Julian K.-C. Ma

The production of recombinant pharmaceutical proteins in plants

Imagine a world in which any protein, either naturally occurring or designed by man, could be produced safely, inexpensively and in almost unlimited quantities using only simple nutrients, water and sunlight. This could one day become reality as we learn to harness the power of plants for the production of recombinant proteins on an agricultural scale. Molecular farming in plants has already proven to be a successful way of producing a range of technical proteins. The first plant-derived recombinant pharmaceutical proteins are now approaching commercial approval, and many more are expected to follow.

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.

Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants

The use of genetically modified (GM) plants to synthesize proteins that are subsequently processed, regulated and sold as pharmaceuticals challenges two very different established regulatory frameworks, one concerning GM plants and the other covering the development of biotechnology-derived drugs. Within these regulatory systems, specific regulations and guidelines for plant-made pharmaceuticals (PMPs)--also referred to as plant-derived pharmaceuticals (PDPs)--are still evolving. The products nearing commercial viability will ultimately help to road test and fine-tune these regulations, and might help to reduce regulatory uncertainties. In this review, we summarize the current state of regulations in different countries, discuss recent changes and highlight the need for further regulatory development in this burgeoning, new industry. We also make the case for the harmonization of international regulations.

Passive immunity in the prevention of rabies

Prevention of clinical disease in those exposed to viral infection is an important goal of human medicine. Using rabies virus infection as an example, we discuss the advances in passive immunoprophylaxis, most notably the shift from the recommended polyclonal human or equine immunoglobulins to monoclonal antibody therapies. The first rabies-specific monoclonal antibodies are undergoing clinical trials, so passive immunisation might finally become an accessible, affordable, and routinely used part of global health practices for rabies. Coupled with an adequate supply of modern tissue-culture vaccines, replacing the less efficient and unsafe nerve-tissue-derived rabies vaccines, the burden of this disease could be substantially reduced.

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.

Molecular Pharming: future targets and aspirations.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George’s, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

Immunogenicity of chloroplast‐derived HIV‐1 p24 and a p24‐Nef fusion protein following subcutaneous and oral administration in mice

High-level expression of foreign proteins in chloroplasts of transplastomic plants provides excellent opportunities for the development of oral vaccines against a range of debilitating or fatal diseases. The HIV-1 capsid protein p24 and a fusion of p24 with the negative regulatory protein Nef (p24-Nef) accumulate to ∼4% and ∼40% of the total soluble protein of leaves of transplastomic tobacco (Nicotiana tabacum L.) plants. This study has investigated the immunogenicity in mice of these two HIV-1 proteins, using cholera toxin B subunit as an adjuvant. Subcutaneous immunization with purified chloroplast-derived p24 elicited a strong antigen-specific serum IgG response, comparable to that produced by Escherichia coli-derived p24. Oral administration of a partially purified preparation of chloroplast-derived p24-Nef fusion protein, used as a booster after subcutaneous injection with either p24 or Nef, also elicited strong antigen-specific serum IgG responses. Both IgG1 and IgG2a subtypes, associated with cell-mediated Th1 and humoral Th2 responses, respectively, were found in sera after subcutaneous and oral administration. These results indicate that chloroplast-derived HIV-1 p24-Nef is a promising candidate as a component of a subunit vaccine delivered by oral boosting, after subcutaneous priming by injection of p24 and/or Nef.

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.

Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

BackgroundCommon oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guys 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials.ResultsThe antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans.ConclusionsHumanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

Exploring the vaccine potential of Dec-205 targeting in Mycobacterium tuberculosis infection in mice

Protein subunit vaccines are an attractive mode of immunisation against infectious diseases but the approach is hampered by the lack of suitable adjuvants for human use. We investigated if antigen targeting to the endocytic cell receptor Dec-205 on dendritic cells (DCs) could induce a protective immune response to Mycobacterium tuberculosis (MTB) infection in the absence of conventional adjuvants. Dec-205 receptor expressed by several subsets of DC has been shown in previous studies to be an efficient endocytic receptor for inducing both humoral and cellular immune responses, but this immunisation approach has not been tested in an experimental model of infection. We therefore prepared chemical conjugates of an anti-mouse Dec-205 monoclonal antibody (mAb) and the highly immunogenic antigen 85B (Ag85B) of MTB and showed that they bound efficiently to bone-marrow derived DC. Moreover, DC stimulated in vitro with Dec-205 conjugates could induce proliferation of splenocytes from Ag85B-immunised mice, while the negative control conjugates failed to do so. Following immunisation of mice with the anti-Dec-205-Ag85B conjugates administered together with a co-stimulatory anti-CD40 mAb, antigen-specific humoral and cellular responses were detected. Although the conjugates induced a strong Ag85B-specific humoral response, T cell proliferation and interferon-γ production were observed only when the conjugates were used to boost BCG vaccine. Importantly though, the conjugate vaccine did not offer significant protection against MTB challenge when used on its own or as a boost to BCG. Therefore, we conclude that Ag85B-based vaccine targeting to Dec-205 alone is not a sufficiently robust vaccination strategy for tuberculosis, although this approach might be more successful with other antigens or infections.