FAIR-CT96-5068 Molecular farming of therapeutic antibodies in plants

Contacts




To find similar Items, click on a keyword below:
Crops for Pharmaceuticals/Cosmetics : FAIR Area 1.2 - Green Chemicals and Polymers Chain : FAIR Marie Curie Research Training Grants : Pharmaceuticals/Cosmetics

Objectives:

The enormous potential of antibodies in human health has created demand for large amounts of functionally active forms of recombinant antibodies (rabs) produced at low cost with no risk to the patient during therapeutic applications. In this project, the tumour specific antibody T84.66 was to be studied since it recognises a major target of colon, breast and lung adenocarcinomas, the carcinoembryonic antigen (CEA), and is suitable for use in diagnosis and therapy. One aim of the project was to generate fusion proteins comprising a single chain antibody scFvT84.66, derived from T84.66, genetically fused to the biological response modifier Interleukin 2 or Pseudomonas exotoxin. These fusion proteins were then to be expressed in tobacco. In addition, genetic linkage of the tumour specific scFvT84.66 to another scfv with specificity for cytotoxic T-cell markers would be carried out to potentially enhance the tumour killing properties of the rAb.

Activities and results: Eight different plant expression constructs were obtained by cloning the scFv84.66 gene using a combination of the following elements: a 5'UTR (CHS or Omega), a leader sequence (LPH or LPL - codon optimised leader peptides of the heavy chain and light chain genes of mAb24), a 3' tag (His6 or a KDEL EER retrieval signal), and the 3' UTR of tobacco mosaic virus. These eight constructs have been cloned into the pSS plant expression vector. Functional expression of scFv84.66 using these constructs has been tested by Agrobacterium mediated transient expression in tobacco leaves. Plant expressed scFvT84.66 competed against the mAb T84.66 for binding to CEA/NA3 protein in competition ELISA, indicating that the recombinant protein is functionally expressed. Sufficient quantities of the proteins were generated for affinity purification of scFvT84.66, coupled to a 6-histidine tag, and its characterisation. Tobacco plants have been transformed with the constructs and shown to be stable. The antibody is functionally expressed up to the then analysed T2 generation. To develop a direct assay for detecting scFvT84.66 expression, chicken IgY antibodies have been raised using plant expressed or bacterial scFv84.66.

Conclusions:

Fusion proteins between IL2 and scFvT84.66 have been generated and tested by transient expression in tobacco leaves, where the antibody domain of the fusion protein is functional and the activity of the IL12 domain is under investigation. Stably transformed tobacco plants expressing this fusion protein were generated as were fusions of scFvT84.66 with Pseudomonas exotoxin and CD8.

Keywords: antibody, engineering, plant expression, single chain antibodies

Contacts

Scientific Officer

• Rainer FISCHER / RWTH Aachen Molecular Biotechnology / GERMANY