Molecular Farming, Antigene, Edible Vaccine, Industrial Enzymes

1. Plant molecular farming for
recombinant therapeutic proteins
Ph.D. (Genetics & Plant Breeding)
K. Satish

2. 2
Outlines
 Introduction
 General strategy in Molecular farming
 Different production systems
 Applications
 Case studies
 Biosafety issues
 Conclusions
 Future thrust

3. 3
Introduction
 Plant molecular farming is the production of pharmaceutically
important and commercially valuable diagnostic proteins and/or
industrial enzymes in plants.
 Combination of biotechnology and agriculture to produce new
biomolecules for the benefits of human being
 It is also known as biopharming or gene pharming.
 Molecular farming started about 20 years ago with the promise to
produce therapeutic molecules.
 Some therapeutic molecules are very expensive to produce.
 Falls under the category of green biotechnology.
Sahu et al., 2014

4. 4
Molecular Farming Milestones
1989- First
plant made
antibody
IgG1 in
tobacco
1986- First
plant derived
protein
rhGH in
tobacco
1992- HBsAg
in tobacco
1995- Oral
vaccine
potato
1998- First clinical
trail of plant derived
pharmaceutical
protein
2003-
Commercialization
Trypsin- Maize
1997-
Commercialization
Avidin - Maize
2000- HGH
produced in tobacco
chloroplasts
2006- Plant – made
vaccine approved by
USDA
2006- First
Commercialized
plant-made Antibody
2012- Plant – made
vaccine approved for
human use
Brief history of molecular farming
Fischer et al. 2013

5. 5
General strategy in molecular farming
Sahu et al., 2014

6. 6
Why plants for Molecular farming?
 Plants had the potential to produce complex mammalian proteins
of medical importance.
 Low cost of production.
 Stability – storage by freezing or drying leaves.
 Safety - free from animal and human virus.
 Arabidopsis thaliana is used as model plant.
 Easily reproducible in consumable form viz. cereals, leafy crops,
fruits and vegetables.
Sahu et al., 2014

7. SPECIES BENEFITS LIMITATION
MODEL PLANT :
Arabidopsis thaliana
 Mutant available
 Small genome
 Accessible genetics
 Low biomass
LEAFY CROPS:
Tobacco
 High biomass
 Rapid scale up
 Non-food/ feed crop
 Low protein stability
 Presence of toxic
alkaloids
Alfalfa
 High biomass yield
 Harvested up to six times/yr.
 Fixes the atmospheric N2 .
 Low protein stability
Lettuce  Can take as edible part  Low protein stability
CEREAL SEED :
Maize, Rice, Wheat
 High protein stability during
storage.
 High yield.
 Easy transformation and
scalability.
 Difficulty in downstream
processes
Plants most often used
Sahu et al., 2014

8. LEGUME SEED:
Pea and Soybean
 High protein stability
during storage.
 High yield.
 Easy transformation and
scalability.
 Fixes the atmospheric N2 .
 Difficulty in downstream
processes
FRUITS AND
VEGETABLES:
Potato
 Edible
 Bulk antibody production
 High biomass yield
 Cooking or boiling degrade
the protein
Tomato
 High biomass yield
 Edible
 Greenhouse require
 Low protein stability
Banana
 Edible
 Less costly
 Low protein stability
FIBER AND
OIL-SEED CROPS:
Flax, Cotton,
Safflower
 Oleosin fusion protein  Fiber and oil can interfere
with downstream processing
Sahu et al., 2014

9. 9
Comparison of expression systems
Expression
system
Yeast Bacteria Plant cell
culture
Transgenic
plants
Transgenic
animal
Animal
cell
culture
Cost of
maintaining
Inexpensive Inexpensive Inexpensive Inexpensive Expensive Expensive
Type of
storage (ͦ C)
-2.0 -2.0 -2.0 RT N2 N/A
Gene(protein)
size
Unknown Unknown Limited Unlimited Limited limited
Production
cost
Medium Medium Low Low High High
Protein yield High Medium Very high High Medium High
Ramalingam et al., 2014Coimbatore

10. 10
Different plant based expression systems
1. Stable nuclear transformation
 Most common
 Used in a species with a long generation cycle
 Foreign genes are transfer via Agrobacterium tumefaciens or
particle bombardment
 Genes are taken up and incorporated in a stable manner
 Large acres can be utilized with the lowest cost- grains
 Long-term non-refrigerated storage of the seed up to 2 yrs
 Manual labor required
 Lower yield and out-crossing
Obembe et al.,2011

11. 11
2. Plastid transformation
 First described by Svab et al. (1990)
 No transgenic pollen is generated
 Very high expression levels can be achieved
 Protein – up to 70% on dry weight but relatively stable
 No out-crossing
 Protein unstable
 Extraction and purification at specific time
 Edible vaccine is not feasible since tobacco is highly regulated
Scotti et al., 2010

12. 12
3. Transient transformation
 Recombinant plant viruses to infect host plants, like TMV,
CaMV, PVX
 Agroinfiltration through recombinant A. tumefaciens
 Small amounts target protein is obtained in weeks
 Infection process is rapid
 Protein accumulate in the interstitial spaces
 Target protein is temporary express in the plant
 No long term storage due to tissue damage
 No stable transgenic plants are generated
 Low scalability and expression levels
Komarova et al., 2010

13. Transient transformation
13
Komarova et al., 2010

14. 4. Stable transformation for hydroponics
 Transgenic plants are grown on hydroponic medium
 Desired products are released as part of root fluid into a
hydroponic medium
 Plants are contained in greenhouse
 Easier purification but expensive to operate
 Not suitable for large scale production
14

15. Downstream processing and recovery
15

16. 16
Applications
Parental therapeutics
and pharmaceutical
intermediates
Industrial proteins and
enzymes
Monoclonal antibodies
Biopolymers
Antigens for edible
vaccines

17. 17
Monoclonal antibody (mAb)
 Antibody that is produced by genetically engineered Plant i.e.
insertion of antibodies into a transgenic plant; referred to as
plantibody
 Biolex (North Carolina) is the trademark for Monoclonal antibody
 No risk of spreading diseases to humans
 Hiatt. et al (1989): First time demonstrate the production of
antibodies in tobacco as therapeutic protein and plant protection
against diseases
 Daniel (2002) was reported that due to the lack of glycosylation,
chloroplast transformation is ideal for single chain fragment(scFv)
 Agrofiltration is ideal for transient expression of heavy and light
chain genes
 All current therapeutic antibodies are of the IgG class
 Purification is done through processes such as filtration,
immunofluorescence, and chromatography

18. Plant Cell
Transformation
Agrobacterium Mediated Transfer
of Antibody Genes
Direct Gene Transfer by Gene
Gun
Plant Development and propagation in
fields
Insertion of Transformed Cell into
Plant tissue
Harvested and downstream processing
Method of antibody production
18

19. Antibodies from transgenic plants
Plant Antibody type Purpose References
Tobacco IgG Catalytic antibodies Hiatt et al., 1989
Tobacco IgG-colon cancer Systemic injection Verch et al., 1998
Alfalfa IgG-human Diarrhea Diagnostic Khoudi et al., 1999
Tobacco IgG-rabies virus Anti rabies virus Ko et al., 2003
Tobacco IgG-hepatitis B virus hepatitis B surface antigen Yano et al., 2004
Tobacco IgG-Anthrax Monoclonal antibody Hull et al.,2005
Tobacco IgG-rabies virus Human anti rabies virus Girard et al., 2006
Tobacco BoNT antidotes Botulinum neurotoxins
(BoNTs)
Almquist et al., 2006
Tobacco mAb 2F5 Activity against
HIV-1
Sack et al., 2007
Tobacco LO-BM2, IgG Therapeutic De Muynck et al.,
2009
Tobacco mAb H10 Tumour-associated
antigen tenascin-C
Villani et al., 2009
Obembe et al., 201119

20. 20
Production costs for antibodies
Production cost Cost in $ per gram
Hybridomas 1000
Transgenic animals 100
Transgenic plants 10
Daniell et al., 2001
E. coli & yeast Tr. animals and
animal cells
Transgenic plants

21. 21
Edible vaccines
 A vaccine developed by engineering a gene for an antigenic protein
into a plant.
 The concept of edible vaccine got incentive after Arntzen et al.
(1992) expressed hepatitis B antigen in tobacco.
 Expressed in the edible portion like tubers, fruits etc.
 Due to ingestion, it releases the protein and get recognized by the
immune system.
 Stimulate both humoral and mucosal immunity.
 It is feasible to administer unlike injection.
 Heat stable - no need of refrigeration.
Kumar et al., 2013

22. 22
Method for the production edible vaccine
Figure : Edible vaccine production methods

23. 23
Plant-derived oral vaccines
Chan et al., 2015
Pathogen Antigen Plant References
Major capsid protein VP6 Potato Langridge et al.
(2003)
Hepatitis B virus Surface antigen Potato Youm et al. (2010)
Human immunodeficiency
virus (HIV-1)
p24-Nef Tobacco Gonzalez-Rabade et
al. (2011)
HIV-1 C4(V3)6 multi-
epitopic protein
Lettuce Govea-Alonso et al.
(2013)
Human papillomavirus (HPV) HPV16-L1 Tobacco Liu et al. (2013)
HPV HPVL1-E6/E7 Tomato Monroy-Garcıa et al.
(2014)
Influenza virus H3N2 nucleoprotein Maize seeds Nahampun et al.
(2015)
Vibrio cholerae CTB Rice seeds Tokuhara et al. (2010)
Rabies virus G protein Tomato hairy
roots
Singh et al. (2015)

24. 24
Outline

25. 25
Industrial enzymes
Seon et al., 2002
Enzyme Plants Applications
α- Amylase Tobacco Food processing
Avidin Corn / maize In diagnostic kits
Cellulase Brassica Production of alcohol from
cellulose
β- glucanase Brassica In brewing industry
β- glucuronidase Brassica In diagnostic kits
Lignin peroxidase Wheat In paper manufacture
Phytase Rice Improved phosphate
utilization
Trypsin Maize Pharmaceutical
Xylanase Brassica Biomass processing, paper
and textile industries

26. 26
Industrial products close to market
Product Company Uses References
Trypsin ProdiGene pharmaceutical Woodard et al., 2003
GUS ProdiGene Reagent for
diagnostics
Kusnadi et al.,1998
Avidin ProdiGene Immunological
reagent
Hood et al.,1997
Aprotinin Large scale Biology Wound closure Zhong et al.,1999
Collagen ProdiGene,
Medicago
Gel cap Ruggiero et al., 2000
Lipase Meristem
therapeutics
Exocrine pancreatic
insufficiency
Gruber et al.,2001
Lactoferrin Ventria Natural defense Samyn-petit et al.,
2001
TGEV edible
vaccine
ProdiGene Swine Lamphear et al., 2002

27. 27
Case Studies

28. Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm
•Cholera is an extremely epidemic diarrheal disease, which continues to devastate
many developing countries.
•Synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized
codon usage of plant genes,
•Introduced into a plant expression vector and expressed under the control of the
Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing
an intron of the rice act1.
•The recombinant vector was transformed into rice plants using a biolisticmediated
transformation method.
Hungary Oszvald et al., 200828

29. • The synthetic CTB gene (sCTB) fragment was removed from
pMYO114 via digestion with KpnI and NcoI. It was inserted into the
plant expression vector, pMYN317, under the control of the Bx17
endosperm specific promoter with the first intron of rice act1 and
the terminator of nopaline synthase (NOS) gene.
Construction of Plant Expression Vector
• The plant expression vector was transformed into rice cells (Oryza
sativa L.) via a biolistic-mediated transformation method
Plant Transformation
• The presence of synthetic CTB was verified via PCR analysis. The
primer pairs were sCT1-F and sCT5-dk-R.
Detection of sCTB Gene in Transgenic Plants
• Total RNA was extracted from the mature rice seeds of transgenic
plants harboring the sCTB gene and wild-type plants.
• The blots were hybridized with a 32P-labeled random-primed.
Northern Blot Analysis
• The membranes were incubated for 2 h with 1:7,000 dilutions of
anti-rabbit IgG conjugated with alkaline phosphatase (Promega
S3731) in TBST buffer.
Immunoblot Detection of CTB Protein in Transformed Rice
Seeds
Quantification of CTB Protein Level in Transgenic Rice Seeds
GM1-Binding Assay29

30.  Synthetic CTB gene (sCTB) fused with SEKDEL was under the control of Bx17
HMW endosperm-specific promoter (HMW-Bx17-p) with the first intron of rice act1
gene (Act1-i).
Hygromycin phosphotransferase gene (hpt) as a selection marker gene is under the
control of Agrobacterium tumefaciens nopaline synthase gene promoter (NOS-p) and
terminator (NOS-t).
BSP is bacterial signal peptide from enterotoxigenic E-coli heat-labile enterotoxin B
subunit (LTB)
Plant expression vector
30

31. Fig: PCR and Northern blot analysis of transgenic rice plants
(A)PCR analysis of transgenic and wild-type plants was conducted to amplify the sCTB
gene. Lane PC is plant expression vector used as a positive control for PCR; lane
WT is wild type plant used as a negative control; lanes 1–7 are PCR products
amplified from the DNA templates of independent transgenic lines.
(B) Northern blot analysis of transgenic and wild-type plants using a 32P-labeled sCTB
probe. Lane WT is wild-type plant as a negative control; lanes 1–7 are transgenic
lines
PCR and Northern blot analysis
31

32. Western blot analysis of CTB protein expressed in the endosperm of transgenic rice
plants. Total soluble protein extracts (15 µg) from the endosperm of wild type (WT)
and transgenic plants along with 80 ng of purified bacterial CTB protein were separated
on 12% SDS-PAGE.
Denatured proteins in B were boiled for 10 min prior to loading on the gel. Arrows
indicate monomer or pentamer of plant-produced CTB
Western blot analysis
32

33. (A)The CTB expression level (% of TSP) in the mature endosperm of transgenic rice
plants #2, 5, and 6 showing high expression levels of CTB transcripts in Northern blot
analysis was determined by ELISA in triplicate.
(B) GM1 binding assay of plant-produced CTB proteins. The GM1 -ELISA was conducted
with coated GM1 ganglioside as receptor molecules or BSA (bovine serum albumin)
as a negative control.
ELISA quantification
33

34. 34
Production of highly concentrated, heat stable hepatitis B surface
antigen in maize
Celine A. Hayden1, Erin M. Egelkrout1, Alessa M. Moscoso1, Cristina Enrique1, Todd K.
Keener1, Rafael Jimenez-Flores2, Jeffrey C. Wong3, and John A. Howard1
Plant Biotechnology Journal. 2012 October ;
10(8): 979–984.
Hayden et al., 2012California
• Over 350 million people are chronically infected with the hepatitis B virus
worldwide.
• HBsAg is a membrane-bound protein, a class of proteins that are typically
difficult to express in heterologous systems, so they have used a new construct.
• These DNA constructs demonstrate improved accumulation of HBsAg over
previously reported material and deliver maize grain suitable for oral
vaccination that is cost effective, heat stable, and highly concentrated.

35. 35
Construct design
Construct design for the production of HBsAg in Zea mays. glb1, 1.4kb
globulin1 promoter; 3kbglb1, extended globulin1 promoter; 3xglb1,
tandemly repeated extended globulin1 promoter; BAASS, barley alpha
amylase signal sequence; Vac, Vacuolar targeting sequence; HB,
hepatitis B surface antigen; PinII, potato proteinase inhibitor II
termination sequence. All constructs also contained an herbicide
resistance gene following the PinII termination sequence.
Hayden et al., 2012

36. 36
Single Seed Desent
method
Enzyme Linked Immuno
Sorbent Assay (ELISA)
Comparison
Construct Total Soluble
Protein (%)
Construct target site
HBE
(Standard)
0.12
Cell wall targetting signal
HBF 0.31
Cell wall targetting signal
HBG 0.41
HBJ 0.51
HBK 0.15 Vacuolar targetting signal
T- DNA
with construct
A. tumifaciens
with T- DNA Hi II Maize
Germplasm
Non – transgenic
Hi II Parent
Backcross
Hayden et al., 2012

37. 37
HBsAg accumulation in single seeds
from the first generation
Hayden et al., 2012
0.12%
0.31%
0.41%
0.51%
0. 15% 0.05%
0.17%
0.27% 0.26%
HBsAg concentration in second
generation (T2) ears with highest
antigen accumulation, as
determined by ELISA
Antigen detection by ELISA

38. 38
Effect of maize processing and temperature treatments
Total soluble protein and HBsAg protein content in HBsAg maize seed
stored at −20°C, 55°C, and 80°C for one week.
Total soluble protein (mg
protein/g maize material ±
S.D)
HBsAg (μg antigen/g maize
material ± S.D)
−20°C 55°C 80°C −20°C 55°C 80°C
Full fat 21.7 ± 1.7 21.1 ±
0.3
2.9 ± 0.6 55.8±5.5 27.8±12.2 <0.1
Hexane-
treated
20.2 ± 0.4 19.6 ±
0.2
12.4 ±
4.4
51.2±3.7 41.1±5.0 0.3±0.6
SFE-treated 20.4 ± 1.5 19.5 ±
0.7
14.9 ±
5.1
45.9±4.4 48.5±4.4 1.7±1.7
Hayden et al., 2012
Effect of oil extraction and
temperature on maize-
produced HBsAg, as
determined by immunoblot.

39. 39
A- Full Fat:
High level of lipids
B- Hexane treated :
Medium level of lipids
C- SFE treated :
Low level of lipids
Confocal microscopy :Presence of protein(fast Green) and
lipids(Nile Red)
Hayden et al., 2012

40.  Avian influenza is one of the most dangerous diseases to domestic
poultry. Mass vaccination of domestic and wild birds is the best
method for preventing its spread.
 Antigenic variation of this virus hinders vaccine development.
 Extracellular domain of the virus-encoded M2 protein (peptide M2e)
is nearly invariant in all influenza A strains, enabling the
development of a broad-range vaccine against them.
Aleksey et al., 2015Jerusalem, Israel
High-Yield Expression of M2e Peptide of Avian Influenza
Virus H5N1 in Transgenic Duckweed Plants
Aleksey Firsov • Irina Tarasenko • Tatiana Mitiouchkina •
Natalya Ismailova • Lyubov Shaloiko •
Alexander Vainstein • Sergey Dolgov
40

41. • plasmid (pBIM130) was transferred into Agrobacterium
tumefaciens CBE21 ->used for transformation of
duckweed pBI121 in the translational fusion upstream
of the β-Glucuronidase gene
Construction of
the
Transformation
Vector
• The calluses were used for agrobacterial
transformation
Agrobacterial
Transformation
of Duckweed
• The activity of β-Glucuronidase in duckweed was
analyzed
using the histochemical method
GUS-
Expression
Assays
• PCR analysis of putatively transgenic plants was
performed
using primers M130F and uidAlowR
PCR Analysis
• Duckweed genomic DNA (50µg) + 100U EcoRI + 37 C,
Overnight
• pBI121 and digested with EcoRI and HindIII was used
as a positive control
Southern Blot
Analysis
• Total proteins (25 µg) from each transgenic line were
separated by 12 % SDS-PAGE and transferred onto an
NC membrane + ELISA Quantification of M130–β-
Glucuronidase accumulation.
Western Blot
Analysis
41

42. Fig. Schematic depiction of the expression cassette of plasmid pBIM130.
A. Nucleotide sequence of the DNA fragment encoding the peptide M130.
B. Expression cassette obtained after cloning the M130-encoding sequence
into plasmid pBI121
42

43. A: Frond regeneration from kanamycin-resistant callus after 10 weeks of growth
on NPM regeneration medium.
B–F: X-Gluc staining of nontransformed control and kanamycin-resistant
duckweed plants.
B & C: Transgenic lines 16 and 54, respectively, with high GUS expression.
D & E: Transgenic lines 19 and 34, respectively, with moderate GUS expression.
F: Nontransformed duckweed plants.
G: Transgenic duckweed plants (line 54) growing on LHFM medium with

44.  PCR analysis
A: K- nontransformed plant, K+ DNA of plasmid
pBIM130. The expected length of the amplified
fragment was 1024 bp. Numbers denote
independent transgenic lines.
B: Southern blot analysis of transgenic duckweed
lines. 1 transgenic duckweed line 54, 2
nontransformed plant, 3 duckweed plants
transformed with pBI121, 4 transgenic
duckweed line 72, M molecular size marker
 Western blot analysis
Western blot analysis using
A: anti-β-Glucuronidase antibody and
B: anti-M2e antibody.
K- nontransformed duckweed plants,
gus β -Glucuronidase from E. coli (25
µg), M molecular size marker.
Numbers denote transgenic lines;
arrow indicates M130–β-
Glucuronidase fusion protein

45. Fig. Quantification of M130–β-glucuronidase fusion protein in
transgenic duckweed plants. K- nontransformed plants.
Numbers denote transgenic lines. Error bars indicate ± SD
45

46. There are two separate categories of risk.
1. Gene and protein pollution- Transgene and their encoded
proteins could spread in the environment and could be affect
non-target organisms. Ultimately, humans could be affected
by the consumption of food containing such genes or
recombinant proteins.
2. Product safety- Such pharmaceutical products and concerns
the risk that such products could be harmful in the human or
animal patients to which they are administered.
Biosafety issues in molecular farming
46

47. Vertical gene transfer
 Gene flow from transgenic to non-transgenic populations of the
same crop could occur by this method if the two populations were
close enough for wind- or insect mediated pollen transfer.
 In this case, transgene pollution would occur via seed dispersal,
either during growth, harvesting or during transport.
Horizontal gene transfer
 Risk that horizontal gene transfer from transgenic plants to
bacteria in the soil or in the digestive systems of herbivores could
yield new bacterial strains.
 These traits could have unpredictable effects on relationships
between different organisms.
47

48. Conclusion
 Plants are effective and efficient bioreactors for the production of
pharmaceutically valuable recombinant proteins. Variety of plant
species that are being explored to serve as green bioreactors, each
with its own advantages and disadvantages.
 Transgenic plant shows low production cost, high productivity, no
risk of contamination and easy storage compared to transgenic
animal. Thus it serves as an alternative to conventional
fermentation systems that use bacteria, yeast or mammalian cells.
 PMPs have already achieved preclinical validation in a range of
disease models like hepatitis B, rabies etc.
48

49.  Plant-derived pharmaceuticals will need to meet the same safety
and efficacy standards as those products obtained from non-
plant sources.
 We must ensure that the potential benefits are not outweighed
by risks to human health.
 Plant based recombinant therapeutics can neither commercially
succeed nor be accepted without addressing proper biosafety
and immunogenicity issues.
 Efforts are required to make this technology non-allergic and
free from side effects.
Future thrust
49