Recombinant proteins in plants have problems and prospects. Dr. Ramanjini Gowda delivered a guest lecture at Tamil Nadu Agricultural University on recombinant proteins in plants. Low expression levels, challenges with post-translational modifications, difficulty conducting animal and human studies, and formulation of vaccines are some limitations of producing recombinant proteins in plants. However, plants allow for cost-effective mass production and some proteins have already been expressed successfully in plants.

1. Recombinant Proteins in Plants
Problems and Prospects
Dr.P.H.Ramanjini Gowda
Professor and Co ordinator
Department of Biotechnology
UAS.GKVK
Bangalore 560065
Guest lecture delivered at Tamil Nadu Agricultural University, Coimbatore on 6.2.2013

2. Why Do We Need Vaccines?
Currently, we have vaccines for TWENTY-SIX different
infectious diseases.
The average American child receives about ten different
vaccinations before the age of 2!
Many different diseases are rarely seen anymore; in some cases
the diseases have disappeared completely!
Vaccinations save countless people all over the world from
severe and even fatal diseases.

3. Before
the 1700’s 1796 1970’s 1994 Today
The Chinese Edward Smallpox Polio was Research
developed Jenner was era- eradicated. is being
the first developed dicated. done all
form of the over the
vaccines - smallpox world in
variolation. vaccine order to
from improve
cowpox. vaccines.

4. Costs
Vaccine production is not at all
efficient for mass production.
http://whyfiles.org/166plant_vaccines/2.html
Their use in many parts of the world
is limited because of the high costs.

5. “Combining a cost-effective production
system with a safe and efficacious
delivery system, edible vaccines
provide a compelling solution.”
– Plants and Human Health: Delivery of Vaccines Via
Transgenic Plants (2003)

6. What exactly are
“edible vaccines?”
• Biopharmaceuticals
• Plants or crops that produce human
vaccines
• The next generation of vaccines

7. Biopharming
• Biopharming is the crop based production of
industrial or therapeutic biomolecules.
• Vaccines are the therapeutic biomolecules.
• Plants are amenable for large scale biomass
production.
• Plants have good system of post
translational modification of proteins.

8. Contd….
• Complex multemeric proteins can be
produced in plants.
• The engineered edible vaccine can be
consunmed orally without alteration.
• The cost of vaccine is cheap since it needs
no purification.
• Needs no refrigeration.

9. Growing plants is much cheaper
The plants that produce the
than producing vaccines.
edible vaccines could be
grown in third world
countries. Targeted expression in plant storage
tissues provides stability and
Advantages
accumulation
Plants are already regularly
Agricultural used in pharmaceuticals, so
products there are established
can be purification protocols.
transported
around the Plants can’t host most
world human pathogens, so the
relatively vaccines won’t pose
cheaply. dangers to humans.

10. Limitations
• Low expression levels.
• Glycosilation and post transcriptional
modifications.
• Animal and human studies are difficult.
• Formulations of the vaccines.

11. For the last decade, scientists have known how to genetically engineer a plant
to produce a desired protein. The two most common tools used to do this are:
Cut out the selected Infect the plant with
region of the plasmid. the agrobacteria and
DNA is coated on
grow it in a medium. microscopically tiny gold
Agrobacteria have a circular beads that are placed in a
form of DNA called plasmids. vacuum chamber. The
The plasmids are easily gene gun then allows
manipulated because they compressed gas to expand,
pushing the beads down
naturally have two “cut” Grow the plant like
Add the desired gene. until they hit a filter. The
points where a gene can beregular crop.
a
DNA then flies off of the
taken out and replaced with beads down into the tissue,
one of the scientist’s choice. where some will enter a
nucleus and become
incorporated.

13. Plant-derived Vaccine Strategies
Gene encoding an antigenic protein from a pathogen.
Incorporate into a plant transformation vector for optimized
expression in plant cells.
Stable expression: Stable expression:
Nuclear genome Chloroplast genome
integration. integration.
Integrate into a viral coding sequence for expression as a
“by product” of viral replication.
Transient expression: Modify viral genome to
Infect plant to initiate adapt it into a plant
viral replication. transformation vector for
subsequent regulated
release as a replicon in
transgenic plants.
Identify protective Create viral replicon coding
epitope within sequence with epitope
antigenic protein. fusions to coat protein.

14. Choice of the Plant System
• Plant product should be eaten raw.
• The plant is amenable for regeneration.
• Should be rich source of protein.
• Fast growing
• Should grow under tough weather
conditions.
• Banana,cantaloupes,Peanut,Papaya.

15. Antigen Expression in Plants
The cumulative number of antigens from pathogens of humans and/or
animals which have been expressed in plants, based upon published reports
(original compilation of the reports was detailed in Khalsa G, Mason H, Arntzen C. Plant-
derived vaccines: progress and constraints. In: R Fischer and S Schillberg (eds.) Molecular
Farming: Plant-made Pharmaceuticals and Technical Proteins. John Wiley and Sons, In
press in 2005).

16. Pharmaceutical Production in Plants
Genetically modified plants have been used as “bioreactors” to
produce therapeutic proteins for more than a decade. A recent
contribution by transgenic plants is the generation of edible
vaccines.
Edible vaccines are vaccines produced in plants that can be
administered directly through the ingestion of plant materials
containing the vaccine. Eating the plant would then confer immunity
against diseases.
Edible vaccines produced by transgenic plants are
attractive for many reasons. The cost associated
with the production of the vaccine is low,
especially since the vaccine can be ingested
directly, and vaccine production can be rapidly up
scaled should the need arises. Edible vaccine is
likely to reach more individuals in developing
countries.
The first human clinical trial took place in 1997.
Vaccine against the toxin from the bacteria E.coli
was produced in potato. Ingestion of this
transgenic potato resulted in satisfactory
vaccinations and no adverse effects.

17. Edible Vaccines
One focus of current vaccine effort is on hepatitis B, a virus responsible for
causing chromic liver disease. Transgenic tobacco and potatoes were engineered
to express hepatitis B virus vaccine. During the past two years, vaccines against
a E.coli toxin, the respiratory syncytial virus, measles virus, and the Norwalk
virus have been successfully expressed in plants and delivered orally. These
studies have supported the potential of edible vaccines as preventive agents of
many diseases.
There is hope to produce edible vaccines in bananas, which are grown extensively
throughout the developing world.
Vol. 19, No. 3 Feb.
1, 1999

18. Transgenic Plants; Nuclear
Transformation

19. Why use this technology?
Familiar Production Systems
• Genes introduced into field crops
• New productions systems not needed
• Producer can use traditional growing strategies
Reduced End-Product Cost
• Animal system: $1000 - $5000 per gram protein
• Plant System: $1 - $10 per gram protein

20. Edible Vaccines – A Biopharming Dream
Biotech Plants Serving Human Health Needs
• A pathogen protein gene is cloned
• Gene is inserted into the DNA of plant (potato, banana, tomato)
• Humans eat the plant
• The body produces antibodies against pathogen protein
• Human are “immunized” against the pathogen
• Examples:
Diarrhea
Hepatitis B
Measles

21. Future Health-related Biotech Products
Vaccines
 Herpes
 hepatitis C
 AIDS
 malaria
Tooth decay
 Streptococcus mutans, the mouth bacteria
 releases lactic acid that destroys enamel
 engineered Streptococcus mutans
does not release lactic acid
destroys the tooth decay strain

36. Rabies Neutralizing antibody titers(IU/ml)inmice immunized with plant extracts
Plant Extract and route Neutralizing antibody titer on Key words
Day 30 Day60
Plant 1 IM 0.4 0.8 IM=Intramuscular
IM+FA 0.7 1.2 IP=Intraperitoneal
IP 0.6 1.0 FA=Compleate
Plant 2 IM 0.5 1.0 Freaunds adjuvant
IM+FA 1.0 1.5 ND=Not detected
IP 0.8 1.2 IU=International units
Plant 3 IM 0.7 1.2
IM+FA 1.2 1.6
IP 0.8 1.5
Control Plant IM ND ND
IM+FA ND ND
IP ND ND

37. Protein expression in crop
plants
 Earlier we have expressed ERA strain of rabies glycoprotein
gene in Tobacco and Muskmelon and also obtained an Indian
patent. The ERA strain obtained from Thomas Jefferson
University has a patent on this gene. Hence, we have designed
our own gene construct.
 The CVS glycoprotein gene will be subcloned to plant expression
vector (pPS1)
 The pPS1 vector containing CaMV 35S promoter with the rabies
glycoprotein gene will be transferred to Agrobacterium strain
EHA105 and will be used for developing transgenic crop plants
expressing Rabies Glycoprotein.

40. Alternative means of pilot production
Production facility    bioreactor    refined product

41. Plants as bioreactors for
pharmaceutical proteins
PRESENT STATUS
• Useful human proteins produced in plants
– Human antibodies & other blood proteins
– Protein and peptide hormones
– Enzymes
– Subunit vaccines
• Proteins from plants are in the clinical pipeline
– Human antibodies
– Subunit vaccines
– Enzymes
• Regulatory Environment is evolving

42. Plants as bioreactors for
pharmaceutical proteins
FUTURE APPLICATIONS
• Clinical unmet needs in cancer, infectious
disease, cardiovascular disease, CNS
disease, metabolic disorders, inflammatory
disease, biowarfare agents
• Options for injectable, oral and topical
application
• Treatment and prevention modalities

43. What is the challenge?
• Developing drugs to treat human disease;
protein based drugs are the fastest growing
class of new drugs for treatment and
prevention of human disease. But we face
these barriers:
• Capacity:
– Insufficient capacity for drugs in the pipeline
• Cost
– Cost of goods
– Capital for manufacturing facilities
• Safety and Efficacy

44. Advantages of plants as
bioreactors
• Plants are the most efficient producers of
proteins on earth
– Plants are scalable bioreactors
– Plants provide cost advantages
• Plants cells are similar to human cells
– Similar protein synthesis machinery
– Read the same genetic code
– Assemble, fold and secrete complex proteins

45. Antibodies:
A Compelling Success Story
• Inherently stable human proteins
• High specificity; low toxicity
• High drug approval rates
• Injectable, topical and oral
applications
• Appropriate for chronic conditions
• Potential long-lasting benefits

46. Antibodies: Natural Defense
• Circulating antibodies protect us from
invading viruses, bacteria and toxins
• Secretory antibodies protect our vulnerable
surfaces from pathogens and toxins,
preventing entry and colonization
• Passive antibodies in colostrum and milk
provide passive immunity to neonates and
infants
• We make ~3g of antibodies a day
• Like most animals, we surrender most of
our antibodies to the environment

47. Antibodies: Natural Defense
ORAL/GASTROINTESTINAL NASAL GENITO-URINARY
TRACT PULMONARY TRACT
TRACT

48. Emerging Antibody
Opportunities
Therapeutic areas requiring high
quantities of antibodies, and low cost
– Inflammatory diseases
– CNS diseases
– Cardiovascular diseases
– Infectious diseases
_ topical applications

49. Plant-produced Antibodies work
• Anti-Streptococcus mutans (Guy’s 13)
– Prevents dental caries in humans
– Plant sIgA 10X more stable than IgG
• Nature Medicine 1998
• Anti-Herpes simplex virus (HSV8)
– Prevents vaginal transmission of herpes
– Proved in mice with rice and soybean PAb’s
• Nature Biotechnology 1998

50. Plants Produce Assembled
Antibodies
Site of production: corn
endosperm (starch and
protein)
Nature 342:76-78 (1989)
Science 268: 716-9 (1995)
Nature Biotechnology: 16:1361-
1364 (1998)

51. Comparison of Plant and
Mammalian Derived Antibodies
• Peptide sequence: identical
• Affinity: identical
• Antibody types: Plant system more
versatile
– Can make any isotype including secretory
IgA
• Post-translational processing: different
– core glycan identical, terminal sugar
different
– antigenicity & clearance: apparently
identical

52. A Plant-produced antibody is
scheduled for clinical development
• Clinical trials planned to begin in 2003
• Clinical importance: herpes simplex virus
– Over 50 million chronic sufferers
– Over 1.5 million new cases/year in U.S.
• Antibodies provide promising application in
both prevention and treatment
• Plant-produced antibodies are ideal
– High quantities required
– Scalable and have lower costs than traditional
production

53. Capacity Shortage
60000
50000
40000
Kg of MAb
30000
20000
10000
0
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Mammalian Cell Culture Protein Capacity in Kg
Optimistic MAb Demand (Dain Rauscher '00)
Realistic MAb Demand (CSFB '01)

54. Processing Comparison
*After harvest, the seeds can be stored indefinitely; therefore, when the protein is
needed, the purification process can begin immediately.
Source: Cline, M.,”Plant-Made Pharmaceuticals: Overview of Technology and
Stewardship,” Fifth Biotechnology Roundtable, American Bar Association, St. Louis,
May 2003.

55. Plant-derived pharmaceuticals have a full load of technology.
Can the load be moved to benefit public health?
• Finalize regulatory regime through commercialization
• Currently pilot scale; commercial scale-up required
• Secure public acceptance of technology

56. First Pant Made Drug on the
Market
• US FDA approved drug produced in
carrots
• The drug Taliglucerase alfa produced for
the rare lysosomal storage disorder
(Gaucher disease).
• Israeli Biotech Protalix Biotherapeutics
developed the method.
• This drug can replace the avialble drug
Cerezyme.

57. THANK YOU