Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 3.19

RATIONAL DESIGN AND PRODUCTION OF INTRACELLULAR ANTIBODIES WITH DESIRED SPECIFICITY

M. DONINI*, V. MOREA**, A. DESIDERIO*, D. PASHKOULOV*, M. E. VILLANI*, A. TRAMONTANO***, E. BENVENUTO*

*) ENEA, UTS Biotecnologie, Sezione Genetica e Genomica Vegetale, C.R. Casaccia,-00100 Roma, Italy

**) CNR Center of Studies of Molecular Biology, Piazzale Aldo Moro 5, 00185 Rome, Italy

***) Department of Biochemical Sciences 'A. Rossi-Fanelli', University 'La Sapienza', Piazzale Aldo Moro 5, 00185 Rome, Italy

ScFv, intrabodies, CDR grafting, immunotherapy

The ability of antibodies to recognise a virtually infinite number of molecules with high affinity and specificity makes them invaluable tools for both experimental and therapeutic applications. In particular recombinant mammalian antibodies and their derivatives, have been expressed in plants to provide the so called “plantibody-based resistance” (Schillberg et al., 2001, Transgenic Res. 10, 1-12) against a number of different pathogens, including viruses, bacteria, fungi and invertebrates.

The first antibody-mediated virus protection in plants was reported by Tavladoraki et al. (Tavladoraki et al., 1993, Nature 366, 469-472). A single chain Fv (scFv(F8)) fragment (specific for artichoke mottled crinkle virus, AMCV) was constructed from a monoclonal antibody and expressed in bacteria showing to retain antigen-binding specificity. The recombinant antibody was then expressed in the plant cytoplasm and both transgenic plants and protoplasts proved to be protected from virus infection.

We wanted to investigate whether reshaped antibodies with new specificity can be rationally designed that retain cytoplasmic solubility and functionality of the stable scFv(F8) molecule.

We present here the design and production of a 'reshaped' antibody obtained by grafting the antigen binding site of the anti-lysozyme D1.3 antibody into the framework regions of the scFv(F8) fragment. The original scFv(F8) neutralises its cognate antigen, the plant virus AMCV, in the plant cytoplasm and it binds the AMCV coat protein in the cytoplasm of bacterial and eukaryotic cells (Tavladoraki et al., 1999, Eur. J. Biochem., 262, 617-624.). These are very peculiar features, in that the large majority of antibodies and antibody fragments are neither stable or functional in the highly reducing cytoplasmic environment. At the same time, these features are very desirable, in view of the very large number of potential applications of intracellular antibodies in research studies and human therapy (Desiderio, A., et al., 2001, J. Mol. Biol. 310, 603-615).

The reshaped scFv(F8)-D1.3 molecule that we designed was found to be able to bind lysozyme with affinity comparable to that of the antigen binding site donor D1.3 both in the oxidized form, obtained by periplasmic extraction, and in the reduced state, obtained by cytoplasmic extraction under reducing conditions. This demonstrates that the framework regions of the scFv(F8) are able by themselves to produce the cytoplasmic stability of the original scFv(F8). These regions can therefore be used as a scaffold to engineer antibody fragments that are stable and functional in the cytoplasm and endowed, in principle, with specificity against any particular plant pathogen.