Carmen Vaquero

Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies.

This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30xa0μg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

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 3.8u2009μgu2009g−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.

A carcinoembryonic antigen-specific diabody produced in tobacco

The feasibility of using tobacco for production of a recombinant antibody (T84.66/GS8 diabody) directed against the carcinoembryonic antigen (CEA) and used for tumor imaging was investigated. Two constructs were generated for targeting the protein either to the apoplast or to the endoplasmic reticulum. Expression of the diabody in tobacco leaves after vacuum‐assisted infiltration of engineered Agrobacteria (agro‐infiltration) and in regenerated transgenic tobacco plants was analyzed and compared. Results in terms of protein expression and accumulation between both systems showed a good correlation. His6‐tagged T84.66 diabody was readily purified from agro‐infiltrated tobacco leaves and from transgenic plants by immobilized metal ion affinity chromatography. The purified protein was analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, electrospray mass spectrometry, direct and competition ELISA, electrophoretic mobility shift assay, and staining of CEA‐positive colon adenocarcinoma cell line LS174T. Our results demonstrate that tobacco is a competent production system for this clinically relevant diabody.

Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy

Field pea (Pisum sativum L.) appears well suited for the production of high-value molecules such as recombinant antibodies, with well-established agricultural practices world-wide and seeds that are easily stored and distributed. In order to evaluate the suitability of this grain legume for the production of biologically active antibodies, we transformed peas with a cDNA encoding the single-chain Fv fragment scFvT84.66. This scFv is derived from the monoclonal antibody T84.66, which recognises the well-characterised tumour-associated carcinoembryonic antigen. The antibody is useful for inxa0vitro immunodiagnosis and inxa0vivo imaging of human cancers. We expressed scFvT84.66 cDNA under the control of the seed-specific legumin A promoter. We targeted the antibody to the endoplasmic reticulum for better stability and high accumulation. Transgenic plants produced up to 9xa0μg per gram fresh weight of functional scFvT84.66 in their seeds. The transgene was stably inherited and expressed in the progeny, and the antibody remained active after storage in dried transgenic seeds for two months at room temperature. Our results demonstrate the suitability of grain legume seeds to produce biologically active recombinant antibodies, and the utility of field pea seeds as production vehicles for recombinant pharmaceutical macromolecules.