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Friday, March 27, 2015
Co-sponsored by the ASU Biodesign Institute and the Arizona Farm Bureau
Charles J. Arntzen
[email protected]
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”.
August 4, 2014 – Bloomberg News broke the story in the U.S.
Ebola Drug Made From Tobacco
Plant Saves U.S. Aid Workers
August 4, 2014 7:52pm MT
Two American Missionaries
received ZMapp – A “cocktail”
of three anti-ebola antibodies.
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.
Zmapp worked
quickly and
dramatically.
The media was fascinated by ZMapp
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 desig ned 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 μg). A version with typical heterogenous mammalian glycoforms (ED50 = 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED50 = 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactu
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?
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.
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.
1st Generation Human Clinical Trials
(The “Edible Vaccine” Concept; Vaccine in Food)
Milli-International units (anti-HBsAg)
• Pre-clinical studies with mice
• Volunteers eat diced, raw potato expressing HBsAg
Weeks: 0
HBsAg boosting trial
Average mean IgG
serum antibody titres
for all volunteers
1
2
3
4
5
6
7
Virus-based pharmaceutical production – the key
innovation after 2005
1. Convert RNA virus to DNA sequence, and reengineer 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.
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?”
Back to back papers published in 2011:
December 20, 2011
• Mapp’s antibody protection
• ASU’s vaccine protection
Both with a mouse model of disease
Animal studies conducted with US AMRIID
PNAS 2011 108 (51) 20690-20694
PNAS 2011 108 (51) 20695-20700
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.)
Host plant
(tobacco)
engineering for
human
glycoslyation
Collaboratio
n among
groups was
essential
Antibody
engineering
(creating mAbs
with human
primary structure)
What was
missing?
Scale-up
manufacturing
expertise.
Accelerating Critical Therapeutics
By 2009, the US Military (DARPA) had
recognized that plant-made
Angel Project
pharmaceutical production was limited The Blue
Col. Alan McGill, MD.
by manufacturing capacity.
Project Mngr.
Nine member
They invested over $80 Million in “bricks
Scientific Advisory
and mortar” in three U.S. companies to
Board
build non-redundant capacity.
How to make monoclonal antibodies (mAbs) at industrial scale
Genes encoding light and heavy chains
“Redesigned” plant virus
Infect plants
Viral “infection” (mAb
production) for several
days; then harvest.
Purify
mAbs
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 Mapp’s anti-Ebola
mAbs.
Dr. Anthony Fauci, Director of NIH
Treatment could be given
up to 5 days after initial
Ebola infection
WSJ January 16, 2015