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Charles J. Arntzen charles.arntzen@asu.edu Co-sponsored by the ASU Biodesign Institute and the Arizona Farm Bureau Friday, March 27, 2015

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After the 9/11/2001 Terrorist Attack on the World Trade Center in New York, and the Pentagon, military funding of counterterrorism increased in the U.S. In 2002 the Army funded a project for Mapp and ASU. Title: Plant Production of Vaccines and Antibodies for Protection Against Biowarfare Agents. Co-Principle Investigators: C. Arntzen and L. Zeitlin Goal determine if viral expression vectors could be used for production of either subunit vaccines or monoclonal antibodies (mAbs) in tobacco. In our 2005 final report we said: we have demonstrated that Ebola virus-specific humanized monoclonal antibodies can be produced using plant biotechnology.

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The tiny company is Mapp BioPharmaceuticals a collaborator of ASU for the last 12 years. Larry Zeitlin (President) and Kevin Whaley (CEO) are adjunct ASU faculty via the Biodesign Institute. August 4, 2014 Bloomberg News broke the story in the U.S. Two American Missionaries received ZMapp A cocktail of three anti-ebola antibodies. Ebola Drug Made From Tobacco Plant Saves U.S. Aid Workers August 4, 2014 7:52pm MT

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Zmapp worked quickly and dramatically.

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No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED50 = 33 g). A version with typical heterogenous mammalian glycoforms (ED50 = 11 g) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED50 = 3 g). Binding studies using Fc receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics. The media was fascinated by ZMapp The stories were: How is ZMapp made? Why tobacco? When will more ZMapp be available? Who will pay for it? Who will decide who gets it?

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1992. Mason, H.D., M.-K. Lam and C. J. Arntzen. Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA 89:11745-749. Let me give you a personal history of the work leading up to Zmapp. This starts over 20 years ago with our work on Plant-Made Pharmaceuticals. Let me give you a personal history of the work leading up to Zmapp. This starts over 20 years ago with our work on Plant-Made Pharmaceuticals.

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Pre-clinical studies with mice Volunteers eat diced, raw potato expressing HBsAg Pre-clinical studies with mice Volunteers eat diced, raw potato expressing HBsAg Milli-International units (anti-HBsAg) Weeks: 0 1 2 3 4 5 6 7 HBsAg boosting trial Average mean IgG serum antibody titres for all volunteers 1st Generation Human Clinical Trials (The Edible Vaccine Concept; Vaccine in Food)

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Virus-based pharmaceutical production the key innovation after 2005 1.Convert RNA virus to DNA sequence, and re- engineer it to express new gene(s). 2.Inject genetic construct into cellular spaces within the leaf. 3.Allow the reconstructed virus to replicate for 4-10 days. 4.Extract and purify the protein drug.

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Let me return to how I became involved in Ebola therapy.. Transgenic plants; minimal processing, oral delivery Viral (transient) expression, GMP processing 2001 was a turning point for research funding in the U.S. Terrorism had become real. After the Sept. 11, 2011 attack on the World Trade Center, Bioterrorism became a stronger military interest. The U. S. Military asked: Do we have the technology to respond quickly? 2001 was a turning point for research funding in the U.S. Terrorism had become real. After the Sept. 11, 2011 attack on the World Trade Center, Bioterrorism became a stronger military interest. The U. S. Military asked: Do we have the technology to respond quickly?

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December 20, 2011 PNAS 2011 108 (51) 20690-20694 PNAS 2011 108 (51) 20695-20700 Back to back papers published in 2011: Mapps antibody protection ASUs vaccine protection Both with a mouse model of disease Animal studies conducted with US AMRIID Back to back papers published in 2011: Mapps antibody protection ASUs vaccine protection Both with a mouse model of disease Animal studies conducted with US AMRIID

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By 2009, there were multiple research centers providing key elements of the Ebola Antibody Project. Creation of a plant molecular toolbox (for engineering plant viruses, controlling protein production, etc.) Antibody engineering (creating mAbs with human primary structure) Host plant (tobacco) engineering for human glycoslyation Collaboratio n among groups was essential What was missing? Scale-up manufacturing expertise. What was missing? Scale-up manufacturing expertise.

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Accelerating Critical Therapeutics Col. Alan McGill, MD. Project Mngr. Nine member Scientific Advisory Board By 2009, the US Military (DARPA) had recognized that plant-made pharmaceutical production was limited by manufacturing capacity. They invested over $80 Million in bricks and mortar in three U.S. companies to build non-redundant capacity. By 2009, the US Military (DARPA) had recognized that plant-made pharmaceutical production was limited by manufacturing capacity. They invested over $80 Million in bricks and mortar in three U.S. companies to build non-redundant capacity. The Blue Angel Project

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Genes encoding light and heavy chains Infect plants Redesigned plant virus How to make monoclonal antibodies (mAbs) at industrial scale Viral infection (mAb production) for several days; then harvest. Purify mAbs

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In 2012-present, KBP has worked to optimize manufacture of anti-Ebola mAbs under cGMP. These were used in preclinical drug characterization. 18 monkeys were infected with high doses of Ebola virus. All survived when treated with Mapps anti-Ebola mAbs. Treatment could be given up to 5 days after initial Ebola infection Dr. Anthony Fauci, Director of NIH

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WSJ January 16, 2015