Making cheap proteinbased drugs in plants

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Plant Factories
Making cheap protein-based drugs in plants
Chris Hawes School of Life Sciences Oxford
Brookes University
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With loads of help from Prof. Julian Ma, St
Georges Hospital Medical School leader of the
Pharma Planta consortium
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Who is in favour of GM technology?
Who would eat GM foods?
Who would take a GM medicine?
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Genetic modification of plants

• Disease resistance
• Weed killer resistance
• Desirable traits -
  ripening, colour, fragrance

• Nutritional benefits
• Medical advances

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The pressures of increasing population
1.7 (70M) per yr 1 billion in 1800, 6 billion
in 2000
Malnutrition and infectious disease are the major
health concerns
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A MINOR GLOBAL PROBLEM????
800 MILLION PEOPLE DO NOT HAVE ACCESS
TO SUFFICIENT FOOD TO MEET THEIR
NEEDS MALNUTRITION PLAYS A SIGNIFICANT ROLE IN 6
MILLION DEATHS OF CHILDREN AGED UNDER 5 EACH
YEAR
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CAUSES OF DEATH (WHO)
Population - 6,000,000,000 Total
deaths - 55,965,000 1. Cardiovascular
disease 17,000,000 2. Infectious disease
14,000,000 3. Cancer 7,000,000 4.
Injuries 5,000,000
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Extended Programme on Immunisation vaccines
Global statistics (WHO)
Tuberculosis Diphtheria Measles Pertussis
Polio Tetanus
8M cases 2M deaths/yr (major cause of death in
HIV) 50,000 cases 4000 deaths/yr 30M cases
777,000 deaths/yr 45M cases 400,000
deaths/yr 2,836 cases in 2000, 537 cases in
2001 164,000 cases 110,000 deaths/yr in Africa
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75 of the worlds population depends almost
exclusively on plants for treating illnesses
30M children born every year are not adequately
immunised
How do we make vaccines for people who cannot pay?
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The cost of new pharmaceuticals

• New pharmaceuticals take on
  average 10-15 years to develop
• For every 10,000 compounds currently under
  development, only 1 will be approved for sale to
  the public
• Approx. 1bn (500m) to bring a new medicine
  to the market

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Vaccine Investment Decisions
Figure adapted from Rappuoli et al., Science, 2002
Vaccines
Social value
Pharmaceuticals
Economic value

• Disincentives
• High costs of development
• Demand for new vaccines at lowest prices

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Major Problems
Worldwide production capacity is limited not
enough fermentors to grow microbial or animal
cells 4 molecules consume 75 of production
capacity Increasing demand 40 growth per
annum since 1995 Production facilities cost
250-500 million Developing world cannot afford
Western drugs
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30 of women with breast cancer overexpress the
HER2 receptor (human epidermal growth factor
receptor 2). This is associated with an
aggressive form of disease. Herceptin works by
targeting HER2-overexpressing tumors and blocking
the HER2 receptors. Herceptin is a monoclonal
antibody
40,000 patients in Britain each year, 13,000
eventually die Cost is 21,800 per course of
treatment Herceptin is licensed for late-stage
breast cancer. In HER-2 positive cancer,
Herceptin reduces the risk of disease recurrence
by 46 Not licensed for early stage treatment
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What is molecular pharming?
The production of recombinant protein-based
pharmaceuticals in transgenic microbes, yeasts,
animals and plants
Best known - insulin
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Production Systems for rProteins
E. coli yeast
Animal cells
Transgenicplants
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Phyto-pharming the production of modern
medicines in genetically modified plants. An
unprecedented opportunity to produce valuable
molecules economically and on a massive scale.
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A genetically modified plant A plant that
contains a gene that has been inserted
artificially. The inserted gene may come from
another plant or a different species and is
called the transgene.
A TYPICAL TRANSGENE
Gene
Terminator
Promoter

Marker gene
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GM Plants as Green Factories

• Plants cells work like human cells, and they can
  make complex proteins.
• Plants are the only feasible production system
  for some proteins that are required at massive
  scale.
• Cost
• Oral vaccines

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Molecular Pharming
Candidate Protein
Introduction
Transgenic Plants
Plant Expression Vector
Structural Gene
Plantation
Pharmaceuticals
Harvest
Extraction Purification
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Production host plants
Benefits
Limitations

• Leafy Crops Large amount of biomass Low
  protein stability
• Seed crops Protein very stable Lower protein
  yields
• possible food chain
• contamination
• Fruit veg Edible Lower protein yields
• possible food chain
• contamination
• Oil containing Containment, ease of
  extraction Lower yield
• Plants
• Suspension Containment, batch consistency,
  Running costs
• cultures GMP production

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Extraction of MAbs from transgenic plants
Purification of MAbs from plants
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Extraction of an AIDs (HIV) antibody from tobacco
plants
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Antibodies a major success story

• Highly specific
• Very stable proteins
• Low toxicity
• High drug approval rates
• Injectable, topical and oral applications

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What is an antibody?
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Antibodies (secreted by B cells) bind antigens
(antibody generator)
Antigen-antibody complex destroyed by macrophages
in the blood stream
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Production costs for antibodies
Cost in per gram
Hybridomas (cultured animal cells)
1000 Transgenic animals
100 Transgenic plants
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The global problem of HIV /AIDS
8000 people die every day from AIDS
40M people are infected with HIV
14,000 new infections every day predominantly in
sub-Saharan Africa
Could we produce enough vaccines with our current
facilities for the global community?
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HIV microbicides are reagents that neutralise
HIV. They can be applied topically in a gel (in
the vagina or rectum) to prevent sexual
transmission of HIV.
Various antibodies have been raised to the HIV
virus coat proteins As part of the Pharma Planta
project we have been working on one HIV
monoclonal antibody called 2G12. It is produced
in tobacco plants or maize seeds
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Where to target your molecule of interest

• Chloroplasts - genes not transferred by pollen
  no contamination of crops
• Oil bodies oleosins are proteins that insert
  into oil body membranes which have a single lipid
  layer. Use oleosin targeting signals to get
  protein of interest to oil body. Harvest by
  floating on water.
• Endoplasmic reticulum
• Cell surface outside the cell
• Vacuole

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The Plant Secretory Pathway
V
V
N
PM CW
?
ER
GA
N, nucleus ER, endoplasmic reticulum GA, Golgi
stack V, vacuole PM plasma membrane CW, cell
wall
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Where to target your molecule of interest
Use the secretory pathway

• Apoplast (outside the cell)

SP
Foreign Gene

• Endoplasmic Reticulum

• Vacuole/protein bodies

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The northern pacific jellyfish, Aequoria
victoria has a fluorescent protein that glows
green with UV or blue light
The protein is known as GREEN FLUORESCENT PROTEIN
or GFP for short. It was first purified in 1962
by Osamu Shimamura
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The Green Fluorescent Protein has revolutionised
biology
Some uses

• Monitoring the expression of genes

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The Structure of a GFP construct
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Fluorescent Tobacco Plant
Tobacco Plant
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Antibody heavy chain retained in the endoplasmic
reticulum
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Antibody light chain in the vacuole
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Antibody light chain secreted out of the cell
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A matter of cost

• Pharmaceutical factories cost 300-500M

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GM Plants as Green Factories

• Plants cells work like human cells, and they can
  make complex proteins.
• Plants are the only feasible production system
  for some proteins that are required at massive
  scale.
• Cost
• Oral vaccines

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Oral immunisation, a better strategy for
vaccination in developing countries.
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Creating GM Plants for Hepatitis B Vaccine
1. The Hep B surface antigen gene, is
transferred from yeast into a plant cell (potato
is used as a prototype).
2. Potato plants are regenerated from transformed
cells
3. Hepatitis vaccine is correctly expressed by
potato plants
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Hepatitis B Human Clinical Results
Average mean IgG titers for all volunteers
Milli-International units (anti-HBsAg)
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Delivering Oral Vaccines by GM plants

• Molecular Pharming products will not be foods.

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Major plant vaccines in development - proof of
concept
Tetanus (UK/USA) Measles (Australia) HIV (Pharma
Planta) Hepatitis B (USA) Cholera, E. coli ,
Norwalk virus (USA) Rabies (UK/USA) Rotavirus
(USA) Respiratory syncytial virus (UK)
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Will plant derived vaccines and medicines be safe?
Health concerns
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World Health Organisation WHO is not aware of
scientifically documented cases in which the
consumption of these foods has had negative human
health effects
The European Commission .81 projects which have
received a total EU funding of 70M Euros, and
have involved over 400 scientific teams. Research
on GM plants and derived products .has not shown
any new risks to human health or the environment,
beyond the usual uncertainties of conventional
plant breeding. Indeed, the use of more precise
technology and the greater regulatory scrutiny
probably make them even safer than conventional
plants and foods
The Royal Society (UK) consumption of DNA
poses no significant risk to human health and
additional consumption of GM DNA has no effect.
American Medical Association National Research
Council The Academies of Science from 7
nations US Food and Drug Administration Organisati
on for economic co-operation and development
(OECD) The Society of Toxicology
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SAFETY
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MEDICINAL SAFETY
All pharmaceuticals undergo a rigorous series of
safety tests. They are also produced in an
approved and regulated manner (Good Manufacturing
Practice).
Pharmaceuticals produced in plants will be
subject to the same regimes used for all drugs
and medicines.
There is no reason why medicines produced in GM
plants should be any different in terms of
safety, to other recombinant medicines. These
include GM bacteria GM yeast and GM hamster cells
that are currently used to manufacture human
insulin, the hepatitis vaccine and herceptin.
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Genetic containment
Physical or geographic containment
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Contained cultivation security and control
Southern Spain

• Large (200 acre) greenhouses are commonly used
  for horticulture

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Genetic identification of GM medical crops

• Potatoes
• - Clonally propagated
• - Cooking inactivates proteins under food use
  conditions
• - Use male sterile plants

Tomatoes - Use seedless varieties or ensure
seedless production with plant hormone
application - utilize colorless line for
identity preservation
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A biotechnology solution to GM seed identification
Expression of a recombinant HIV MAb in maize is
linked to expression of a fluorescent
marker (jellyfish red fluorescent protein)
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Development of pharmaceuticals in GM plants