Plant molecular farming for recombinant therapeutic

proteins

Ph.D. (Genetics & Plant Breeding)

K. Satish

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Outlines

Introduction

General strategy in Molecular farming

Different production systems

Applications

Case studies

Biosafety issues

Conclusions

Future thrust

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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

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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 vaccinepotato

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

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General strategy in molecular farming

Sahu et al., 2014

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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

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

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

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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., 2014 Coimbatore

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Different plant based expression systems

1. Stable nuclear transformationMost commonUsed in a species with a long generation cycle Foreign genes are transfer via Agrobacterium tumefaciens or

particle bombardmentGenes are taken up and incorporated in a stable mannerLarge acres can be utilized with the lowest cost- grains Long-term non-refrigerated storage of the seed up to 2 yrsManual labor requiredLower yield and out-crossing

Obembe et al.,2011

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2. Plastid transformationFirst described by Svab et al. (1990) No transgenic pollen is generated Very high expression levels can be achievedProtein – up to 70% on dry weight but relatively stableNo out-crossing

Protein unstableExtraction and purification at specific timeEdible vaccine is not feasible since tobacco is highly regulated

Scotti et al., 2010

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3. Transient transformationRecombinant plant viruses to infect host plants, like TMV,

CaMV, PVXAgroinfiltration through recombinant A. tumefaciensSmall amounts target protein is obtained in weeksInfection process is rapidProtein accumulate in the interstitial spaces

Target protein is temporary express in the plantNo long term storage due to tissue damageNo stable transgenic plants are generatedLow scalability and expression levels

Komarova et al., 2010

Transient transformation

13 Komarova et al., 2010

4. Stable transformation for hydroponicsTransgenic 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

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Downstream processing and recovery

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Applications

Parental therapeutics and pharmaceutical

intermediates

Industrial proteins and enzymes

Monoclonal antibodies

Biopolymers Antigens for edible

vaccines

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Monoclonal antibody (mAb)Antibody that is produced by genetically engineered Plant i.e. insertion

of antibodies into a transgenic plant; referred to as plantibodyBiolex (North Carolina) is the trademark for Monoclonal antibody No risk of spreading diseases to humansHiatt. et al (1989): First time demonstrate the production of antibodies

in tobacco as therapeutic protein and plant protection against diseasesDaniel (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 classPurification is done through processes such as filtration,

immunofluorescence, and chromatography

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

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Antibodies from transgenic plantsPlant Antibody type Purpose ReferencesTobacco 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 againstHIV-1

Sack et al., 2007

Tobacco LO-BM2, IgG Therapeutic De Muynck et al., 2009

Tobacco mAb H10 Tumour-associatedantigen tenascin-C

Villani et al., 2009

Obembe et al., 201119

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Production costs for antibodies

Production cost Cost in $ per gramHybridomas 1000Transgenic animals 100Transgenic plants 10

Daniell et al., 2001

E. coli & yeast Tr. animals andanimal cells

Transgenic plants

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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

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Method for the production edible vaccine

Figure : Edible vaccine production methods

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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)

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Outline

Candidates for edible vaccine

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Industrial enzymes

Seon et al., 2002

Enzyme Plants Applicationsα- 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

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Industrial products close to marketProduct 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

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Case Studies

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

• 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 SeedsGM1-Binding Assay29

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

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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

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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

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(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

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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.

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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

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Single Seed Desent method

Enzyme Linked Immuno Sorbent Assay (ELISA)

Comparison

Construct Total Soluble Protein (%)

Construct target site

HBE (Standard)

0.12Cell wall targetting signal

HBF 0.31

Cell wall targetting signalHBG 0.41

HBJ 0.51

HBK 0.15 Vacuolar targetting signal

T- DNA with construct

A. tumifaciens with T- DNA Hi II Maize

GermplasmNon – transgenic Hi II Parent

Backcross

Hayden et al., 2012

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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

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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.

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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

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

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•plasmid (pBIM130) was transferred into Agrobacteriumtumefaciens CBE21 ->used for transformation of duckweed pBI121 in the translational fusion upstream of the b-glucuronidase geneConstruction

of the Transformatio

n Vector

•The calluses were used for agrobacterial transformation

Agrobacterial Transformati

on of Duckweed

•The activity of b-glucuronidase in duckweed was analyzedusing the histochemical methodGUS-Expression

Assays

•PCR analysis of putatively transgenic plants was performedusing primers M130F and uidAlowR

PCR Analysis

•Duckweed genomic DNA (50ug) + 100U EcoRI + 37C, Overnight

•pBI121 and digested with EcoRI and HindIII was used as a positive control

Southern Blot Analysis

•Total proteins (25 lg) from each transgenic line were separated by 12 % SDS-PAGE and transferred onto an NC membrane + ELISA Quantification of M130–b-Glucuronidase accumulation.

Western Blot Analysis

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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

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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 kanamycin

PCR analysis

A: K- nontransformed plant, K+ DNA of plasmidpBIM130. 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

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

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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

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Vertical gene transferGene 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.

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ConclusionPlants 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.

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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

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EDIBLE VACCINE

PARENTERAL VACCINE

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