Produzione in pianta del recettore rErbB2 e valutazione delle sue proprietà immunogeniche

The rat ErbB2 (Her2/neu) gene encodes a 185-kDa glycoprotein belonging to the epidermal growth factor related proteins (ErbB) of receptor tyrosine kinases. Through its heterodimerization with other ErbB receptors, ErbB2 can mediate signal transduction from all ErbB receptors and thus be involved in a variety of cell functions, including cell proliferation, differentiation and apoptosis. ErbB2 also plays a role in human malignancies. It is amplified and/or overexpressed in approximately 25% of human breast carcinomas and in many other malignancies, such as breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 has immunogenic properties and represents an ideal oncoantigen and a candidate for cancer immunotherapy. We investigated the possibility to express the extracellular (EC) domain of rat ErbB2 (653 amino acids, aa) in plants, using different types of synthetic genes to evaluated the influence of the 23 aa trans-membrane (TM) domain. Synthetic variants of the rErbB2-EC gene with human codon optimization and linked to the full TM domain (rErbB2-TM, 676 aa), to a portion of it (rErbB2-pTM, 662 aa), or alone (rErbB2-noTM, 653 aa) were cloned fused to the 5'-terminal His tag of the pEAQ-HT expression vector. pEAQ is a small non-replicating binary vector, provided with 5'- and 3'- regulatory untranslated regions from the Cowpea mosaic virus RNA-2 and including a potent P19 silencing suppressor (Sainsbury et al., 2009). Following construct delivery to Nicotiana benthamiana plants by agroinfiltration (a technique that permits a transient expression of a gene), expression was evaluated using anti-ErbB2 monoclonal antibody. The rErbB2 (about 73 kDa) could be detected by Western blots only when TM was absent or partially deleted. When soluble extracts from plant leaves expressing ErbB2-noTM were used to immunize BALB/c mice, specific antibodies against ErbB2 expressed from a 3T3/NKB cell line, as measured by flow cytometry. Moreover, these extracts displayed a potent anti-tumor activity when vaccinated mice were challenged with TUBO carcinoma cells expressing H-2Kd and ErbB2 molecules. The immunisation with rErbB2-noTM significantly prolonged tumour-free survival in injected mice. This presents the first report of expression of ErbB2 proteins in plants and of their efficacy in inducing specific anti-ErbB2 antibodies and in protecting from tumor development, opening interesting perspectives for further immunological testing.