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FAIR-CT96-3110
Production of diagnostic and therapeutic antibodies in plants by molecular farming |
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Type of Project | Shared Cost |
| Contract No | FAIR-CT96-3110 | |
| Total Cost | 1,298,382 ECU | |
| EC Contribution | 950,000 ECU | |
| Start Date | 01/10/1997 | |
| Duration | 48 Months |
Production of diagnostic and therapeutic antibodies in plants by molecular farming
Objectives
Technical Approach
Antibodies have enormous potential as therapeutic and diagnostic agents in human health care. This has created a demand for low cost production of large amounts of active recombinant antibody (rAb) forms. Importantly, the uses of antibodies in diagnostics, therapeutics and vaccines require that recombinant antibodies can be used at no risk to patients or consumers and that antibodies will have to be engineered to increase the range of available applications.
It is planned that this will be achieved by preparing different rAb forms through protein engineering and molecular farming. This will provide rAbs with improved performance and stability when compared to rAbs produced in microbes, mammalian cultures or transgenic animals.
Antibodies will be fused to enzymes to create single fusion proteins that can be directly used in diagnostic assays. This will be evaluated as an alternative to the time and cost intensive use of enzyme labelled secondary antibodies.
Also, rAb-fusions using IL-2 as a biological response modifier will be tested for their improved properties. Finally, genetic linkage of a target-specific single chain antibody (scFv) to a second scFv with specificity for the cytotoxic CD8-T-cell marker should improve efficacy by recruiting the cellar immune arsenal.
Although some of these recombinant proteins have been expressed in microbes or mammalian cells, results were disappointing in terms of yield, cost, low amount of correctly folded proteins or safety issues (presence of pathogenic viruses or oncogenic sequences in products). Therefore, alternative expression systems that avoid these problems are needed. To solve these problems, we will fine tune and evaluate rAb production using molecular farming, to generate rAbs with improved efficacy.
Antibodies were selected for evaluation of heterologous expression in plants for several reasons:
Two antibodies that are important in human health were selected for evaluation of the molecular farming concept: Guy's 13, which can be used for topical therapy of human oral pathogens and the adenocarcinoma tumour-specific antibody T84.66.
Upon modification of different rAb molecules for plant expression vectors, plant expressing macromolecules of interest at high levels will be identified and yields further improved through optimisation of gene expression, targeting to different plant cell compartments, and sexual crossing.
Pea was selected as a production vehicle for a number of important reasons:
Recombinant proteins will be compared with their natural counterparts to evaluate biological and immunological activities, differences in protein and carbohydrate structures, biochemical properties, product stability, conformity and safety. Function and lot-to-lot conformity of plant-expressed rAbs and fusion proteins will be tested in vitro and in vivo in animal models through the proposed interdisciplinary research project that will merge expertise from molecular immunology, plant molecular biology, protein engineering, medicine and structural biology. Our results will lead to the identification of the most potent recombinant proteins and their production at high yields and low cost for improved future applications in diagnostics, therapeutics and vaccines.
Results to Date
To test the feasibility molecular farming therapeutic antibodies in plants, expression of scFvGuy's 13 scFvT84.66 and a human/mouse chimeric antibody in plants was assayed by Agrobacteria mediated transient transformation of tobacco leaves. Expression of ER targeted scFvs containing a C-terminal KDEL retrieval signal resulted in ~ 10 fold higher production of functional protein than when the KDEL was replaced with a His6 tag. Affinity purified scFvs retained both affinity and specificity when compared to the original murine parental antibody.
After demonstrating the feasibility of scFvGuy's13 and scFvT84.66 expression in tobacco plants, genetically stable transformed pea plants expressing both scFvs under the control of the seed specific leg promoter are being generated.
To generate therapeutic antibodies with a reduced HAMA response, human/mouse chimeric full size T84.66 antibodies were generated. Both heavy and light chain retained their original murine leader peptides and a KDEL retention signal was inserted at the C-terminal of the heavy chain. Full-size cT84.66 antibody was produced in tobacco leaves by simultaneous expression of both the heavy and light chain constructs using Agrobacteria mediated transient expression. Affinity purified full-size cT84.66 retained the same function and specificity as the parental murine antibody. This construct is currently being used for stable transformation of pea.
Contacts
Coordinator
EC Scientific Officer
Participant
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