FAIR-CT96-3110 Production of diagnostic and therapeutic antibodies in plants by molecular farming

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Biotechnology : FAIR Area 3 - Generic Science and Advanced Technologies for Nutritious Foods : Pharmaceuticals/Cosmetics

	Contract No:	FAIR-CT96-3110
	Date Prepared:	September 1999
	Source:	Second Annual Progress Report Abstract

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Second Annual Progress Report Abstract

Objectives

The key objectives for Year 2 were to transform pea with antibody gene constructs prepared in year 1, and to evaluate significant numbers of independently-derived transgenic pea plants at the biochemical and molecular level. The analyses would address the influence of molecular parameters such as transgene copy number, linear versus circular plasmids and targeting signals on recombinant antibody (rAb) expression levels. Raw material for downstream processing and detailed analysis of rAbs should be provided and the initiation of sexual crossings was required to accumulate the four genes of the sigA into a single host plant.

Engineering of scFvs, scFv-fusion proteins and diabodies should be completed, and constructs should be verified by sequencing and analyse either by bacterial or transient expression in plant cells. These constructs should also be provided for stable transformation of pea.

Downstream processing of rAbs from plant leaves and seeds using perfusion chromatography based ion-exchange, protein-A or IMAC-based affinity chromatography techniques was anticipated. Also, the improvement of procedures for homogenising raw material and extraction were envisaged. Biochemical and immunological characterisation of rAbs and carbohydrate analysis of murine derived mAbs was initiated. The optimisation of gene constructs of alpha-CEA and alpha-S. mutans rAb cDNA constructs for improved MRNA stability and enhanced translation should be initiated, and retention signals as well as targeting sequences for improved expression of rAbs should be incorporated.

Activities

Antibody engineering Cloned genes can be modified by replacing or deleting certain amino acids using site directed mutagenesis or PCR. Cloned recombinant antibodies can be converted into Fab- or F(ab')₂ -fragments by introducing stop codons at the appropriate site. Effector functions can be eliminated by deleting the C_H2 domain and new, or better, effector functions introduced by converting mouse immunoglobulins into chimeric mouse/human polypeptides. Furthermore, alteration of the hinge region, carbohydrates or the inactivation of Fc-receptor binding sites can change the performance, biodistribution and stability of rAbs.

Chimeric antibodies

Upon administration of mAbs into humans, human anti-mouse antibodies (HAMA) develop in most immunocompetent individuals. This response can markedly accelerate clearance of murine monoclonal antibodies from the blood, interfere with mAbs before they reach the target cell and block antigen binding. An immediate application of antibody engineering is to redesign mouse and rat monoclonal antibodies to remove xenogenic portions and to replace them with their human counterparts. Many cloned murine antibodies have been chimerized by domain swapping (e.g. antibodies against CEA, CD20, CD7, IL-2 receptor, P-glycoprotein and hepatitis surface antigen). By exchanging human for murine constant domains, these chimeric antibodies frequently gain functional activity in ADCC (antibody-dependent cell-mediated cytotoxicity), compared to their murine counterparts. Many of these chimeric antibodies have been used successfully for immunotherapy and only a minority of patients mounted an immune response against these molecules.

Secretory antibodies

Immunological prevention of infections at mucosal surfaces has been difficult. Active immunisation to induce mucosal antibodies has not been straightforward, and frequently leads to low titre and short lived antibody responses. Passive immunisation has been hampered by technical difficulties in producing monoclonal secretary antibodies because sIgA is the product of both plasma cells (that produce IgA and J chain) and epithelial cells (that produce the secretary component precursor molecule). Until the recent demonstration of sIgA assembly in plants, it had not been possible to produce significant quantities of these antibodies for analysis. Enhancing the mucosal immune system by topical administration of monoclonal sIgA could be an important prophylactic measure, to be used alone or in combination with systemic immunisation to provide both levels of immunological protection.

Antibody Fragments (size reduction)

The size of antibodies can be reduced by enzymatic treatment, with Fab or Fv fragments generated in the process. The elimination of a Fc-portion(s) of the antibody results in desirable changes, such as improved tissue penetration or radioimaging of tumours compared to whole antibodies. This feature may be critical in therapy studies to reduce bone marrow toxicity and improve treatment of poorly vascularized solid tumours. However, smaller fragments will be cleared from the body much faster than whole antibodies. Single chain Fv fragments (scFv) are a form of recombinant antibody with a reduced size that incorporates the complete antigen-binding Fv domain of an antibody into a single polypeptide by joining the variable domains (V_L and V_H) with a synthetic linker peptide of 14 to 25 residues. Recombinant antibody technologies have considerably improved the production of either diabodies, that can be obtained by making scFvs with very short linker peptides (< 15 residues); or single polypeptide bispecific scFvs, that can be produced by incorporating a long and flexible linker to connect two scFvs with different specificities. These two rAb forms lack the antibodies' Fc portion to avoid unwanted activation of cells expressing Fc receptors and their reduced size should improve tumour penetration.

Fusion proteins

The combination of recombinant DNA technologies and the novel information on members of the immunoglobulin superfamily opened possibilities for generation of new hybrid molecules. Portions of a cloned immunoglobulin gene can be substituted with sequences from other molecules, or fused to foreign sequences. The host used to produce the antibody will then express both genes as a single fusion protein, which gives improved stability compared to chemical coupling. Antibody fusion proteins fall into two categories: (1) Fc-fusion proteins (also called immuno-adhesion molecules) and (2) antigen-binding fusion proteins, which will be described here. Antigen-binding fusion proteins are recombinant proteins in which the antigen-binding portion of an antibody is linked to a ligand. For example, chimeric fusion proteins have been made by maintaining the antibody variable domains and replacing the constant domains with toxins, biological response modifiers (here IL-2), enzymes, prodrugs and radionucleotides. Recombinant calorimetric antibodies are a novel class of fusion proteins which circumvent the inefficient coupling of enzymes, such as alkaline phosphatase (AP), to the antibody and are an interesting alternative to existing immunodiagnosties. An scFv-AP or Fab-AP fusion protein can be produced in heterologous expression systems and the expressed chimeric recombinant proteins retain the binding affinity of the original antibody. The chimeric protein resembles a "complete IgG-molecule" in which the Fc-part is replaced by the dimeric form of the alkaline phosphatase enzyme, making the recombinant protein bifunctional. This recombinant protein can be directly used in immunoassays.

Progress

Gene constructs encoding mouse-human chimeric A-CEA (T84.66) and sigA alpha-S. mutans (Guy's 13 with modified heavy chain + J-chain) cDNA constructs for plant transformation have been prepared using existing vectors. The sequence of all modified genes and expression constructs has been confirmed and provided to generate constructs for stable expression and for transient expression studies. Engineering of scfv, scfv-fusion proteins (IL-2) {PI} with C-terminal purification tags and diabodies has been initiated. Transient plant transformation, as well as bacterial expression of scfvs are in progress. For stable transformation of pea plants appropriate cassettes, with suitable selectable markers (bar and hph) targeting signals and optimal promoters for constitutive and seed specific expression of target proteins (provided in form of modified cDNAs have been generated.

In brief, the status against the projected deliverables and milestones is as follows:

• Modification of first CDNA constructs. Completed
• Initiation of transient (bacteria and plant cells) and stable transformation. Completed
• Completion of transiently transformed pea with first cDNAs for molecular and biochemical characterisation. Completed
• First transgenic pea plant lines for extraction, months purification and initial characterisation of recombinant antibodies. Completed
• Finalisation of all modified cDNAs. Completed
• First analysis of plant-derived antibodies to implement strategies for improvement of plant production systems by crossing experiments, optimisation of expression constructs, Incorporation of targeting and retention signals. Completed
• Finalisation of all expression constructs. Completed
• First improved plant production systems for downstream processing Ongoing
• Completion of quality control to monitor and validate months production and purification processes of recombinant proteins; initiation of efficacy studies. Ongoing
• Final evaluation of approaches to improve rAb production in plants even further. Ongoing
• Completion of structural and functional studies in vitro and in vivo. Ongoing
• Final report of evaluation of cost and feasibility of molecular farming project. Ongoing

Achievements

The transformation of the target species pea with constructs prepared in year 1 has been completed. The evaluation of significant numbers of independently-derived transgenic pea plants molecularly and biochemically, as well as the influence of molecular parameters such as transgene copy number, linear versus circular plasmids, targeting signals on levels of expression was initiated. Raw material containing rAbs for downstream processing was analysed in detail. Downstream processing of rAbs from plant leaves and seeds using perfusion chromatography based ion-exchange and protein-A or IMAC- based affinity chromatography techniques was carried out and procedures for homogenising raw material and extraction were improved. Sexual crossings required to accumulate the four genes of sIgA into a single host plant were initiated. The engineering of scFvs, scFv-fusion proteins and bi-scFvs or diabodies was completed. Biochemical and immunological characterisation of rAbs and carbohydrate analysis of murine derived mAbs and mouse/human chimeric rAbs was initiated and first conclusive data were provided.

Future actions

The objectives of year 3 will cover:

• The transformation of the remaining constructs for pea prepared in year
• Immunolocalisation experiments to ascertain effects of targeting signals. The initiation of crossing experiments between selected transgenic pea plants to evaluate role of genetic component(s) in maximising levels of expression.
• Continued crossing experiments of plants required for production of sIgA.
• Evaluation of significant numbers of transgenic pea plants for levels and stability of expression of target proteins.
• Continued molecular analysis for all transgenic plants.
• Scale up production of A-CEA and alpha-S. mutans rAbs by planting highest producing lines from pea in contained greenhouses.
• Continued optimisation of extraction and downstream processing.
• The analyses of specificity, affinity, off- and on-rates and rAb stability in vivo , and product conformity and transgene stability over an extended cultivation.
• Determination of performance of purified rAbs in diagnostic assays of analysis of plant derived carbohydrate structure and comparison with murine glycosylation.
• Comparison of influence of carbohydrates on stability and immunogenicity in animals in collaboration with other project members.
• Complete optimising gene constructs of A-CEA and a-S. mutans rAb CDNA constructs (improved MRNA stability, enhanced translation, retention signal, targeting sequences).
• Confirmation of all modified gene and expression constructs by sequencing.
• Use of optimised expression constructs for transient plant gene expression.