1. Guy A. Cardineau, Ph.D.
Higher Accumulation of
F1-V Recombinant Fusion
Protein in Plants After
Induction of Protein Body
Formation
Director, Centro de Agrobiotecnología
Departamento Agrobiotecnología y Agronegocios
Tecnológico de Monterrey, Campus Monterrey
ASASU Centennial Professor, Emeritus
Research Professor, Emeritus & Faculty Fellow
Center for Infectious Disease and Vaccinology
The Biodesign Institute,
The School of Life Sciences and
The Sandra Day O’Connor College of Law
Arizona State University
2. Biotechnology Drug Approvals 1982-2008
While the number of approved biotech-based products
approved per year is variable, the trend is upward.
Biotechnology drugs appear the fastest-growing sector for
drug development, and it is predicted that biotech drugs will
comprise over 50% of all drug approvals by 2015 and more
than 75% by 2025. These predictions are supported by the
expected benefits of increased understanding of drug targets
and the molecular and genetic bases of disease, as well as
the declining conventional small-molecule drug pipelines in
most major pharma companies. BioWorld Today Sept 1,2009
The table to the left represents information
from an article published in BioWorld Today in
late August 2009, written by Michael Harris,
2
late August 2009, written by Michael Harris,
Executive Editor, about the top 25 biotech
drugs currently on the market. The data
provided includes revenues for each of these
biotech drugs in 2008 (>$70B US), the date
each drug product was first approved by the
FDA and when patents protecting each drug
are due to expire. It should be kept in mind
that one feature of all these drugs is that they
have been approved for more than one
ndication; Harris reports that Genentech's
Avastin is being tested in more than 450
clinical trials for treating more than 30 different
types of cancer. It should also be kept in mind
that 7 of the 25 "biotech" drugs are small
molecules, and another 6 are antibodies.
3. Historically, Plants Have Been Routinely Used to
Produce Pharmaceuticals, Naturally
Global over-the-counter sales of plant-derived drugs are estimated
at $40 billion per year
Well established regulatory systems are in place for these products
Estimated one-quarter of the prescription drugs sold in the
US, Canada and Europe contain active ingredients derived from
plants
Tens of thousands of plants are used for medicinal purposes
Well established regulatory systems are in place for these products
Drug/Chemical Action/Clinical Use Plant Source
Cocaine Local anaesthetic Erythroxylum coca
Codeine Analgesic Papaver somniferum
Digitalin, Digitoxin Cardiotonic Digitalis purpurea
Quinine Antimalarial Cinchona ledgeriana
Taxol Antitumor agent Taxus brevifolia
Vinblastine, Vincristine Antitumor, Antileukemic Catharanthus roseus
SUMMARY from Large Scale Biology, Inc.
4. • Hormones and immune modulators
• Monoclonal antibodies - IgG
• Subunit vaccines
• Enzymes
Classes of New Protein Drug ProductsClasses of New Protein Drug Products
Production Systems in UseProduction Systems in UseProduction Systems in UseProduction Systems in Use
• Bacterial fermentation
• Mammalian cells in fermentation
• Yeast
• Insect cells (GSK’s cervical cancer vaccine; 2005/6)
• Green plants – Stable and Transient Transformation,
Whole Plants and Plant Cells
One approved product in the market in plant cells
• Bacterial fermentation
• Mammalian cells in fermentation
• Yeast
• Insect cells (GSK’s cervical cancer vaccine; 2005/6)
• Green plants – Stable and Transient Transformation,
Whole Plants and Plant Cells
One approved product in the market in plant cells
5. Early Patent Filings on
Plant Made
Pharmaceuticals
5
Original Concepts of
Therapeutic Protein,
Vaccine Antigen, and
Antibody Expression in
Plants
6. Dow AgroSciences/ASU collaboration
developed a Newcastles Disease Virus
subunit vaccine in tobacco NT1 cells.
United States Patent 7,132,291, Cardineau, et al., November 7, 2006 (Canadian counterpart CA 2524293)
Vectors and cells for preparing immunoprotective compositions derived from transgenic plants
Abstract
The invention is drawn towards vectors and methods useful for preparing genetically transformed plant cells that express
immunogens from pathogenic organisms which are used to produce immunoprotective particles useful in vaccine preparations. The
invention includes plant optimized genes that encode the HN protein of Newcastle Disease Virus. The invention also relates to
methods of producing an antigen in a transgenic plant.
7. WHY ORALLY DELIVERED
PLANT-MADE VACCINES?
Plant-derived vaccines are cost-effective and
stable at room temperature.
Plants provide both an encapsulated antigen
and an oral delivery system that stimulates
the mucosal immune system resulting in both
secretory and circulating antibodies.
The mucosal immune system is the first line
of defense against most pathogens.
Oral vaccines are potentially safer, require no
needles and may not require trained medical
personnel to administer.
Several Phase I Human Clinical Trials with
plant-made vaccines have been run resulting
in positive immune responses.
8. WHY INCREASE F1-V FUSION PROTEIN
ACCUMULATION IN PLANTS?
Our primary objective is to produce plant-derived heat
stable vaccines that can be delivered orally.
We have been using F1-V, a fusion between two
antigens from the plague bacterium Yersinia
pestis, as our model antigen in production
improvement studies.
pestis, as our model antigen in production
improvement studies.
We are assessing parameters that affect expression
of F1-V fusion protein in plants and plant cells to be
used as both a production and delivery system of
vaccines and potentially other biopharma proteins.
High antigen accumulation is required to compensate
for partial proteolysis in the gut upon oral delivery.
9. Protein accumulation in plant tissues reflects a
balance between protein synthesis and degradation
• To date, most efforts have focused on increasing protein
synthesis.
– enhanced transgene expression can be obtained by optimizing
regulatory elements including stronger promoters, transcriptional
enhancers, translational enhancers, alternative polyadenylation signals,
using synthetic genes with codons that have been optimized for gene
expression in target plants, overcoming RNAi and silencing
• Unfortunately, high transgene expression does not always
guarantee high levels of recombinant protein accumulation
since proteins may be expressed successfully but
subsequently degraded.
• It has been demonstrated that post-synthesis and/or post-
secretion instability and degradation are critical factors
contributing to low foreign protein yield.
10.
11. 25000
30000
35000
preboost
postboost
ANIMAL TRIALS: PRIME-BOOST STRATEGY
PRIME: s.c. 15 µg
bacterially derived
F1-V
BOOST: 2 g non-transgenic
tomato (n = 5) on days
BOOST: 2 g F1-V transgenic
tomato (n = 6) on days
21, 28, 35 (300 ug) and 42
(1200 ug)
[Ug/ml]
250
300
350
preboost
postboost
[Ug/ml]
0
5000
10000
15000
20000
25000
30000
35000
F1-specific IgG1 V -specific IgG1
preboost
postboost
[Ug/ml]
F1-specific IgG1 V-specific IgG1
0
50
100
150
200
250
300
350
F1-specific IgG2 V-specific IgG2
preboost
postboost
[Ug/ml]
F1-specific IgG1 V-specific IgG1
0
5000
10000
15000
20000
F1-specific IgG1 V -specific IgG1
Combined F1-V and V-specific IgG1 titers
correlate with protection in mouse model
(Williamson et. al., Clin. Exp.
Immunol., 1999, 116; 107-114.)
tomato (n = 5) on days
21, 28, 35 and 42)
F1-specific IgG1 V-specific IgG1
0
50
100
150
200
F1-specific IgG2 V-specific IgG2F1-specific IgG2a V-specific IgG2a
CHALLENGE (s.c. 20 LD50 Y. pestis)
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Days post-infection
%survival
TG
WT
CONTROLS
Challenge of the
vaccinated mice
with s.c. Y. pestis
Alvarez & Cardineau
Biotechnology Advances
2010, 28 (1): 184-196
%ofsurvival
Days post-infection
CHALLENGE (s.c. 20 LD50 Y. pestis)
13. Protein accumulation in plant tissues reflects a
balance between protein synthesis and degradation
• There are several possible sites and mechanisms of foreign
protein degradation in plants. Cytoplasmic proteases contribute
significantly to product losses within plant cells.
• Proteolytic degradation of foreign proteins can be minimized by
targeting synthesis to the endoplasmic reticulum (ER) rather than
the cytosol, but this doesn’t always work.the cytosol, but this doesn’t always work.
– ER retention of soluble transport-competent proteins is inducible by the
carboxy-terminal retention/retrieval signal KDEL or HDEL, which is
recognized by a receptor located in the Golgi complex.
– Upon binding, the receptor retrieves C-terminal tagged proteins back into the
ER. Localization within the ER via the addition of KDEL or HDEL increases
the accumulation of foreign proteins in transgenic plants.
– However, the ER retention via KDEL is mediated by a KDEL receptor.
When the receptor is saturated with KDEL ligands, the KDEL-tagged
recombinant protein either secretes or is transported to the lytic vacuole
14. Protein accumulation in plant tissues reflects a
balance between protein synthesis and degradation
• Some KDEL-tagged recombinant protein can be also misfolded
and delivered for degradation through an ER-dependent
mechanism named ‘‘unfolded protein response’’ or UPR, which
functions for both endogenous or heterologous proteins
• The K/HDEL system is common to all eukaryotes, but plants can
use a different ER localization system in seeds consisting of
specialized organelles called protein bodies (PB), which stablyspecialized organelles called protein bodies (PB), which stably
accumulate seed storage proteins within the ER.
• The maize 27 kD γ-zein seed protein is not secreted even though
it bears an N-terminal signal sequence and lacks a canonical
KDEL/HDEL ER-retention signal; it is able to form ER-localized
PB not only in maize endosperm but also when expressed in
storage or vegetative tissues of transgenic Nicotiana tabacum,
Hordeum vulgare and Arabidopsis thaliana plants, respectively.
• PB formation can lead to higher protein accumulation in the ER
possibly because of the exclusion from the normal ER turnover
16. WHAT IS ZERA®®®®?
Cereal grains have evolved to store large amounts of proteins:
γ-Zein is the major storage protein in maize.
Zera® (γ-Zein ER-accumulating domain) is the N-terminal
proline-rich domain of γ-zein that is sufficient to induce the
assembly of protein bodies.
Zera® adopts an extended helix conformation where polarZera® adopts an extended helix conformation where polar
residues (histidines) are located on one side of the helix and
hydrophobic residues (leucines and valines) on the opposite
side of the helix.
This conformation provides high solubility in aqueous media
and the ability to self-assemble both in hydrophobic and
hydrophilic environments.
17. The Zera® domain retains its ability to develop
protein bodies after being fused with an exogenous
protein of interest.
Zera® contains two targeting signals:
ZERA®®®® PROTEIN BODIES
Organelles surrounded by a membrane derived from the ER.Organelles surrounded by a membrane derived from the ER.
Zera® contains two targeting signals:
1- A signal peptide that internalizes Zera® fusion
protein inside the ER
2- The Zera® domain itself that oligomerizes coating
the ER membrane and inducing the protein body
formation.
19. The benefits of Zera®®®® induced protein bodies
(PBs)
Zera® fusion proteins inside PBs escape the ER degradation
pathway allowing higher accumulation rates.
The accumulation of the Zera® fusion proteins in PBs also
protect the plant cell from toxic proteins.protect the plant cell from toxic proteins.
Post-translational modifications of Zera® fusion proteins inside
PBs: ER classical processing (N-glycosylation). Absence of
Golgi complex glyco-modifications.
The easy isolation of the protein body-like organelles makes
them an extraordinary enrichment tool.
21. TRANSIENT EXPRESSION OF F1-V FUSION
PROTEIN IN N. benthamiana
pCaSFV
5’ CsVMV3’ Ag7 5’ NOS
LB RB
NPT2 F1-V fusion
5’ vspA SP
3’ vspB
pCaSFV
5’ CsVMV3’ Ag7 5’ NOS
LB RB
NPT2 F1-V fusion
5’ vspA SP
3’ vspB5’ CsVMV3’ Ag7 5’ NOS
LB RB
NPT2 F1-V fusion
5’ vspA SP
3’ vspB5’ CsVMV3’ Ag7 5’ NOS
LB RB
NPT2 F1-V fusion
5’ vspA SP
3’ vspB
pCFV
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7 NPT2
pCFV
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7 NPT2
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7 NPT2
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7 NPT210 2
35S:Zera-
F1-V
35S:F1-V
CsVMV-F1-V
CsVMV-SP-
F1-V
ng bacterial
rF1-V
W.TRB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7 NPT2
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7 NPT2
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7 NPT2
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7
RB
5’ CsVMV 3’ vspB5’ NOS
LB
F1-V fusion3’ Ag7 NPT2
p35SF1V
RB
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LB
Bar F1-V fusion
TEV-5’ UTR
p35SF1V
RB
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LB
Bar F1-V fusion
TEV-5’ UTR
RBRB
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LB
Bar F1-V fusion
TEV-5’ UTR
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LB
Bar F1-V fusion
TEV-5’ UTR
p35S:Zera®®®®
-F1V
TEV-5’ UTR
RB
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LB
Bar F1-V fusionZera®®®®
p35S:Zera®®®®
-F1V
TEV-5’ UTR
RB
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LB
Bar F1-V fusionZera®®®®
TEV-5’ UTRTEV-5’ UTR
RB
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LB
Bar F1-V fusionZera®®®®
RB
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LB
Bar F1-V fusionZera®®®®
10 2
2
V
W.T
.
Zera-F1-V
(67 kDa)
F1-V
(56 kDa)
Zera-F1-V
dimers
22. NT1 TRANSFORMATION:
Zera®®®®-F1-V vs. F1-V
3-week old
Selection of the
healthiest NT1 calli
3-week old
calli
Liquid culture of
NT1 cells
Freeze-dried
NT1 cell culture.
Selection of the elite
lines by Western-blot
25. F1-V FUSION PROTEIN
ACCUMULATION IN NT1 CALLI
0
5000
10000
15000
20000
25000
Zera-F1-V NT1 F1-V NT1
Bandintensity[A.U.]
F1-V fusion protein
accumulation: >3X
higher in Zera®®®® -
F1-V than in F1-V
NT1 calli
26. ALFALFA TRANSFORMATION: ZERA®®®®-F1-V vs.
F1-V
Zera®®®®F1-V
F1-V
1 month after transformation
ZERA®®®®-F1-V F1-V
day 0
1 month
19 elongated leaves
(5% of explants)
144 elongated leaves
(58% of explants)
Zera®®®®F1-V
2 months
1 month
4-5
months
27. F1-V FUSION PROTEIN
ACCUMULATION IN ALFALFA
F1-V fusion protein
accumulation: >3X
higher in Zera®®®®-F1-V
than in F1-V alfalfa.
0
5000
10000
15000
20000
25000
30000
Zera-F1-V F1-V
Bandintensity[A.U.]
28. ANALYSIS OF NT1 CALLI AND ALFALFA
BY F1-V SOUTHERN-BLOT ANALYSIS
ALFALFA
30. CONCLUSIONS
The F1-V fusion protein accumulation in NT1 cells and
alfalfa was at least 3X higher using Zera® technology.
The accumulation of F1-V in ER-derived PB-like structures
induced by Zera® was confirmed by EM.
The regeneration of alfalfa or NT1 calli expressing Zera®-
F1-V was delayed compared to F1-V likely due to the PB-
like formation.
These results confirm the potential of Zera® technology as
a strategy to increase value-added proteins in plants.
40. Acknowledgements
Boyce ThompsonBoyce ThompsonBoyce ThompsonBoyce Thompson
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