(gardening) Production of Therapeutic Proteins in Plants.pdf

AGRICULTURAL BIOTECHNOLOGY IN CALIFORNIA SERIES

Publication 8078

Production of Therapeutic

Proteins in Plants

BRUCE R. THOMAS, Researcher, Seed Biotechnology Center, University of California,

Davis; ALLEN VAN DEYNZE, Biotechnology Specialist, Seed Biotechnology Center,

University of California, Davis; KENT J. BRADFORD, Professor of Vegetable Crops and

Director, Seed Biotechnology Center, University of California, Davis

UNIVERSITY OF

CALIFORNIA

Division of Agriculture

and Natural Resources

http://anrcatalog.ucdavis.edu

INTRODUCTION

Until recently, pharmaceuticals used for the treatment of diseases have been based

largely on the production of relatively small organic molecules synthesized by

microbes or by organic chemistry. These include most antibiotics, analgesics, hor-

mones, and other pharmaceuticals. Increasingly, attention has focused on larger and

more complex protein molecules as therapeutic agents. Proteins are large molecules

composed of long chains of subunits called amino acids (see Suslow, Thomas, and

Bradford 2002). Just as words are composed of the 26 letters of the alphabet, pro-

teins are composed of different combinations of the 20 or so amino acids, except that

the length of proteins is often 100 to 1,000 amino acids (“letters”) long. The struc-

ture and functionality of a given protein is determined by its sequence of amino

acids, which, in turn, determines its three-dimensional conformation, or structure.

Internal bonds (sulfur and hydrogen bonds) among the amino acids give the protein

its final shape and form. Complex proteins undergo further processing such as the

addition of phosphate groups (phosphorylation) or carbohydrate molecules (glycosy-

lation), which modify the proteins’ functions. Information stored in DNA directs the

protein-synthesizing machinery of the cell to produce the specific proteins required

for its structure and metabolism. Since proteins play critical roles in cell biology,

they have many potential therapeutic uses in preventing and curing diseases and dis-

orders. The first protein used to treat disease was insulin, a small peptide that revo-

lutionized the treatment of diabetes. In addition, the antigens used in vaccinations

to induce immune responses are often proteins.

While short peptide chains (containing fewer than 30 amino acids) can be syn-

thesized chemically, larger proteins are best produced by living cells. The DNA that

encodes the instructions for producing the desired protein is inserted into cells, and

as the cells grow they synthesize the protein, which is subsequently harvested and

purified. Since 1982, more than 95 therapeutic proteins, or peptides (“biologics”),

have been licensed for production using bacterial, fungal, and mammalian cells

grown in sterile cultures, and hundreds of additional therapeutic proteins are cur-

rently being developed and tested. In fact, many analysts anticipate that in the near

future the capacity of cell culture facilities will fall far short of demand, as aug-

menting cell culture facilities requires large investments in buildings and equip-

ment. Recently, transgenic (i.e., plants engineered to produce specific proteins) plant

expression systems (Suslow, Thomas, and Bradford 2002) were developed as alter-

native sources for the production of biologics, known as plant-made pharmaceuticals,

or PMPs. In general, the use of plants means a lower cost of production and easier

expansion for large-volume production than cell culture systems. Instead of a large

capital investment in cell culture facilities, plant production systems can be expand-

ed simply by growing and harvesting additional plants. However, about 50 percent

of the total cost of production is in extraction and purification of the proteins, which

is required in both systems. This reduces the potential cost advantage for PMPs.

Produced by

Seed Biotechnology

Center, UC Davis

http://sbc.ucdavis.edu

ANR Publication 8078

PRODUCTION OF THERAPEUTIC PROTEINS IN PLANTS

Nonetheless, plant expression systems can potentially produce hundreds of kilograms

per year of a purified protein whereas the cost of a similar production capacity using

mammalian cell cultures may be prohibitive.

Like mammalian cells, plant production systems have the advantage over microbial

systems of being able to produce active forms of complex proteins with appropriate

post-translational modifications (e.g., glycosylation). However, mammalian cells used

for the production of biologics must be managed carefully to avoid unintentional trans-

mission of viral diseases that infect humans; this is not a risk for biologics produced in

plant systems. Nevertheless, production of proteins using plant expression systems

poses some unique challenges, such as containment to prevent gene transfer to con-

ventional crops during plant growth and possibly higher costs to extract the desired

protein due to the presence of interfering compounds in plants.

This publication describes the types of biologics produced in plants, the plant-

based production systems in use, the government agencies responsible for regulation

of biologics, and some agricultural practices that are required to safely produce bio-

logics in crop plants.

UNIVERSITY OF CALIFORNIA RESEARCH HIGHLIGHTS

Bob Buchanan and Peggy Lemaux, Department of Plant and Microbial Biology,

University of California, Berkeley, are studying the use of thioredoxin to decrease

the allergenicity and increase the digestibility of foods. Food allergies continue

to be a major problem worldwide. The magnitude of the problem becomes appar-

ent when one considers that 2 percent of the total population shows clinical

symptoms of the problem, and up to one-third of all adults believe they have

food allergies. Buchanan and Lemaux have begun to address the problem using

a ubiquitous protein, thioredoxin. When milk and wheat preparations are treat-

ed in vitro with thioredoxin, they became less allergenic. Buchanan and Lemaux

are currently attempting to lower allergenicity in wheat and other grains by

enriching thioredoxin in the starchy part of the grain with the use of a strong

promoter to drive its expression specifically in the protein storage organelles.

The modified grain has been successfully grown for several generations in the

greenhouse and most recently in the field. The thioredoxin-enriched grains are

now being analyzed for allergenic as well as digestibility properties. The results

obtained so far indicate that it may be possible to obtain wheat that shows

decreased allergenicity and increased digestibility. For more information please

refer to http://www.aspb.org/downloads/foodallerg.pdf

Bo Lonnerdal at the Department of Nutrition, University of California, Davis,

studies the biological activity of anti-infective and nutritional human milk pro-

teins produced in rice seeds. Incorporation of these proteins into transgenic food

crops may help to prevent human diseases and improve human nutrition. These

proteins may be particularly useful in rice-based infant formulas by providing

important components normally found in human milk for infants that cannot be

breastfed.

ANR Publication 8078

PRODUCTION OF THERAPEUTIC PROTEINS IN PLANTS

THERAPEUTIC PROTEINS

Antibodies

Passive immunizations using monoclonal antibodies are the largest category of

biotechnology-derived drugs. They are a result of the development of humanized

antibodies that do not activate an immune response when administered. In passive

immunization, rather than injecting an antigen and inducing the body to produce

antibodies against it, an antibody targeted toward the specific antigen is adminis-

tered directly as a therapeutic. For example, multiple doses of Herceptin, a mono-

clonal antibody effective against breast cancer, are introduced intravenously to

patients to boost the body’s ability to suppress the cancer cells. Antibody therapies

for non-Hodgkin’s lymphoma, rheumatoid arthritis, and respiratory syncytial virus

have been approved for use in recent years. Clinical trials are underway for hundreds

of additional antibody drugs directed against various cancers, heart disease, infec-

tious diseases, inflammation, transplantation rejection, and skin, blood, neurologi-

cal, respiratory, allergic, and autoimmune disorders. The annual market for these

antibody drugs may grow to $8 billion by 2004.

Transgenic plants have been used for the production of antibodies directed

against dental caries, rheumatoid arthritis, cholera, E. coli diarrhea, malaria, certain

cancers, Norwalk virus, HIV, rhinovirus, influenza, hepatitis B virus, and herpes sim-

plex virus. Some of these have demonstrated preventative or therapeutic value and

are currently in clinical trials. The most advanced product to date is an anti-

Streptococcus mutans secretory antibody for the prevention of dental caries that is

currently in Phase II clinical trials. Plants offer the only viable, large-scale produc-

tion system for this antibody (Gavilondo and Larrick 2000, Larrick and Thomas

2001). Links to further information and a comprehensive table of antibody targets

in clinical trials can be accessed through the Seed Biotechnology Center at the

University of California, Davis. (See “For Additional Information,” below.)

Vaccines

Protein antigens from various pathogens have been expressed in plants and used to

produce immune responses resulting in protection against diseases in humans.

Plant-derived vaccines have been produced against Vibrio cholerae , enterotoxigenic

E. coli , hepatitis B virus, Norwalk virus, rabies virus, human cytomegalovirus,

rotavirus, and respiratory syncytial virus F. Insulin expression in plants produced a

vaccine useful for protection against insulin-dependent autoimmune mellitus dia-

betes. Plant virus particles expressing multiple antigens from various pathogens

have been useful as vaccines against pulmonary infections of Pseudomonas aerugi-

nosa , opportunistic infections of Staphylococcus aureus , malaria, HIV, hepatitis B

virus, and respiratory syncytial virus F. A company in California has developed a

virus-based system in tobacco to produce personalized vaccines against cancer.

Antigens specific to an individual patient’s tumor are expressed in tobacco, harvest-

ed, purified, and administered to the patient. This entire process can take as little as

4 weeks, compared to 9 months for the same process using mammalian cell culture.

Many of these plant-derived antigens were purified and used as injectable vac-

cines, but oral delivery of these vaccines within foods has also been successful.

Edible vaccines may be particularly valuable as a low-cost delivery mechanism for

immunization against various diseases in developing countries. They circumvent the

need for injections and sterile needles and do not require refrigeration. Edible vac-

cines have successfully immunized test animals against enterotoxigenic E. coli ,

ANR Publication 8078

PRODUCTION OF THERAPEUTIC PROTEINS IN PLANTS

Vibrio cholerae , hepatitis B virus, Norwalk virus, rabies virus, respiratory syncytial

virus F, and rotavirus. The concentration of vaccine proteins produced in edible plant

tissues is currently relatively low. Research is underway to increase the production of

vaccine in targeted plant tissues that are best for human consumption. The choice of

crop is also important. Edible vaccines are being tested in potatoes, tomatoes, bananas,

and carrots. Potatoes are usually cooked for consumption, which may inactivate the

vaccine. Short storage life and length of production cycle may hinder vaccine produc-

tion in tomatoes and bananas. Carrots have few storage problems and can be eaten raw,

and carrots modified to produce the antigen used in hepatitis B vaccines are currently

entering preclinical trials.

Although several challenges need to be overcome, including standardizing expres-

sion and dosage levels and immune responses to food-based vaccines, several products

are currently undergoing clinical testing. It is not anticipated that such edible vaccines

would ever be marketed through standard food distribution channels. Rather, they

would be distributed through health service channels to be consumed under the super-

vision of health professionals. Nonetheless, edible vaccines could greatly reduce the

cost of immunizing children in many parts of the world. While not strictly a disease,

vitamin A deficiency affects over 120 million children, causing blindness and 1 to 2

million deaths each year in countries where rice is the primary staple food. Rice seeds

have been engineered to synthesize and accumulate beta-carotene, which is converted

into vitamin A in the body. This Golden Rice is being developed into commercial culti-

vars at the International Rice Research Institute to help alleviate vitamin A deficiency.

Other Proteins

Plants have been tested as production systems for a range of therapeutic proteins to be

used either directly in foods or after purification. Expression in plants of milk proteins

such as lactoferrin and beta-casein may contribute the therapeutic values of these pro-

teins to other food products. Expression of thioredoxin in foods such as cereal grains

would increase the digestibility of proteins and thereby reduce their allergenicity. (See

page 2, “University of California Research Highlights.”) Other proteins that have

potential therapeutic applications after expression and purification from plants are list-

ed in table 1 .

PROTEIN EXPRESSION SYSTEMS

To achieve specific protein production in plants, the DNA that encodes the desired pro-

tein must be inserted into the plant cells. This can be done as a stable transformation

when foreign DNA is incorporated into the genome of the plant. A promoter associat-

ed with the inserted DNA then directs the cells to produce the desired protein, often

targeting it to accumulate only in specific tissues such as the seed. Alternatively, a plant

virus can be used to direct expression of a specific protein without genetically modify-

ing the host plant. The transformation and expression systems used to engineer these

proteins in plants affect the stability, yield, cost of purification, and quality of the pro-

teins produced. In addition, the methods used affect the procedures needed to prevent

the spread of the engineered traits to other plants during their growth in the field.

Plant Transformation Systems

Foreign genes may be inserted, or transformed, into plants via a number of methods.

Stable transformation into the nuclear genome is done primarily using Agrobacterium -

mediated transformation or particle bombardment methods (Suslow, Thomas, and

Bradford 2002). In each case, the DNA coding for the protein of interest and an asso-

ciated promoter to target its expression to a particular tissue or developmental stage is

integrated into the genome of the plant. Thus, when the plant is propagated, each plant

ANR Publication 8078

PRODUCTION OF THERAPEUTIC PROTEINS IN PLANTS

Table 1. The production in transgenic plants of biopharmaceuticals for human health

Potential application or

Plant host

Protein

human protein

anticoagulant

tobacco

protein C

thrombin inhibitor

canola (Brassica napus)

hirudin

neutropenia

tobacco

granulocyte-macrophage

colony-stimulating factor

growth hormone

tobacco

somatropin, chloroplast

anemia

tobacco

erythropoietin

antihyperanalgesic by opiate activity

arabidopsis

enkephalins

wound repair and control of

tobacco

epidermal growth

cell proliferation

hepatitis C and B

rice, turnip

interferon-

tobacco

interferon-

liver cirrhosis, burns, surgery

tobacco

serum albumin

blood constitute

tobacco

hemoglobin

collagen

tobacco

homotrimeric collagen

cystic fibrosis, liver disease

rice

-1 antitrypsin trypsin inhibitor for

transplant surgery maize aprotinin

antimicrobial

potato

lactoferrin

Non-human proteins

hypertension

tobacco, tomato

angiotensin-converting enzyme

HIV therapies

tobacco

-tricosanthin from TMV-U1 sub-genomic

coat protein

Gaucher’s disease

tobacco

glucocerebrosidase

Source:Daniell, Streatfield, and Wycoff 2001.

will transmit this property to its progeny and large numbers of plants containing the

transferred gene are readily generated. It is also possible to deliver genes into the

separate genome of plastids (chloroplasts and mitochondria) in plant cells. As plants

have multiple copies of chloroplasts per cell, chloroplast transformation has the

potential for high expression levels and high yields of recombinant proteins.

Chloroplast transformation has been successful in tobacco and potato, and research

is being done to expand to other crops. Because genes in chloroplast genomes are

not transmitted through pollen, recombinant genes are easier to contain, thereby

avoiding unwanted escape into the environment.

Viral Expression Systems

A second method of engineering plant protein expression is transduction, the use of

a recombinant plant virus to deliver genes into plant cells. The DNA coding for the

desired protein is engineered into the genome of a plant virus that will infect a host

plant. A crop of the host plants is grown to the proper stage and is then inoculated

with the engineered virus. As the virus replicates and spreads within the plant, many

copies of the desired DNA are produced and high levels of protein production are

achieved in a short time. The entire plant is then harvested to extract the protein.

This method does not result in the transfer of DNA into the plant genome, and the

viral particles are generally excluded from pollen and egg cells, so the recombinant

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