Matthew J. Paul

Plant‐made pharmaceuticals: Leading products and production platforms

The number of approaches to recombinant protein production in plants is greater than ever before. Development of these new and improved technologies as production platforms for plant‐made pharmaceuticals has and will continue to create new commercial opportunities in the pharmaceutical sector. However, it is inevitable that no single system will be optimal for the production of all recombinant proteins of interest in plants due to both the physical characteristics and the envisaged therapeutic application of each product. Here, we review a range of promising product/platform pairs emphasizing synergies during production and in clinical trials.

Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco

Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plantderived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guys 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin‐N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator α‐naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 Hg/g root dry weight/24 h for Guys 13, 10.43 μg/g root dry weight/24 h for 4E10, and 766 μg/g root dry weight/24 h for cyanovirin‐N, the highest figures so far reported for a full‐length antibody and a recombinant protein, respectively. The plant growth regulators indolebutyric acid, 6–benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guys 13. The effect of the growth regulators differed, as α‐naphthalene acetic acid and indole‐butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guys 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guys 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guys 13 antibody. In addition to the attractiveness of controlled cultivation within a contained environment for pharmaceutical‐producing plants, this study demonstrates advantages with respect to the quality and downstream purification of recombinant proteins.—Drake, P. M. W., Barbi, T., Sexton, A., McGowan, E., Stadlmann, J., Navarre, C., Paul, M. J., Ma, J. K.‐C. Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. FASEB J. 23, 3581–3589 (2009). www.fasebj.org

Antibody degradation in tobacco plants: a predominantly apoplastic process

BackgroundInterest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem.ResultsIn this study, we investigated the dynamics of the assembly and breakdown of a human IgG1κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space.ConclusionsThe results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions.

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.

Plant-made immunogens and effective delivery strategies

Plant systems for the production of recombinant immunogens have the potential to overcome obstacles currently impeding the delivery of vaccines to poorer, more remote populations by localizing production and reducing associated costs. The nature of the future plant-derived vaccine candidates will have an important impact on the extent to which universal access to vaccines can be achieved using these technologies. In this article, we examine approaches taken to design immunogens, expression systems and delivery strategies that are medically feasible and immunologically effective while retaining key benefits of a plant production platform. We identify three ‘target areas’ in which plant-made immunogens may offer particular advantages over conventional production systems.

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.

Immune-Complex Mimics as a Molecular Platform for Adjuvant-Free Vaccine Delivery

Protein-based vaccine development faces the difficult challenge of finding robust yet non-toxic adjuvants suitable for humans. Here, using a molecular engineering approach, we have developed a molecular platform for generating self-adjuvanting immunogens that do not depend on exogenous adjuvants for induction of immune responses. These are based on the concept of Immune Complex Mimics (ICM), structures that are formed between an oligomeric antigen and a monoclonal antibody (mAb) to that antigen. In this way, the roles of antigens and antibodies within the structure of immune complexes are reversed, so that a single monoclonal antibody, rather than polyclonal sera or expensive mAb cocktails can be used. We tested this approach in the context of Mycobacterium tuberculosis (MTB) infection by linking the highly immunogenic and potentially protective Ag85B with the oligomeric Acr (alpha crystallin, HspX) antigen. When combined with an anti-Acr monoclonal antibody, the fusion protein formed ICM which bound to C1q component of the complement system and were readily taken up by antigen-presenting cells in vitro. ICM induced a strong Th1/Th2 mixed type antibody response, which was comparable to cholera toxin adjuvanted antigen, but only moderate levels of T cell proliferation and IFN-γ secretion. Unfortunately, the systemic administration of ICM did not confer statistically significant protection against intranasal MTB challenge, although a small BCG-boosting effect was observed. We conclude that ICM are capable of inducing strong humoral responses to incorporated antigens and may be a suitable vaccination approach for pathogens other than MTB, where antibody-based immunity may play a more protective role.

Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana : influence on expression yield and antibody degradation

We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which β1,2-xylosyltransferase and α1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10XylT/FucT) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10XylT/FucT demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of β1,2-xylose and α1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible ‘fragile’ sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.

Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

Summary In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen‐presenting cells. These self‐adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII‐PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII‐PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII‐PIGS also induced a significant cellular immune response, IFN‐γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof‐of‐principle study shows that the potent antibody Fc‐mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad‐ranging immune response.

Commercialization of new biotechnology: a systematic review of 16 commercial case studies in a novel manufacturing sector.

The 1980s and 1990s saw a major expansion of biotechnology into new areas of science including genomics and recombinant technologies. This was coupled to the widespread emergence of academics into the commercial sector as they were encouraged to spin out companies or commercialize their intellectual property. There were many opportunities to raise investment, and extraordinary success stories were prominent across many areas of technology. The field of plant biotechnology for manufacturing recombinant pharmaceuticals (molecular pharming) emerged and was developed in this period. Like other biotechnologies, this was an exciting new development which offered some very obvious benefits and commercial advantages. In particularly, plant molecular pharming represented a highly novel and potentially disruptive manufacturing technology for recombinant proteins. Twenty-five years on, a series of interviews with senior members of sixteen of the most prominent companies involved in the field provides insight into the original drivers for commercialization, strategic thinking and planning behind key commercial decisions and an insider view into the major reasons for commercial success or failure. These observations and recurring themes identified across a number of commercial ventures remain relevant today, as new biotech companies continue to spin out of the world of academia.