The rat ErbB2 (rErbB2) protein is a 185-kDa glycoprotein belonging to the epidermal growth factor-related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast， lung， pancreatic， bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids， aa) in Nicotiana benthamiana plants， testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain， optimized with a human codon usage and eith... More
The rat ErbB2 (rErbB2) protein is a 185-kDa glycoprotein belonging to the epidermal growth factor-related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast， lung， pancreatic， bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids， aa) in Nicotiana benthamiana plants， testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain， optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM， 676 aa)， to a portion of it (rErbB2-pTM， 662 aa)， or deprived of it (rErbB2_noTM， 653 aa) were cloned in the pEAQ-HT expression vector as 6X His tag fusions. All rErbB2 variants (72-74.5 kDa) were transiently expressed， but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice， specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2(+) mammary carcinoma cells. To our knowledge， this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response， opening interesting perspectives for further immunological testing.
