 2165–2175.
Zhu Q., Zeng C., Huhalov A., Yao J., Turi T.G., Danley D., et al. (1999) Extended half-life and elevated steady-state level of a single-chain Fv intrabody are critical for specific intracellular retargeting of its antigen, caspase-7. J. Immunol. Methods 231, 207–222.
Ramm K., Gehrig P., and Pluckthun A. (1999) Removal of the conserved disulfide bridges from the scFv fragment of an antibody: effects on folding kinetics and aggregation. J. Mol. Biol. 290, 535–46.
Visintin M., Tse E., Axelson H., Rabbitts T.H., and Cattaneo A. (1999) Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc. Natl Acad. Sci. USA 96, 11,723–11,728.
Rizzuto R., Simpson A.W.M., Brini M., and Pozzan T. (1992) Rapid changes of mitochondrial Ca++ revealed by specifically targeted recombinant æquorin. Nature 358, 325–327.
Harrison J.L., Williams S.C., Winter G., and Nissim A. (1996) Screening of phage antibody libraries. Methods Enzymol. 267, 83–109.
Dower W.J., Miller J. F., and Ragsdale C. W. (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16, 6127–6145.
Kipriyanov S. M., Moldenhauer G., and Little M. (1997) High level production of soluble single-chain antibodies in small-scale Escherichia coli cultures. J. Immunol. Methods 200, 69–77.
Bradbury A., Ruberti F., Werge T., Amati V., Luzio A.D., Gonfloni S., et al. (1995) The cloning of hybridoma V regions for their ectopic expression in intracellular and intercellular immunization. in Antibody Engineering. Borrebaeck C.A., ed., Oxford University Press.
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Nam, C.H., Moutel, S., Teillaud, JL. (2002). Generation of Murine scFv Intrabodies from B-Cell Hybridomas. In: O’Connell, J. (eds) RT-PCR Protocols. Methods in Molecular Biology, vol 193. Humana Press. https://doi.org/10.1385/1-59259-283-X:301
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DOI: https://doi.org/10.1385/1-59259-283-X:301
Publisher Name: Humana Press
Print ISBN: 978-0-89603-875-2
Online ISBN: 978-1-59259-283-8
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