Recombinant Proteins in PlantsRecombinant Proteins in Plants Problems and Prospects Problems and Prospects

Dr.P.H.Ramanjini GowdaDr.P.H.Ramanjini Gowda Professor and Co ordinatorProfessor and Co ordinator

Department of BiotechnologyDepartment of Biotechnology UAS.GKVKUAS.GKVK Bangalore 560065Bangalore 560065

Guest lecture delivered at Tamil Nadu Agricultural University, Coimbatore on 6.2.2013

Why Do We Need Vaccines?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.

Why Do We Need Vaccines?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.

The Chinese developed the first form of vaccines - variolation.

Before

the 1700’s

Edward Jenner developed the smallpox vaccine from cowpox.

1796

Research is being done all over the world in order to improve vaccines.

Today

Smallpox was era-dicated.

1970’s

Polio was eradicated.

1994

Costs

Vaccine production is not at all efficient for mass production.

Their use in many parts of the world is limited because of the high costs.

http

://w

hyfi

les.

org/

166p

lant

_vac

cine

s/2.

htm

l

““Combining a cost-effective production Combining a cost-effective production system with a safe and efficacious system with a safe and efficacious delivery system, edible vaccines delivery system, edible vaccines provide a compelling solution.” provide a compelling solution.”

– – Plants and Human Health: Delivery of Vaccines Via Plants and Human Health: Delivery of Vaccines Via Transgenic Plants (2003)Transgenic Plants (2003)

What exactly are

“edible vaccines?”

• Biopharmaceuticals

• Plants or crops that produce human

vaccines

• The next generation of vaccines

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.

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.

AdvantagesAdvantages

The plants that produce the edible vaccines could be grown in third world countries.

Growing plants is much cheaper than producing vaccines.

Plants are already regularly used in pharmaceuticals, so there are established purification protocols.

Agricultural products can be transported around the world relatively cheaply.

Plants can’t host most human pathogens, so the vaccines won’t pose dangers to humans.

Targeted expression in plant storage tissues provides stability and accumulation

Limitations

• Low expression levels.

• Glycosilation and post transcriptional modifications.

• Animal and human studies are difficult.

• Formulations of the vaccines.

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:

Agrobacteria have a circular form of DNA called plasmids. The plasmids are easily manipulated because they naturally have two “cut” points where a gene can be taken out and replaced with one of the scientist’s choice.

DNA is coated on microscopically tiny gold beads that are placed in a vacuum chamber. The gene gun then allows compressed gas to expand, pushing the beads down until they hit a filter. The DNA then flies off of the beads down into the tissue, where some will enter a nucleus and become incorporated.

Cut out the selected region of the plasmid.

Add the desired gene. Grow the plant like a regular crop.

Infect the plant with the agrobacteria and grow it in a medium.

Plant-derived Vaccine Strategies

Gene encoding an antigenic protein from a pathogen.

Modify viral genome to adapt it into a plant transformation vector for subsequent regulated release as a replicon in transgenic plants.

Transient expression: Infect plant to initiate viral replication.

Identify protective epitope within antigenic protein.

Create viral replicon coding sequence with epitope fusions to coat protein.

Integrate into a viral coding sequence for expression as a “by product” of viral replication.

Stable expression: Chloroplast genome integration.

Stable expression: Nuclear genome integration.

Incorporate into a plant transformation vector for optimized expression in plant cells.

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.

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).

Pharmaceutical Production in PlantsGenetically 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.

Vol. 19, No. 3 Feb. 1, 1999

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.

Edible Vaccines

There is hope to produce edible vaccines in bananas, which are grown extensively throughout the developing world.

Transgenic Plants; Nuclear Transformation

Familiar Production Systems

Why use this technology?

• 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

Edible Vaccines – A Biopharming DreamBiotech 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:

DiarrheaHepatitis BMeasles

Tooth decay

Future Health-related Biotech Products

Vaccines Herpes hepatitis C AIDS malaria

Streptococcus mutans, the mouth bacteria releases lactic acid that destroys enamel engineered Streptococcus mutans does not release lactic acid destroys the tooth decay strain

Rabies Neutralizing antibody titers(IU/ml)inmice immunized with plant extracts

Plant Extract and route Neutralizing antibody titer onDay 30 Day60

Key words

Plant 1 IM IM+FA IP Plant 2 IM IM+FA IP Plant 3 IM IM+FA IP Control Plant IM IM+FA IP

0.4 0.80.7 1.20.6 1.00.5 1.01.0 1.50.8 1.20.7 1.21.2 1.60.8 1.5ND NDND NDND ND

IM=IntramuscularIP=IntraperitonealFA=Compleate Freaunds adjuvantND=Not detectedIU=International units

Protein expression in crop Protein expression in crop plantsplants

Earlier we have expressed ERA strain of rabies glycoprotein Earlier we have expressed ERA strain of rabies glycoprotein gene in Tobacco and Muskmelon and also obtained an Indian gene in Tobacco and Muskmelon and also obtained an Indian patent. The ERA strain obtained from Thomas Jefferson patent. The ERA strain obtained from Thomas Jefferson University has a patent on this gene. Hence, we have designed University has a patent on this gene. Hence, we have designed our own gene construct.our own gene construct.

The CVS glycoprotein gene will be subcloned to plant expression The CVS glycoprotein gene will be subcloned to plant expression vector (pPS1) vector (pPS1)

The pPS1 vector containing CaMV 35S promoter with the rabies The pPS1 vector containing CaMV 35S promoter with the rabies glycoprotein gene will be transferred to glycoprotein gene will be transferred to AgrobacteriumAgrobacterium strain strain EHA105 and will be used for developing transgenic crop plants EHA105 and will be used for developing transgenic crop plants expressing Rabies Glycoprotein.expressing Rabies Glycoprotein.

Alternative means of pilot production

Production facility bioreactor refined product

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

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

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

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

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

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

ORAL/GASTROINTESTINALORAL/GASTROINTESTINALTRACTTRACT

NASALNASALPULMONARYPULMONARYTRACTTRACT

GENITO-URINARYGENITO-URINARYTRACTTRACT

Antibodies: Natural Defense

Emerging Antibody Opportunities

Therapeutic areas requiring high quantities of antibodies, and low cost– Inflammatory diseases– CNS diseases– Cardiovascular diseases– Infectious diseases

_ topical applications

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

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)

• 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

Comparison of Plant and Mammalian Derived Antibodies

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

Capacity Shortage

0

10000

20000

30000

40000

50000

60000

2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Kg o

f M

Ab

Mammalian Cell Culture Protein Capacity in KgOptimistic MAb Demand (Dain Rauscher '00)Realistic MAb Demand (CSFB '01)

*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.

Processing Comparison

• Finalize regulatory regime through commercialization

• Currently pilot scale; commercial scale-up required

• Secure public acceptance of technology

Plant-derived pharmaceuticals have a full load of technology. Can the load be moved to benefit public health?

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.

THANK YOU