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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 2.18

PLANT AND BACTERIAL PRODUCTION OF A SCFV-aHER2 FOR PHARMACOLOGICAL USE IN ONCOLOGY

LOMBARDI A.*, PIETRAFORTE I.*, NOVELLI F.*, SPERANDEI M.*, FRAIOLI R.**, GIACOMINI P.**, CANTALE C.*, GALEFFI P.*

*) ENEA CR Casaccia - UTS BIOTEC, Via Anguillarese 301, 00060 Rome, Italy

galeffi@casaccia.enea.it

**) IFO-IRE Immunology lab., Via delle Messi d’oro 156, 00158 Rome, Italy

engineered antibodies, Herb2, plant factory, single chain fragment

The aim of this proiect was to use plants as a biomachine to produce a large amount of antibodies (phytoantibody) correctly synthesized and folded, that are able to bind their antigenes. We applied this approach to obtain an engineered antibody specific for the Herb 2 oncogene strongly related to the breast and ovary cancers. Single chain antibody (scFv) seems to be particularly adapt for their sizes and immunogenetic characteristics to be used in diagnostic kits and in several pharmacological applications.

Two monoclonal antibodies (800E6 and 100A4 clones) were used to derive engineered antibodies (scFv-aHER2) by PCR amplification system. Specific oligos and primers and addressed strategies of cloning and subcloning were carried out. All molecular biology steps were successfully accomplished and expression, recovering, stability and affinity experiments were carried out on scFvs-aHER2 protein. Analyses of nucleotide and aminoacid sequences were also done in order to evaluate theoretical stability by a structural approach.

ScFvs extracted and purified from E.coli expression system were tested for functionality by FACS on several cell lines (SKBR, Mat10 and others) showing the antigen Her2. Several experiments were carried out and positivive results were obtained. . ScFVs partially purified both from bacteria have shown positive results in immunohistochemical (IHC) recognition with human breast cancer frozen tissues.

The entire constructs scFvs were introduced into plant vector and in vitro transgenic plants of Nicotiana tabacum were obtained. All transgenic plants were analysed by RT-PCR and the positives transgenic plants were grown for mass production of scFvs as protein. Experiments are in progress to test the behaviour of scFvs produced in plants both in FACS and in IHC. There are initial indications that they are highly specific and represent a good chances for application and use in diagnosis and therapy. Particular attention has paid to set up the extraction/purification protocols for large scale recovery of the engineered antibody obtained from plants growth in greenhouse.