Therapeutic proteins such as vaccines, antibodies, hormones, and cytokines are generally produced in bacteria or eukaryotic systems, including chicken eggs and mammalian or insect cell cultures, with high production yield according to well-defined regulatory guidelines (1). The use of plants for the production of therapeutic proteins, called molecular farming, was proposed as an alternative biomanufacturing method in 1986. The first and only plant-derived therapeutic protein for human use was approved in 2012 for the treatment of Gaucher disease. In 2019, a plant-produced influenza virus vaccine completed phase 3 clinical trials, with encouraging results (2). More recently, phase 3 trials for an adjuvanted plant-made vaccine (CoVLP) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (NCT04636697) began in March 2021. These successes have revived interest in plant-produced pharmaceuticals for human use, which could include edible drugs.
Molecular farming was initially proposed because growing plants only requires light, water, and soil (or artificial support). Procurement of greenhouses is cheaper than bioreactor suites, which are required for bacterial, mammalian, and insect cell culture systems, making molecular farming particularly attractive for developing countries. Furthermore, production can be scaled up or down based on the number of plants grown. Additionally, unlike in the traditional production systems, zoonotic pathogens are unable to infect plants and therefore cannot be a source of contaminant in molecular farming–derived products.
Technological progress, such as codon optimization, the inclusion of organelle-specific promoters, humanization of N-glycans, and transient transfection systems, has increased the performance of molecular farming, with yields above 1 mg/g of fresh plant weight. In comparison, yields between 5 and 20 g/liter are commonly reached in mammalian Chinese hamster ovary cell cultures that are used to manufacture monoclonal antibodies. Transient transfection systems have also increased the production speed, with harvest possible within days after transfection of adult plants as opposed to months when stable expression was sought. The production speed of molecular farming is a critical asset. Indeed, plant-produced vaccines can readily be made against new pathogens or emerging strains, with the first batch of vaccine candidates typically produced within 3 weeks (3). The speed of molecular farming is particularly suited for personalized medicine in which pharmaceuticals need to be tailored to individual patients, such as for cancer treatment (4).
Constructing facilities capable of purifying therapeutic proteins produced in plants under good manufacturing practice (GMP)–compliant conditions has also supported clinical evaluation of plant-made vaccines and therapeutics (5, 6). Moreover, the simplicity and low costs associated with plant growth are an advantage, whereas expensive GMP facilities are required for the extraction, purification, and fill processes of molecular farming. By contrast, the lower yield, limited regulatory guidelines available, and limited manufacturing capacity worldwide have dampened enthusiasm toward molecular farming (1).
For vaccine purposes, plant-produced proteins have several advantages over their counterparts produced in bacterial, mammalian, or insect systems. Unlike bacteria, plants are capable of posttranslational modifications. Plants express different glycans, which renders plant-derived proteins more immunogenic than their mammalian counterparts (7). In plant-made vaccines, virus-like particles (VLPs) are produced that comprise the target pathogen’s protein(s) of interest (the immunogen) and plant components within the particles. These plant components of VLPs, such as lectins, glycans, saponins, and heat shock proteins, have adjuvant properties (7) that can further potentiate the immune response against plant-made vaccines and may reduce the need for adjuvants in vaccine formulation.
This increased immune stimulation may lead to hypersensitivity (allergic reactions) toward plant components. However, several clinical trials, including phase 1 trials for CoVLP (NCT04450004) and phase 3 trials for plant-made vaccines against influenza virus (NCT03301051, NCT03739112), have alleviated this concern. In these large phase 3 clinical trials, involving more than 20,000 adults aged 18 to 94 years, a mixture of four separate VLPs, each containing the hemagglutinin proteins from a selected seasonal influenza virus strain, produced in tobacco plants, have shown efficacy similar to that of existing influenza vaccines (2). Notably, immunization with this quadrivalent VLP formulation was not associated with more adverse events or an increase in hypersensitivity reactions compared with individuals receiving a licensed influenza virus vaccine.
The plant-made vaccines against influenza virus and SARS-CoV-2 are expected to be the first therapeutic proteins produced in whole plants for human use. The glucocerebrosidase enzyme, produced as an injectable protein drug called taliglucerase alfa for the treatment of Gaucher disease, is produced in a carrot cell culture rather than in actual plants (8). Several plant-produced veterinary vaccines have also been developed, and one is approved by the US Department of Agriculture for immunization of chickens against Newcastle disease. However, there is competition to reduce the selling price of farm animals, so the cost of plant-made veterinary vaccines needs to be substantially reduced to ensure their widespread adoption (5).
Although the increased immunogenicity of plant-made protein is beneficial for vaccines, it can be detrimental for therapeutic proteins, potentially reducing their in vivo efficacy and contributing to adverse events. Despite these limitations, monoclonal antibodies against HIV (NCT01403792) and Ebola virus (NCT02363322, NCT02363322) have reached clinical stages of development. In human trials, intravenous (Ebola virus) or intravaginal (HIV) administrations of these antibodies were generally well tolerated. Mild adverse events, such as hypotension and fever, were observed following intravenous delivery (9, 10). Life-threatening reactions, such as anaphylactic shock, have only been reported in a single recipient of 238 treated with antibodies against HIV or Ebola virus (10, 11). The administration of antihistamine and antipyretic (fever-reducing) agents before intravenous infusion of plant-produced pharmaceuticals has mitigated the occurrence of severe adverse events (11).
The use of plants engineered to express human glycans also reduces the immunogenicity of plant-made proteins. It is worth noting that independently of the production system, the intravenous injection of grams of antibodies is associated with some adverse events, which are observed even with antibodies produced in mammalian cells. Another hurdle for plant-made monoclonal antibodies is limited manufacturing capacity. Unlike vaccines, monoclonal antibody regimens usually require larger doses (50 to 150 mg/kg) and sometimes with repeated administrations. To achieve the required scale of plant-made antibodies, a substantial expansion of current manufacturing capacity, which is currently limited to less than 15 GMP facilities worldwide, is required. Funding opportunities, similar to that of the US Defense Advanced Research Projects Agency (DARPA), which financed the development of three large GMP facilities for the production of plant-made vaccines in the US, are needed (5).
Using transient expression systems, plant-made vaccine and therapeutic proteins can be produced within weeks. For oral administration, edible plants that express therapeutic proteins require minimal processing after harvest. By combining the reduced cost and ease of administering edible vaccines or therapeutic proteins with the speed of transient expression systems, molecular farming could have a considerable impact on both human and animal health.
GRAPHIC: C. BICKEL/SCIENCE
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Using transient expression systems, plant-made vaccine and therapeutic proteins can be produced within weeks. For oral administration, edible plants that express therapeutic proteins require minimal processing after harvest. By combining the reduced cost and ease of administering edible vaccines or therapeutic proteins with the speed of transient expression systems, molecular farming could have a considerable impact on both human and animal health.
GRAPHIC: C. BICKEL/SCIENCE
Oral administration of drugs is a user-friendly alternative to the intravenous route. Furthermore, oral administration can mitigate the adverse events associated with intravenous administration of pharmaceuticals. Gut immune responses are crucial for tolerance to food and self-antigens and play an important role in ensuring a balanced immune system (12). Moreover, most pharmaceutical proteins currently under clinical evaluation are purified before parenteral injections. Given orally, plant-made therapeutics might only require minimal processing, thus possibly skipping expensive and time-consuming steps in the manufacturing process. Products for oral delivery can also be stored in lyophilized (dehydrated) form at room temperature for an extended period, hence sharply reducing both their cost of production and storage while simultaneously facilitating their administration.
The use of edible plants such as cereal crops, tomatoes, corn, and rice is under development for oral delivery of plant-made therapeutic proteins (12). Parenteral administration of tumor necrosis factor (TNF) antagonists is used for treating autoimmune diseases such as rheumatoid arthritis. In a phase 1 clinical trial, oral administration of lyophilized tobacco plant–derived cells expressing a TNF antagonist was safe and increased the amount of immunosuppressive regulatory T cells (Tregs) in human volunteers, demonstrating the feasibility of this approach (13). In addition to reducing adverse events, the oral route can favor the induction of tolerance to suppress autoimmune or allergic responses. To date, the induction of tolerance by using recombinant proteins within edible plants is restricted to animal models of hypersensitivity or autoimmunity. However, this could provide low-cost and simple solutions to better manage and prevent the growing incidence of allergies to common substances (12, 14).
Edible vaccines are also under development. The safety and feasibility of this approach were demonstrated in proof-of-concept phase 1 clinical trials, to monitor the safety and immunogenicity of edible vaccine candidates against Escherichia coli, hepatitis B virus, rabies lyssavirus, and norovirus, which occurred between 1998 and 2004. In these trials, the proportion of immunized individuals who generated an immune response against the desired target was disappointingly lower than in clinical trials involving standard vaccines administered via the parenteral route. The yield of recombinant proteins produced in plants has since increased substantially, suggesting that new edible plant-made vaccines could now generate meaningful immune responses. The use of edible plant vaccines in prime-boost immunization regimens, with an injectable vaccine as a prime and an edible vaccine as a booster, has also been investigated to improve the clinical relevance of plant-produced oral vaccines against poliovirus (12, 14, 15). However, further optimization is required before clinical acceptance of these vaccine candidates (14). Ensuring that edible plant-made vaccines do not lead to hypersensitivity against the plant used for production is crucial, especially plants that are widely consumed such as rice, cereals, and corn (see the figure).
The plant-made quadrivalent VLP influenza vaccine will likely be licensed for use, given the favorable outcome of the phase 3 clinical trials. Ongoing clinical development will continue to inform regulatory guidelines for plant-made therapeutic proteins. However, because doses for therapeutics are much higher than for vaccines, investment in manufacturing infrastructure must increase and production costs need to further decrease to achieve large-scale manufacturing of plant therapeutic products. Until then, the speed of molecular farming will be useful for preclinical and early clinical evaluation of therapeutic candidates. Edible, plant-made therapeutics are still predominantly in the preclinical stage of development but, if successful, could create new classes of pharmaceutical products. Manufacturing of pharmaceutical proteins may remain dominated by current production systems until economic attractiveness through easy manufacture and technological progress, such as potent edible plant-made therapeutics, shifts the balance toward molecular farming.
Acknowledgments: The authors are supported by the International Development Research Centre (109075-001); the Canadian Department of Foreign Affairs, Trade and Development (BIO-2019-005); and the Canadian Institutes of Health Research. Thanks to L. Zeitlin for useful discussions.