Sunday, September 11, 2011
Pharm Crops Ignoring Health & Environment
By Prof. Joe Cummins
September 11, 2011
Pharmaceutical drug production has undergone major changes following the development and approval of drugs called ‘biologicals’ that are for the most part proteins produced by genetic engineering. Biologicals make up at least a quarter of new drug approved, though they are about twice as likely as chemical drugs to experience regulatory action following approval (see ‘Biologicals’, Wonder Drugs with Problems, SiS 42). The recombinant protein drugs are produced using viruses, bacteria, yeast, and cell cultures from insects, rodents, primates or humans. The use of genetically modified (GM) crop plants to produce biologics has been an attractive prospect because the crops are capable of producing vast quantities of recombinant proteins at low cost. There have been a large number of such transgenic ‘pharm crops’ created in the laboratory and field trialled, though none have been approved for commercial drug production. However, some have now progressed to clinical trials.
The phases of clinical trials
Clinical trials are separated into several phases. Phase 0 is a recent designation for exploratory, first-in-human trials conducted in accordance with the United States Food and Drug Administration (FDA). Phase 0 trials include the administration of single sub-therapeutic doses of the drug to a small number of subjects (10 to 15) to gather preliminary data on the agent’s pharmacodynamics (what the drug does to the body) and pharmacokinetics (what the body does to the drugs). Phase I trials are the first stage of testing in human subjects involving a small (20-100) group of healthy volunteers, and designed to assess the safety (pharmacovigilance), tolerability, pharmacokinetics and pharmacodynamics of a drug. Once the initial safety of the study drug has been confirmed in Phase I trials, Phase II trials are performed on larger groups (20-300) to assess the drug’s efficacy, as well as to continue Phase I safety assessments. When the development process for a new drug fails, this usually occurs during Phase II trials. Phase III studies are randomized controlled multicentre trials on large patient groups (300–3 000 or more depending on the disease/medical condition) and are the definitive assessment of the drug’s efficacy. When the phase III trial is completed and deemed satisfactory, the drug may be released for use in treating the general population.
Pharm crops in clinical trials
The first report of a plant-made therapeutic (PMT) protein reaching Phase II human clinical trials is Locteron of Biolex Therapeutics, Inc., a controlled-release interferon alfa (IFN-α) for treating chronic hepatitis C. The current treatment involves weekly administration of an IFN-α in combination with an antiviral drug ribavarin. In 2005, 32 patients participated in Phase IIa clinical trials of Locteron. The IFN-α produced in the aquatic plant Lemna was administered fortnightly in combination with ribavirin in a randomised double blind study. An early 100 percent virological response was achieved in all 16 hepatitis C patients treated with 480 and 640 μg doses. Early virological response has been established as a pre-requisite for long-term response in hepatitis C patients.
The first plant-made therapeutic directed for human use to reach Phase III clinical trials was a carrot cell suspension Gaucher’s disease therapeutic developed by Protalix BioTherapeutics. Gaucher’s disease is the most common of the lysosomal storage diseases. It is caused by a hereditary deficiency of the enzyme glucocerebrosidase (also known as acid β-glucosidase), which breaks down the glycolipid glucocerebroside, resulting in its accumulation particularly in white blood cells (mononuclear leukocytes), and also in the spleen, liver, kidneys, lungs, brain and bone marrow. The transgenic human glucocerebrosidase (human prGCD) in carrot cells were grown in a bioreactor system. The purified recombinant protein was tested in Phase I/II trials in 2006 before entering Phase III trials in 2009. Thirty-one patients suffering from Gaucher’s disease were tested in a multi-centre, randomised, double blind trial. The primary endpoint (20 percent average reduction of spleen volume) was achieved in treated patients after only 6 months, and safety analysis showed that the treatment was well tolerated with no serious or severe adverse events reported. Patients who successfully completed this study were granted expanded access, some for over two and a half years. On December 1, 2009, Pfizer and Protalix entered into an agreement to develop and commercialize prGCD for the treatment of Gaucher’s disease, giving Pfizer exclusive worldwide licensing rights while Protalix retains commercialization rights in Israel. However, in early 2011, the FDA declined approval for the drug, asking for additional data from existing studies, but not requiring any additional trials.
SemBioSys has also completed a phase I-II trial of safflower-produced insulin, and found it to have a similar safety profile to current recombinant insulin. The insulin is produced in oil bodies allowing for simplified extraction, and the plants have been grown in open fields.
Pharm crop systems
Production of biological drugs in pharm crops is potentially hazardous, particularly if it is undertaken in open fields. They should all be done, if at all, under strictly confined containment. The threat of open field pharm crops ranges from the contamination of the human food supply to impact of the biological drugs on wildlife and farm animals. ISIS has written extensively on this issue over the years (see Pharm crops , ISIS reports since 2002). Unfortunately the emphasis has been on the safety of the purified final biological drug and not on the threat to the human food supply and to wildlife and farm animals. The regulatory framework envisioned by the advocates of pharm crop drug production is far from adequate to prevent catastrophic consequences of amplified open-field pharm crop production.
The main plant-based platforms for production of biological drugs or vaccines are the production of pharmaceutical proteins and vaccines in seeds produced in open fields, and/or transient expression systems in contained environments or bioreactor plant cell cultures. Some examples are given below.
Seed-based production of biological drugs
Seed-based production of pharmaceutical drugs is achieved by linking the promoters and signal sequences for seed proteins with the gene for the pharmaceutical protein. The resulting human or animal pharmaceutical protein or vaccine is usually stable for several years if maintained in a cool dry environment. The most commonly employed seed crops include rice, maize and safflower.
Boothe and colleagues of SemBioSys and Calgary University stated: “Physical containment strategies may be impractical or at least uneconomical for most seed-based systems.” Biological containment such as male sterility or “organelle expression” could be used, but for many species, physical segregation may be the only practical method available. In other words, pharm crops are to be grown in open fields, but at distances sufficient to isolate the plants from any food production areas and from any close relatives that could cross with the pharm crop and spread the gene. They suggested that the use of largely or completely self-pollinating species could provide an additional safeguard against transgene escape. And as the most likely cause of food supply adulteration is through the inadvertent mixing of seed from pharm crop and food crop of the same species, companies engaged in pharm crop production must maintain a tight chain-of-custody over their transgenic seed. For all these reasons, Booth and colleagues think “it is highly unlikely” that pharm crops will ever be approved for unconfined release.
Nevertheless, SemBioSys has begun the final stages of a clinical trial for human insulin produced in safflower.
Little or no mention has been made about the impact of pharm crops on wildlife by those intending grow the crops in open fields. Human insulin-producing crops were even tested in areas with threatened animal species (see GM safflower with human pro-insulin, SiS 35). DNA techniques have identified insulin-like peptide genes in invertebrates, including insects, molluscs and nematodes; these findings clearly establish that insulin is an evolutionarily ancient hormone present in all higher animals. Insulin toxic shock may appear in honey bees, birds and other treasured animals. However, the advocates of open field plant pharmaceutical production have not been obliged to fully evaluate the impact on wildlife, nor have they reported comprehensive ecological studies following open field tests.
Transgenic synthetic approximation of human insulin-like growth factor was produced at high levels in rice seeds and found to be active in rats. Insulin-like growth factor has cancer promoting properties making open field production highly hazardous (see Cancer promoting transgenic rice, SiS 22).
Another seed-based biological in the pipeline is for treatment of Alzheimer’s disease (AD). The main signs of AD are cognitive impairment and plaques composed of amyloid beta (Aβ) protein in patients’ brains. Therefore, therapy for AD focuses on the removal of Aβ. An “edible vaccine” was developed to boost intestinal immunity. Currently there are no published reports on the ecological and health impacts of the oral vaccine produced in open fields.
For the treatment of chronic allergic diseases such as bronchial asthma and rhinitis, biologicals are being used to induce oral tolerance to allergens such as those produced by house dust mites (HDM). HDM allergens are a major cause of such diseases. Oral tolerance is a T cell-mediated phenomenon involving a specific decrease in immune responses to antigens previously encountered via the oral route. Oral tolerance has been demonstrated in numerous species, including humans and mice, and can be achieved using many different antigens. The dust mite allergens are heat labile, acidic glycoproteins present in HDM faeces. To further increase the immunogenicity of this therapy, researchers utilize the native machinery of the host plants. Several plant seeds contain protein bodies (PBs), which function as sites of protein storage and are resistant to proteolytic digestion. Bioencapsulation of allergens into PBs is a strategy to increase the immunogenicity of allergens for oral immunotherapy. However, the inclusion of an antigen for which oral tolerance has already been established such as a previous treatment for rice allergy, may lead to inhibition of responses to other, unrelated antigens included in the immunization. This phenomenon is known as ‘bystander suppression’. The induction of bystander suppression has been demonstrated in multiple allergic diseases, but it has also been shown to enhance susceptibility to infection by inhibiting antibody production. The effect of HDM vaccine was antigen-specific because the levels of specific IgE and IgG in mice secondary immunized with unrelated antigens in transgenic rice seeds were not affected by oral vaccination with HDM rice. The results demonstrate the ability of HDM to induce oral tolerance without associated bystander suppression. The oral rice seed vaccine illustrates several of the problems involved in production of seeds for oral vaccination including the care with which oral tolerance must be considered in the event that the human population is exposed to GM seeds containing antigens which lead to immune suppression. Most dangerous of all is the use of a staple food crop for so many billions of people around the world.
Despite concerns of genetic contamination, maize seeds that produce heat labile E. coli enterotoxin (LT-B) in the seeds of maize have been approved for testing in the open field in Iowa. The National Environmental Policy Act evaluation of the field trial failed to take into account the fact that bees may spread transgenic pollen while birds and small mammals along with careless seed transportation could contaminate the human food chain with LT-B and certainly threaten the human food produced in the state of Iowa. Furthermore, there has been no report on whether or not the LT-B antigen produced in maize causes induction of bystander suppression frequently associated with vaccines subject to oral tolerance (see above).
There have been numerous other studies and field trials on seed based oral vaccines and the studies reported above are typical of such studies and field trials.
Transient expression systems
Transient expression systems are created in plant viruses with Agrobacterium. The transient expression methods are capable of producing higher levels of the recombinant proteins than permanent genetic modifications because many more copies of the recombinant genes are active in each plant cell. A combination of the transient expression methods is called magnifection. Magnifection involves using genetically modified plant virus vectors to transform Agrobacterium strains that are in turn allowed to infect the crop plant in a mild vacuum. The combined use of Agrobacterium and plant virus vectors results in a large number of recombinant RNA virus particles entering the plant cells. These recombinant RNA viruses epigenetically produce high levels of recombinant proteins in a few days, and then decline. The crop plants transfected by the modified viruses are used only once, producing no long term foot print on the environment, it is claimed. The process involves vacuum infiltration of whole plants with dilute suspensions of agrobacteria carrying T-DNAs encoding RNA replicons. The bacteria provide infection and systematic movement throughout the plant while the viral vector provides short distance spread, amplification and high level expression. The speed of the process is such that milligrams and grams of recombinant protein are available in 3 to 4 weeks, and as much as 100 kg within less than a year. Transient expression provides recombinant protein at up to 80 percent of the total cell protein. The process is relatively inexpensive due to the speed of production and high yield of recombinant protein that simplifies purification. The whole process may be contained in a greenhouse; a one hectare greenhouse is capable of producing 500 kg recombinant protein per year. Previous viral vectors require a thousand times more space. The speed at which large quantities of recombinant proteins can be produced means that recombinant proteins can be tailor-made for diseases of individuals (see Magnifection Safe Pharming or Doomsday Device, SiS 42).
However, it is imperative that the strictest containment must be applied to the use of these transient systems. The greenhouse must be closed, not just to insects and rodents, but also outfitted with air filters that prevent virus particles from escaping. Otherwise, it could create and spread catastrophic diseases of plants and animals. Indeed, some of the numerous transient systems being employed include serious human and animal pathogens. Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non-human primates. The US Centers for Disease Control classifies Ebola and Marburg viruses as ‘category A’ pathogens (defined as posing a risk to national security as bioterrorism agents). To generate a vaccine against it, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in a tobacco-related species, Nicotiana benthamiana. This EIC induces an immune response targeting the Ebola glycoprotein GP1 that mediates Ebola viral infection. The vaccine consists of the Ebola GP1 fused to the heavy chain of humanized monoclonal antibody that specifically binds to a linear epitope on GP1. This GP1-heavy chain fusion protein allows the individual immunoglobulins to bind to each other to form the Ebola immune-complex, which in-turn induces a high immune response when used as a vaccine. Subcutaneous immunization of mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. Large quantities of vaccine can be produced in the transient system in a short time.
To assess the quality of antibodies transiently expressed to high levels in plants, the human anti-HIV monoclonal antibody, 2G12, was expressed and characterized using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV) RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue) of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used (HT is a general enhancer of protein expression in plants), and the antibody was retained in the endoplasmic reticulum (ER). The non-replicating system is based on a disabled cowpea mosaic virus RNA-2, and high-level expression may be achieved using Agrobacterium-mediated transient transformation. This non-replicating system contains the virus but does not prevent the modified Agrobacterium from escaping into the environment. The HIV antibody produced in plants proved equivalent to the antibody produced in animal cells. Analysis by mass-spectrometry showed that the glycosylation pattern of the transgenic protein was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used.
To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable a-amylase) was evaluated in numerous species and cultivars of Nicotiana. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, making it the most effective plant host for recombinant protein production. Tobacco has proven to be the most effective plant for producing transiently expressed recombinant proteins.
The transient expression systems are proving attractive for their speed of production, relatively low cost of production and the absence of adverse environmental impact provided that strict containment is applied.
Production in Chloroplasts
Expressing transgenes in chloroplasts has caught the attention of researcher as chloroplasts are claimed to be transmitted by eggs alone and not spread to wild populations through pollen. Scientists are using this strategy to develop vaccines for illnesses including malaria and cervical cancer.
Genetically engineered starch granules for a malaria vaccine were produced containing plasmodial vaccine candidate antigens in the unicellular green algae Chlamydomonas reinhardtii. The C-terminal domains of the apical major antigen (AMA1) from Plasmodium berghei were fused to the algal granule bound starch synthase (GBSS).The peptides are efficiently expressed and bound within the chloroplast. Mice were either injected with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, the B-subunit of heat-labile enterotoxin (LTB) derived from E. coli, and then challenged intraperitoneally with a lethal inoculum of the malaria parasite, P. Berghei. Both experimental strategies led to a significantly reduced parasitemia (parasites circulating in the blood) with an extension of life span including complete cure for intraperitoneal delivery. In the case of the starch-bound vaccine, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly Plasmodium species.
Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. There are also serious doubts over efficacy and safety (The HPV Vaccine Controversy, SiS 41) yet to be addressed. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as a vaccine candidate. Capsomeres form the outer surface of a viral particle. Furthermore, coupling of an adjuvant-like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co-administration of adjuvants. Two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein were expressed in tobacco chloroplasts. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2 percent of total soluble protein. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, the plants reached maturity and produced seeds by pollination with wild-type plants. Production of pharmaceuticals in chloroplasts has been promoted as a way to limit the spread of transgenic pharm crops, however. As this study shows, the transformation of the chloroplast may create toxic side effects on the transformed plant. In spite of the toxic side effects, the tobacco plants produced significant quantities of vaccine peptides.
We have refuted the dogma that proclaims chloroplast genetic modification offers fool proof containment of transgenes. Many plant species transmit chloroplasts through pollen alone while others transmit chloroplasts through both pollen and egg. Even strictly maternal chloroplast transmitters will transmit the chloroplast through pollen under stressful conditions (see Molecular Pharming by Chloroplast Transformation, SiS 27).
Between the years 1991 and 2011 there have been 101 open field trials of crops modified with genes for pharmaceutical proteins in the United States alone. Modified maize was released in 51 of the trials, modified rice in 21 of the trials, modified tobacco in 18 trials, modified safflower in 3 trials, tomato was modified in 1 trial, rapeseed was modified in 1 trial, alfalfa in 1 trial and the Tobacco mosaic virus (TMV) in 6 trials while the Tobacco etch virus (TEV) was modified in 1 trial. The management of the environmental impact of the most of the modified crops cannot be independently evaluated in most releases because the information is withheld as trade secrets. However, in the case of those crops with which environmental impact studies were reported, the safety requirements employed appeared to be very primitive and in most cases ineffective. Immediate efforts should be made to evaluate the level of contamination of the United States maize and rice crops with the genes and proteins from the carelessly managed field trials.
The production of pharmaceuticals appears to be most safely done using transient expression systems contained in greenhouses. But they need to be virus proofed, as well as insects and rodent-proof. The seed based production of pharmaceuticals can be safely undertaken in greenhouses which prevent release of both pollen and seeds (and hence must be rodent and insect proof), but not at all in the open fields. The use of primary food and feed crops such as maize and rice in open fields is pure folly. Furthermore, the horizontal transfer of the genes for pharmaceutical products deserves much full attention (GM DNA Does Jump Species, SiS 47).