Yolande Perrin

Practical considerations for pharmaceutical antibody production in different crop systems

The potential of plant cells to produce functional recombinantantibodies has been demonstrated in a number of different plant systems. Wepresent a comparative study of a well-defined target protein, a single chain Fvantibody, in different transgenic crop species and cultured tissues. The effectof different regulatory elements and signals for subcellular targeting areconsidered. Practical considerations for the choice of a particular cropsystem,such as yield, storage, distribution and containment properties are discussed.

Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies.

We investigated the suitability of transformed rice cell lines as a system for the production of therapeutic recombinant antibodies. Expression constructs encoding a single-chain Fv fragment (scFvT84.66, specific for CEA, the carcinoembryonic antigen present on many human tumours) were introduced into rice tissue by particle bombardment. We compared antibody production levels when antibodies were either secreted to the apoplast or retained in the endoplasmic reticulum (ER) using a KDEL retention signal. Production levels were up to 14 times higher when antibodies were retained in the ER. Additionally, we compared constructs encoding different leader peptides (plant codon optimised murine immunoglobulin heavy and light chain leader peptides) and carrying alternative 5′ untranslated regions (the petunia chalcone synthase gene 5′ UTR and the tobacco mosaic virus omega sequence). We observed no significant differences in antibody production levels among cell lines transformed with these constructs. The highest level of antibody production we measured was 3.8 μg g−1 callus (fresh weight). Immunological analysis of transgenic rice callus confirmed the presence of functional scFvT84.66. We discuss the potential merits of cell culture for the production of recombinant antibodies and other valuable macromolecules.

An antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts

Summary To investigate the potential of antibody derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed a small immune protein (SIP) in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The ɛSIP molecule consisted of a single‐chain antibody (scFv) specific for the porcine coronavirus transmissible gastroenteritis virus (TGEV) linked to the ɛ‐CH4 domain from human immunoglobulin E (IgE). In some constructs, the sequence encoding the ɛSIP molecule was flanked by the leader peptide from the original murine antibody at its N‐terminus and an endoplasmic reticulum retention signal (HDEL) at its C‐terminus to allow the expressed protein to be directed to, and retained within, the endoplasmic reticulum. Western blot analysis of samples from Nicotiana clevelandii or cowpea tissue infected with constructs revealed the presence of SIP molecules which retained their ability to dimerize. The analysis of crude plant extracts revealed that the plant‐expressed ɛSIP molecules could bind to and neutralize TGEV in tissue culture, the levels of binding and neutralization reflecting the level of expression. Oral administration of crude extracts from SIP‐expressing plant tissue to 2‐day‐old piglets demonstrated that the extracts which showed the highest levels of in vitro neutralization could also provide in vivo protection against challenge with TGEV.

Use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies

Abstract To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full‐length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The agr;SIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the α‐CH3 domain from human IgA. To express the full‐length IgA, the individual light and heavy chains from the TGEV‐specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co‐infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant‐expressed αSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody‐expressing plant tissue to 2‐day‐old piglets showed that both the αSIP and full‐length IgA molecules can provide in vivo protection against TGEV.