A Broad and Potent H1-Specific Human Monoclonal Antibody Produced in Plants Prevents Influenza Virus Infection and Transmission in Guinea Pigs.

Although seasonal influenza vaccines block most predominant influenza types and subtypes, humans still remain vulnerable to waves of seasonal and new potential pandemic influenza viruses for which no immunity may exist because of viral antigenic drift and/or shift. Previously, we described a human monoclonal antibody (hMAb), KPF1, which was produced in human embryonic kidney 293T cells (KPF1-HEK) with broad and potent neutralizing activity against H1N1 influenza A viruses (IAV) in vitro, and prophylactic and therapeutic activities in vivo. In this study, we produced hMAb KPF1 in tobacco plants (KPF1-Antx) and demonstrated how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian-produced KPF1-HEK hMAb. KPF1-Antx hMAb showed broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1) in vitro, which was comparable to that observed with KPF1-HEK hMAb. Importantly, prophylactic administration of KPF1-Antx hMAb to guinea pigs prevented pH1N1 infection and transmission in both prophylactic and therapeutic experiments, substantiating its clinical potential to prevent and treat H1N1 infections. Collectively, this study demonstrated, for the first time, a plant-produced influenza hMAb with in vitro and in vivo activity against influenza virus. Because of the many advantages of plant-produced hMAbs, such as rapid batch production, low cost, and the absence of mammalian cell products, they represent an alternative strategy for the production of immunotherapeutics for the treatment of influenza viral infections, including emerging seasonal and/or pandemic strains.

Plant-Made Antibodies: Properties and Therapeutic Applications.

BACKGROUND: A cost-effective plant platform for therapeutic monoclonal antibody production is both flexible and scalable. Plant cells have mechanisms for protein synthesis and posttranslational modification, including glycosylation, similar to those in animal cells. However, plants produce less complex and diverse Asn-attached glycans compared to animal cells and contain plant-specific residues. Nevertheless, plant-made antibodies (PMAbs) could be advantageous compared to those produced in animal cells due to the absence of a risk of contamination from nucleic acids or proteins of animal origin. OBJECTIVE: In this review, the various platforms of PMAbs production are described, and the widely used transient expression system based on Agrobacterium-mediated delivery of genetic material into plant cells is discussed in detail. RESULTS: We examined the features of and approaches to humanizing the Asn-linked glycan of PMAbs. The prospects for PMAbs in the prevention and treatment of human infectious diseases have been illustrated by promising results with PMAbs against human immunodeficiency virus, rotavirus infection, human respiratory syncytial virus, rabies, anthrax and Ebola virus. The pre-clinical and clinical trials of PMAbs against different types of cancer, including lymphoma and breast cancer, are addressed. CONCLUSION: PMAb biosafety assessments in patients suggest that it has no side effects, although this does not completely remove concerns about the potential immunogenicity of some plant glycans in humans. Several PMAbs at various developmental stages have been proposed. Promise for the clinical use of PMAbs is aimed at the treatment of viral and bacterial infections as well as in anti-cancer treatment.

Transient Production of Recombinant Pharmaceutical Proteins in Plants: Evolution and Perspectives.

During the last two decades, the production of pharmaceutical proteins in plants evolved from proof of concept to established technology adopted by several biotechnological companies. This progress is particularly based on intensive research starting stable genetic transformation and moving to transient expression. Due to its advantages in yield and speed of protein production transient expression platforms became the leading plant-based manufacturing technology. Current transient expression methods rely on Agrobacteriummediated delivery of expression vectors into plant cells. In recent years, great advances have been made in the improvement of expression vectors, host cell engineering as well as in the development of commercial manufacturing processes. Several GMP-certified large-scale production facilities exist around the world to utilize agroinfiltration method. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. The great potential of transient expression platform in respect to rapid response to emerging pandemics was demonstrated by the production of experimental ZMapp antibodies against Ebola virus as well as influenza vaccines. This review is focused on current design, status and future perspectives of plant transient expression system for the production of biopharmaceutical proteins.

Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail.

Ebola virus (EBOV) remains one of the most lethal transmissible infections and is responsible for high fatality rates and substantial morbidity during sporadic outbreaks. With increasing human incursions into endemic regions and the reported possibility of airborne transmission, EBOV is a high-priority public health threat for which no preventive or therapeutic options are currently available. Recent studies have demonstrated that cocktails of monoclonal antibodies are effective at preventing morbidity and mortality in nonhuman primates (NHPs) when administered as a post-exposure prophylactic within 1 or 2 days of challenge. To test whether one of these cocktails (MB-003) demonstrates efficacy as a therapeutic (after the onset of symptoms), we challenged NHPs with EBOV and initiated treatment upon confirmation of infection according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization and observation of a documented fever. Of the treated animals, 43% survived challenge, whereas both the controls and all historical controls with the same challenge stock succumbed to infection. These results represent successful therapy of EBOV infection in NHPs.

Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques.

Filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. Here, three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), we found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. In a pivotal follow-up experiment, we found significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful postexposure in vivo efficacy by a mAb mixture and suggest that this immunoprotectant should be further pursued as a postexposure and potential therapeutic for Ebola virus exposure.

Plant production of anti-ß-glucan antibodies for immunotherapy of fungal infections in humans.

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting ß-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the ß-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially ß1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

Secretory IgA antibodies from plants.

Secretory IgA (SIgA) is the antibody type produced in both mammals and birds that protects the body from infection at mucosal surfaces. While monoclonal IgG antibodies, particularly those against tumor antigens, have received a great deal of attention, both scientific and commercial, as immunotherapeutic agents, the potential of SIgA antibodies has only recently begun to be exploited. Part of the reason for this is that SIgA production in vivo normally requires the cooperation of two different cell types, and single animal cell systems for monoclonal SIgA production are inefficient. Transgenic plants are currently the most productive and economical system for making SIgA. The only monoclonal SIgA to be tested therapeutically in a human clinical trial is a product called CaroRx, made in transgenic tobacco, which is designed to block adherence to teeth of the bacteria that causes cavities. This antibody accumulates to high levels in the leaves of tobacco, where it is located primarily in the endoplasmic reticulum. The antibody can be efficiently purified using the affinity reagent protein G. Topical oral treatment in human subjects was safe and effective. Characterization of the expression, secretion, purification and therapeutic use of this antibody serves as a model for additional plant-made therapeutic SIgA antibodies under development.

Recent progress in plantibody technology.

Antibodies are an important class of proteins that can be used for the prevention, treatment and diagnosis of many diseases. Consequently, there is an intense and growing demand for recombinant antibodies, placing immense pressure on current production capacity which is based largely on microbial cultures and mammalian cells. Alternative systems for cost effective antibody production would be very welcome, and plants are now gaining widespread acceptance as green bioreactors with advantages in terms of cost, scalability and safety. Several plant-produced antibodies (plantibodies) are undergoing clinical trials and the first commercial approval could be only a few years away. The performance of the first generation of products has been very encouraging so far. In terms of product authenticity, differences in glycosylation between plantibodies and their mammalian counterparts have been defined, and the scientific evaluation of any possible consequences is underway. Ongoing studies are addressing the remaining biochemical constraints, and aim to further improve product yields, homogeneity and authenticity, particularly where the antibody is intended for injection into human patients. A remaining practical challenge is the implementation of large-scale production and processing under good manufacturing practice conditions that are yet to be endorsed by regulatory bodies. The current regulatory uncertainty and the associated costs represent an entry barrier for the pharmaceutical industry. However, the favourable properties of plants are likely to make the plant systems a useful alternative for small, medium and large scale production throughout the development of new antibody-based pharmaceuticals.

Clinical trial of a plant-derived antibody on recolonization of mutans streptococci.

OBJECTIVE: This double-blinded, placebo-controlled clinical trial tested the safety and efficacy of a topical secretory IgA antibody manufactured in tobacco plants (plantibody) in preventing recolonization of mutans streptococci (MS) in human plaque as measured by whole stimulated saliva samples. METHODS: Following a 9-day antimicrobial treatment with chlorhexidine (CHX), 56 eligible adults (enrollment salivary MS > or = 10(4) CFU/ml; no current caries) were randomized equally to a group receiving 0, 2, 4, or 6 topical applications of plantibody followed by 6, 4, 2, or 0 applications of placebo, respectively, over a 3-week period. RESULTS: Among the 54 subjects who completed the trial, the CHX regimen eliminated salivary MS in 69%. After 6 months, there were no significant differences in MS levels by number of applications, relative to placebo (p > 0.43). No adverse effects were observed. CONCLUSION: Plantibody is safe but not effective at the frequency, concentration, and number of applications used in this study.

Antibodies: an alternative for antibiotics?

In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases.