How Anti-Ebola Drugs Are Produced In Plants….And Why

Ebola Virus (CC BY 2.0) by NIAID

Ebola Virus (CC BY 2.0) by NIAID
Scanning electron micrograph of Ebola virus budding from the surface of a Vero cell (African green monkey kidney epithelial cell line).

“On Thursday, Dr. Kent Brantly thought he was going to die.
It was the ninth day since the American missionary worker came down sick with Ebola in Liberia.
His condition worsening by the minute, Brantly called his wife to say goodbye.
Thankfully, the call was premature.
Brantly is back on his feet — literally — after receiving a last-ditch, highly experimental drug.”
(from Ref. 1 below)

This drug is called ZMappTM.

…having received one dose of ZMapp out of the required three doses, and a blood transfusion from a fourteen-year-old boy who had recovered from Ebola, Kent Brantly walked onto the evacuation plane. At Emory University Hospital, in Atlanta, he received two more doses of ZMapp, which had been sent from the tobacco facility in Kentucky, and was discharged from the hospital after two weeks, free of the virus.” (from Ref. 2 below)

Yes, the ZMappTM that likely saved Kent Brantly’s life was produced in tobacco plants. (see Ref. 3 below)

In the many stories about ZMappTM and the Ebola virus I’ve read in the past few weeks, I’ve never seen a good explanation about how ZMappTM is made in tobacco, and why.

So, what follows, dear reader, is MY take on HOW this drug is manufactured in plants, and WHY.

But first, I think it’s important to find out a bit more about ZMappTM.

What The Heck Is ZMappTM ?

According to the manufacturer of this drug:
ZMappTM is not a serum or serum derived product. It is composed of three monoclonal antibodies directed against the Ebola Zaire virus strain. The component monoclonal antibodies were licensed from Defyrus (Toronto) and USAMRIID, humanized and recombinantly manufactured in a variety of tobacco (Nicotiana benthamiana).” (from: ZMappTM FAQ (PDF) from Mapp Biopharmaceutical)

My interpretation of the above:

  • ZMappTM is not derived from animal or human blood. Simply put, it is produced not by animals, but by plants.
  • ZMappTM = 3 antibody proteins that very specifically bind to the surface of a particular strain of the Ebola virus.
  • These antibodies were first produced (and licensed) by Defyrus and USAMRIID (see “Cast of Characters” below)
  • The monoclonal antibodies were “humanized”. Briefly, this means that the antibody genes (typically developed using animals such as mice) are modified to produce proteins that resemble human antibodies so that they won’t induce a severe allergic reaction when administered to patients.
  • Once so modified, these humanized monoclonal antibody genes are inserted – via recombinant DNA techniques – into a genetic vector. Millions of copies of this vector are then introduced into living tobacco plants.
  • In theory, how does injecting these monoclonal antibodies to Ebola virus into people sick with Ebola work? Briefly, giving a person antibodies against a particular pathogen is an example of passive immunization. This is in contrast to “active immunity”, which is when people produce their own antibodies when exposed to a pathogen or are vaccinated against a pathogen.

    Does ZMappTM work?
    We don’t know. The ZMappTM combination of antibodies was identified in January 2014. As an experimental product only limited supplies were manufactured for testing in animals. ZMappTM has shown efficacy in a monkey model of Ebola in studies conducted by the Public Health Agency of Canada (submitted for publication). Available data in humans are extremely limited. Larger trials are necessary to determine whether ZMappTM is safe and effective.
    ” (from: ZMappTM FAQ (PDF) from Mapp Biopharmaceutical)

    (By the way, you can also read about ZMappTM at Wikipedia.)

    How Is ZMappTM Made In Tobacco Plants?

    First, a bit of background on plant genetic engineering:

    Back in the day, when I was boring undergraduates, I’d tell students that, because plant and animal cells are likely derived from common eukaryotic ancestors, they have many cellular and metabolic processes in common.

    The molecular machinery of plant cells is closely related to that of animal cells. Thus, plant cells are able to synthesize and process relatively complex biologics. Indeed, GE [Genetically Engineered] plants have been shown to be able to produce human antibodies (immunoglobulins). Such so-called “plantibodies” appear to have the same biochemical properties as antibodies produced in animal cell cultures.” (from “Plant Trek”)

    The first proof of concept for functional antibody production in plants was provided in 1989, when two transgenic tobacco plants, each expressing light or heavy chains, were produced by Agrobacterium-mediated transformation of tobacco leaf discs. Crossing these two transgenic tobacco lines led to the expression of assembled functional IgG antibodies, accumulating up to 1.3% of total soluble protein. From then on, numerous antibodies and other proteins have been expressed in plants, demonstrating that plants can express and assemble components into functional, complex multimeric proteins.” (from Ref. 4 below)

    According to ZMappTM FAQ (PDF) from Mapp Biopharmaceutical: “Nicotiana [tobacco] seeds are planted in flats carried on mechanized roller trays similar to those used in large warehouses. The plants are grown for several weeks with careful control of light, temperature, and humidity. The plants are treated with the antibody vector system and grown for an additional week allowing the plants to manufacture humanized antibody proteins. The plants are harvested and homogenized in a large vat followed by separation of the antibody proteins using a series of specialized purification techniques. The resultant antibody is tested for purity and potency before being formulated into the drug.

    By the way, a series of photos of the process at Icon Genetics (see “Cast of Characters” below) can be viewed at at Zimbio.

    Why Is ZMappTM Made In Tobacco Plants?

    Why plants?

    1. “Plant expression systems are an attractive platform for the production of antibodies, for several reasons. Predominantly due to the possibility of production scale-up at a fraction of the costs compared to conventional systems.
    2. “Another advantage is that many plant species have a ‘generally regarded as safe’ status, since they do not contain mammalian viruses or pathogens, or produce endotoxins.
    3. “The ease of purification and downstream processing of plant-made antibodies is often postulated to result in a low cost of the final product, which can be applied parenterally, topically or orally.
    4. “Moreover, the developments in glyco-engineering of plants has made it possible to produce antibodies with desired glycoforms. Modification of glycans has also been perfected in comparative expression systems like mammalian cell cultures, but it has been seen that glyco-engineered plants have a much higher degree of glycan homogeneity.” (all of the above from Ref. 4 below)

    Why tobacco plants?

    Then there is the merit of speed: using the established state of the art tobacco leaf-based transient expression system [see Ref. 5 below, for example], bulk quantities of antibodies can be manufactured in a record time as compared to any other established expression system.” (from Ref. 4 below)

    In summary, the time it takes to grow and to genetically modify tobacco plants is much faster, and way less expensive, than to genetically engineer mice, for example. And plant cells also seem to produce higher quality antibodies, with less complicating factors, than animal cells.

    Cast of Characters:

    Mapp Biopharmaceutical (San Diego, CA, USA) “…was founded in 2003 to develop novel pharmaceuticals for the prevention and treatment of infectious diseases, focusing on unmet needs in global health and biodefense.”

    LeafBio, Inc. (San Diego, CA, USA) is the commercialization arm of Mapp Biopharmaceutical.

    Defyrus Inc. (Toronto, Canada) is “…a private life sciences biodefence company that collaborates with public health agencies and military R&D partners in the United States, United Kingdom and Canada to develop and sell broad spectrum anti-viral drugs and vaccine system as medical countermeasures to bioterrorist threats and emerging infectious diseases.”

    Icon Genetics (Halle, Germany) – pioneered genetic vectors for engineering tobacco (genus Nicotiana) plants to produce biopharmaceuticals.

    Kentucky Bioprocessing (Owensboro, KY, USA) – specializes in the good manufacturing practices (GMP) production of therapeutic proteins in Nicotiana.

    U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) (Frederick, MD, USA) is the “...the Department of Defense’s (DoD) lead laboratory for medical biological defense research. While our core mission is to protect the warfighter from biological threats, we also investigate disease outbreaks and threats to public health.”

    For Further Reading:

    A Possible Ebola Vaccine

    WHO plans for millions of doses of Ebola vaccine by 2015

    1/22/2015 Fast Track on Drug for Ebola Has Faltered

    12/31/2015 Special Report: Ebola’s thin harvest

    2/25/2016 The Ebola outbreak is over, but the hunt for a vaccine continues

    References

    1. Gupta, S. and D. Dellorto (2014) “Experimental drug likely saved Ebola patients.” CNN, August 5, 2014, online. (Full Text)

    2. Preston, R. (2014) “The Ebola Wars: How genomics research can help contain the outbreak.” The New Yorker, October 27, 2014 issue, online.
    (Full Text)

    3. Qui, X., et al. (2014) “Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.” Nature, doi:10.1038/nature13777. (Full Text)

    4. Virdi, V. and A. Depicker (2013) “Role of plant expression systems in antibody production for passive immunization.” The International Journal of Developmental Biology, Vol. 57, pp.587-593. (Full Text PDF)

    5. Sainsbury Laboratory (2012) “High Efficiency Transient Expression System for Plants – Methods to highly express proteins in plants in days not months.” www.pbltechnology.com (Full Text PDF).

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