Plant-Made Pharmaceuticals and

Biomaterials:

Novel Industrial Processes & Products

Relevant to Developing Countries

Yuri Gleba Nomad Bioscience GmbH, Munich/Halle

IPBO Event, Gent, May 17, 2017

“Ultimately, the world will obtain most of its food, fuel, fiber, chemical feedstocks, and some of its pharmaceuticals from genetically altered vegetation and trees.”

P.H. Abelson, “A Third Technological Revolution”,

Science, 279:2019 (1998)

The Future of Plant Biotechnology

New Green Revolution!

• 2015 global area: 180 M Ha (>12% of total 1.5 B Ha), grew at double-digit rate for 19 consecutive years

• 2015 global market value of GM crops: over $15.7 B

• 2015 – 18 million farmers directly benefiting

• 28 countries grow, 90% in just five: USA, Argentina, Brazil, Canada and India, (but also 5 EU countries)

• Americans have transgenics in their diet for 20 years

• No ill effects on health or environment found

• Just four crops: soybean, corn, cotton, rape seed

• Just two traits: herbicide tolerance, insect resistance

• Just two companies making serious money on it ISAAA , 2014

New Green Revolution?

Source: R. Fraley (1994)

agronomic

traits

food

processing

pharmaceuticals

1995 2000 2005 2010

B$10.0

B$ 6.0

B$ 3.0

B$ 2.0

new

products

specialty

chemicals

Plant-Made Pharmaceuticals:Entered Market Phase

Several products advanced:

• Glucocerebrosidase/Protalix, approved

• Anti-caries Mab/Planet, approved

• Fabry Disease Therapy/Protalix, Phase I-II

• Anti-TNF Therapy/Protalix, Phase I

• Lactoferrin/Ventria, Phase I-II

• Anti-HIV Mab PharmaPlanta, Phase I

• NHL Vaccine/Icon-Bayer, Phase I

• Influenza Vaccine/Medicago, Phase I-II

• Influenza Vaccine/iBio, Phase I

• Anti-Ebola Mab, Mapp, Phase I-II

Financial support:

• Protalix, IPO, US$117 M cap, deal with Pfizer

• Icon/Nomad, over US$175 M invested by Bayer

• Icon/Nomad, US$85 M deal with Denka

• Medicago, deal with Philip Morris, US$357 M deal with Mitsubishi Tanabe Pharma

• DARPA program for PMP, over US$250 M

most recent

trials rely on

transient

production

technology!

SWOT Analysis

Animals Yeasts CHO cells Plants E. coli Plant cells

Technology Principle

• Van Larebeke N, Engler G, Holsters M, Van der Elsaker

S, Zaenen I, Schilperoort RA, Schell J, Large plasmid in

Agrobacterium tumefaciens essential for crown-gall-

inducing ability. Nature 252, 169 (1974)

• Van Larebeke N, Genetello C, Schell J, Schilperoort RA,

Hermans AK, Van Montagu M, Harnalsteens JP,

Acquisition of tumour-inducing ability by non-oncogenic

agrobacteria as a result of plasmid transfer. Nature, 255,

742 (1975)

Technology: Deconstructed Virus Delivered by Agrobacterium

viral vector:

magnICON®:

CPPOL MP

POL

agrodelivery:

UV

magnICON®: the Ultimate Yield, Speed

GFP

0

20

40

60

80

100

120

140

160

180

200

0 2 4 6 8 10 12

days post infiltration

flu

orescen

ce u

nits

pICH18711 leaf a

pICH18711 leaf b

pICH16707 leaf a

pICH16707 leaf b

state of the art magnifection

4-7 g protein per /kg of leaf biomass

or up to 80% TSP

4-7 days

TMV-Protein A nanoparticles

Werner et al., 2006

Yersinia antigens

Santi et al., 2006

Aprotinin

Bayer’s first Batch of a PMP:

100mg of purified Aprotinin from Tobacco

magnICON®: Extreme Expression Levels (up to 80% TSP or 7g/kg)

NHL patient IgG1

Bendandi et al., 2010

cGMP Facility in Halle,

Germany

Plant-based

GMP facilities:

Icon/Denka

KBP/R.J. Reynolds

Protalix

Medicago/M-Tanabe

Greenovation

Fraunhofer Aachen

Fraunhofer USA

Caliber/iBio

magnICON®: Large-Scale

Vacuum Infiltration

UV

KBP/Bayer Deal:

Pilot plant,

1.2 ton biomass/day

The ICON process is

fully scalable, requires

growing plants in trays

ideal for high-cost products,

e.g. biopharmaceuticals, vaccinesideal for low-cost products,

e.g. industrial enzymes,

agronomic traits

ideal for high-volume products,

e.g. biomaterials, antimicrobials

ethanol-inducible amplification

transfection using vacuum infiltration transfection using spraying

magnICON®

NOMADIC®

NOMADIC® Technology:

Choice of Production Platforms

magnICON®

transgenic

seed biopriming

ideal for agronomic traits

NOMADIC®

Cost of Enzyme, Capital

and Operating Expenses

SuperPro Designer® v.8.5 (Intelligen)

modeling software:

- process stimulation

- flowsheet development

- mass energy balances

- equipment sizes

- batch scheduling, etc.

Daniel et al., 2014

Green Biomass Versus Seed:

15-100X Better Acre Utilization

Corn seed

Seed promoter

3-6 t/ha seed

max. 15-30kg API/Ha

1st gram: 2 years

Tobacco leaf

CP promoter

100.-300. t/ha leaves

max. 500-1500kg API/Ha

1st gram: 7 days

But: 10X more biomass

to process

Plant-Made Medicines & Foods

• Novel antivirals for rapid response during

outbreaks (e.g. Ebola, Zica)

• Inexpensive plant-made vaccines

• Inexpensive ‘biobetter’ therapeutic

antibodies

• Non-caloric natural sweeteners to replace

sugar

• Natural non-antibiotic antibacterials

new product concepts, low cost and

manufacturing speed essential, greener

Mapp Biopharmaceutical:

Ebola Immunotherapy

Phase I-II studies completed

Mapp therapeutics pipeline:

Ebola virus

Marburg virus

Junin virus

17

new product concept

manufacturing speed essentialsame concept:

antibacterials

Ann Depicker’s group

Rituximab

(6,7 B $)

Trastuzumab

(5,5 B $)

Infliximab

(8,0 B $)

Icon Icon IconOriginator Originator Originator

“It ain’t bragging, if you’ve done it!” attributed to Walt Whitman

Biosimilars/Biobetters From Plants

existing product concept

essential improvements

lower cost of goods

Xyl-/Fuc- Rituximab: Removal of B-cells in Transgenic Mice

%

RNAi 7xKOwt

days

mice model – C57 BL6 containing human CD20 transgene;

mAb doze – 100mkg/day 0;

Flow cytometry measurement of circulating B-cells was

done at day 0; 1; 2 and 7 after treatment with mAb

anti-fucose

anti-xylose

• 22 kDa protein; 207 amino acids; 8 disulphide bonds;

not glycosylated; water soluble; resistant to heating;

stable under acidic pH; easily purified and crystallized

• Natural source: fruit arils of Thaumatococcus daniellii

• 100,000 times as sweet as sucrose on molar basis;

2000-3000 times sweeter than sucrose on w/w basis

• Introduced in early 70ies by Tate & Lyle

• Approved in USA, EU, Japan, etc., as sweetener,

flavour modifier

• Limited supply of natural plant substance (160 t/year

in 2016), price of bulk substance $250/kg

• Microbial production not competitive

• NOMAD’s proposition:

unlimited supply of recombinant Thaumatin;

significantly lower COGs; GRAS approval in USA in 2018

Thaumatin

One third of people are overweight,

0.5 Billion are obese,

primarely due to sugar consumption

leaf tissue (7dpi) extracted in

1.) 3vol 50mM Acetat, 150mM NaCL, pH 4

incubated at 60°C for 0, 5, 10, 20, 40 and 60min

in water bath, vortex before taking sample

2.) 3vol water pH 4

incubated at 60°C for 0, 10, 20, 40 and 60min

in water bath, centrifuge before taking sample

- crude extract mixed 1:2 with 2x Lämmli

- 10µl (corresponding to 1.7 mg leaf tissue )

loaded on 15% SDS gel

S: BSA-Standard (1mg/ml) in Lämmli + ME, 3µl

= 3µg

M NC 0‘ 10‘ 20‘ 40‘ 60‘ S M V

C

THM

2

extracted in water pH 4 Acetat

pH4

lane 10 and 11:

sample from Acetat extraction after 60min at 60°C

(from 16.10.2013, stored at 4°C)

V = vortex before taking sample

C = centrifuged before taking sample

CGE analysis

Calculated yield:

1mg THM / gram leaf tissue (partially purified)

Thaumatin-2: Expression Yield

and Purification by Heat

- 1.5 g thaumatin

per kg fresh leaf

- 30-50% tsp

- correct taste

Per capita Sugar 21 kg/y (34 kg in USA)

Thaumatin per capita 7-10 g/y or >0.05%

2016 global Sugar production: 171.000.000 ton;

Global Thaumatin: 80.000 ton or >0.05%

2016 sugar production area: 12.000.000 Ha

Global Thaumatin area needed: 570.000 Ha or 5%

Bacterial pathogens:

Campylobacter spp.*

Escherichia coli *

Listeria monocytogenes

Salmonella spp. *

Bacillus cereus

Clostridium spp.

Shigella spp.*

Vibrio spp.*

Staphylcoccus aureas

Enterococcus spp.

Yersinia spp.*

Viral pathogens:

Hepatitis A

SARS

Rotaviruses

Norovirus

newly emerging

viruses

Parasitic pathogens:

Nematodes

-Ascaris

-Trichinella

Platyhelmints

Protozoa

-CryptosporidiaNewell et al 2010

Foodborne Diseases

* Gram-negative bacteria

Rise and Fall of Antibiotics

• 1999 US study: the introduction of antibiotics in 1936 caused deaths in

the US to fall by 220 per 100,000 people within 15 years. All other

medical technologies combined over the next 45 years reduced deaths by

only 20 per 100,000 people

• The golden age of antibiotics took place in the 1930s to 1970s, with at

least 11 new classes discovered; since then, only two new classes of

antibiotics

• Antibiotic resistance potentially puts everyone else at higher risk

• Dame Sally Davies: "Antimicrobial resistance poses a catastrophic threat.

Any one of us could go into hospital in 20 years for minor surgery and die

because of an ordinary infection that can’t be treated byantibiotics.“

• About 25,000 patients a year die in the European Union from an

infection caused by an MDR bacterium – and on current trends this is

predicted to grow to 390,000 a year by 2050.

Foodborne Diseases Worldwide, 2010

Pathogen Illnesses Deaths

Norovirus 677 million 214 thousand

E. coli

ETEC

233 million 73 thousand

E. coli

EPEC

81 million 121 thousand

Shigella 188 million 64 thousand

Total 1800 million 600 thousand

Pires et al., 2015

‘The United States spends $500 million

per victim of terrorism, and piddling

$10,000 per cancer death.’

‘Food is a mortal menace. Every year,

one in six Americans gets sick, and 3,000

die from food-borne illness. Your burger

is a bigger threat than radical Islam.’

T. Egan, Intern. N. Y. Times, June 6-7, 2015

CDC USA: E. Coli Outbreaks

• 2016 0121 flour

• 2016 O157 alfalfa sprouts

• 2015 0157:H7 chicken salad

• 2014 0121 raw clover sprouts

• 2014 0157:H7 ground beef

• 2013 0157:H7 ready-to-eat-salads

• 2013 0121 frozen food products

• 2012 0157:H7 organic spinach

• 2012 O26 raw clover sprouts

• 2011 O157:H7 romaine lettuce

• 2011 0104 organic bean sprouts

• 2011 O157:H7 bologna

• 2011 O157:H7 in-shell hazelnuts

• 2010 0157:H7 cheeses

• 2010 O145 romaine lettuce

• 2010 O157:H7 beef

• 2009 O157:H7 beef

• 2009 O157:H7 beef

• 2009 O157:H7 cookie dough

• 2008 O157:H7 beef

• 2007 O157:H7 pizza

• 2007 O157:H7 beef

• 2006 O157:H7 fresh spinach

source: www.cdc.gov/ecoli/outbreaks.html26

Antibacterial Colicin Family

• Colicins are a class of bacteriocins –

antibiotic proteins- produced by and toxic

to some strains of Escherichia coli.

• Colicins are released to reduce

competition from other bacterial strains.

• Pore-forming colicins are trans-

membrane proteins that depolarize the

cytoplasmic membrane, leading to

dissipation of cellular energy.

• Colicins may also act as nuclease to

hydrolyze DNA or RNA of the target cell,

or they inhibit cell wall synthesis.

• Colicins have a 3 domain structural

design:

– N terminus Translocation domain (T)

– Receptor binding domain is at the center of

the peptide (R)

– C terminus Cytotoxic domain (C)

T

C

R

27

Plant Expression of All Known Colicins

12% SDS-PAGE and Coomassie staining, 7.5 µl of TSP extracts corr. to 1.5 mg FW

wt wt

95

72

55

43

34

26

95

72

55

43

34

26

* * * * * *

*

*

*

*

** *

* *

*

*

*

* *

*

all colicins (except colE5) well expressed

Expression range 1.5-8.3 g/kg or 13-50% TSP

in planta colicin

expression –I

(estimated)

in planta colicin

expression –II

(estimated)

%

TSP

Yield(mg colicin/

g FW)

% TSPYield(mg colicin/

g FW)

ColE2/ImmE2 20 2.3 17 2.6

ColE3 10 0.95 10 1.25

ColE5/ImmE5 - - - -

ColE6/ImmE6 38 4.37 25 4.0

ColE7/ImmE7 13 1.5 14 2.0

ColE8/ImmE8 13 1.6 10 1.6

ColE9/ImmE9 13 1.7 17 2.8

ColD/ImmD 7 1.0 10 1.5

DF13/ImmDF13 25 3.25 20 3.3

ColA 15 1.2 15 2.0

ColN 10 0.85 7 0.98

ColS4 20 2.8 19 2.9

ColK 20 2.9 50 7.8

Col5 25 2.75 50 8.3

Col10 20 2.8 38 6.7

ColU 25 2.9 25 3.5

ColR 25 2.37 25 3.3

Col28b 25 2.75 17 2.1

ColY 20 1.7 15 2.0

ColB 10 0.7 25 0.7

ColIa 25 2.13 20 3.9

ColIb 33 3.3 20 3.0

ColM 38 3.99 30 4.7

Colicin M causes a

drastic reduction of living

cells in broth culture for

all analyzed protein

concentration at all

analyzed timpoints!

E. coli O157:H7

3-5 log reduction

in CFU counts!

Colicins: E.coli Growth

Reduction

0

1

2

3

4

Effect of ColM on E. coli O157:H7 on Fresh Steak Meat

log

cfu

/g o

f m

ea

t

1h, 10°C 1d,

10°C

3d, 10°C

Comparison no/carrier treatment -0.073 -0.0248 -0,7

Comparison no/colicin treatment 2.3 2.7 2.6

Comparison carrier/colicin treatment: 2.3 3.0 3.26

Reduction of E. coli O157:H7 cells (Δlog)

30

First GRAS Regulatory Approval

for NOMAD’s Colicins in USA

• "GRAS" is an acronym for the phrase Generally

Recognized As Safe under sections 201(s) and 409 of

the U.S. Federal Food, Drug, and Cosmetic Act.

NOMAD’s R&D Pipeline, 2017

(Antimicrobials)

Antibacterial/antiviral Pathogen Product candidates

Colicins Escherichia coli EHEC Food additives/proc. aids

Salmocins Salmonella enteridis Food additives/proc. aids

Endolysins Listeria monocytogenes Food additives/proc. aids

Endolysins Clostridium perfringens Food additives/proc. aids

Endolysins Campylobacter jejunii Food additives/proc. aids

Pyocins Pseudomonas

aeruginosa

Pharmaceuticals

Endolysins Clostridium difficile Pharmaceuticals

Bacteriocins To be defined Oral Pharmaceuticals

Bacteriocins To be defined Oral Pharmaceuticals

Mabs, anti-Norovirus Norovirus Oral Pharmaceuticals

1977, CPU 1 Mhz, 4 KB memory

GM plant

Transgenic Versus Transient Process

Transiently modified plant

2017, 8.4 GHz CPU, 10 TB memory

2017, Flash drive, 512 GB memory

GM Agrobacterium

3 KB

Where Do We Go From Here?

“I offer a prediction: the early twenty-first century

is going to see a struggle between

information technology and biotechnology on the one hand

and environmental degradation on the other.

Biotechnology is going to be our most powerful tool.

It will let us miniaturize things, avoid waste, and produce

more value without producing and processing more stuff.

The substitution of information for stuff

is essential to sustainability.”

R. Shapiro, Monsanto, CEO,

1997 interview to ‘Harvard Business Reviews’