Lecture 9 - Pharming - Gray

Question 1

How many of the top drugs are biologics?

Answer 1

10 of the top 20

Question 2

How are many of these recombinant protein pharmaceuticals produced?

Answer 2

Chinese Hamster Ovaries (CHOs), E.coli, and yeast cells.

Question 3

What are the advantages of using CHOs?

Answer 3

They are mammalian cells, so post translational modifications such as glycosylation are more likely to be human-like

Question 4

What are the negatives?

Answer 4

Expensive, $100m - 200m
Take time to develop
Difficult to scale up production
Virus and prion and non-human protein contamination (mammalian cells require care)

Question 5

What is the alternative to using these?

Answer 5

Producing drugs using plants! Genetically engineer plants to produce drugs basically

Question 6

Are plant-derived drugs new?

Answer 6

No. Opiates, caffeine etc.

Question 7

What are the adv of using this?

Answer 7

Transgenic plants cheap compared to mammalian system
Small scale experiments relatively sample
Scale up and large biomass production possible
Low infrastructure costs
No contamination with animal virus, prions, or proteins
Plants are eukaryotic, and problems with differences in glycosylation pattern haven’t materialised

Question 8

What other technologies have been tried, and what went wrong?

Answer 8

Expression of proteins in sheep or goat milk, but relatively low yield and product recovery

Question 9

What went wrong with plant biotech early on/

Answer 9

2002 USA ProdiGene
Volunteer GM corn left over from growing for commercial launch was harvested with subsequent Soybean crop
Fined $250,000 and ordered to pay $3M for clean up.
In Iowa, GM crops cross-pollinated with a neighbouring field.
Scared investors, they pulled out, companies went bankrupt.

Question 10

Give examples of some biologics already produced in plants

Answer 10

LACTOFERRIN put in rice - has antimicrobial properties. Used for diarrhoea outbreaks.
ETELYSO (recombinant glucocerebrosidase). Treats rare genetic deficiency in this enzyme. (Gaucher disease). Current drug = Cerezyme, produced in CHO cells, costs $200k/year.
Etelyso produced in carrot cell bioreactors. 25% cheaper.
This was used as proof of concept that human enzyme can be produced in plants.

Question 11

What are plantibodies?

Answer 11

Monoclonal antibodies produced in plants

Question 12

What are the pros of plantibodies?

Answer 12

Very stable, active years later. They can be targeted to be produced in the seeds - easy to store. No cold chain needed.

Question 13

What was the first plantibody?

Answer 13

1989 - Mouse IgG heavy and light chain genes introduced separately into tobacco. Plants crossed. Progeny with both genes produced functional antibodies.

Question 14

What is a single-chain Fv fragment (scFv)?

Answer 14

Plants expressing single chain recombinant antigen-binding antibody fragments (Variable light chain + variable heavy chain = scFv)
Not as effective as IgG but easier to express
Active for 1.5 years

Question 15

Give some examples of antibodies produced in plants

Answer 15

Immunoglobulins in tobacco

Ebola plantibodies

Question 16

How did they manufacture ebola plantibodies?

Answer 16

Magnifection - ZMapp - mixture of three humanised monoclonal antibodies produced transiently in plants using magnifection (Agrobacterium). Had a T-DNA construct - deconstructed viral vector within T-DNA LB and RB.
Strong promoter
Viral RdRP - replicate genome
MP - movement protein - moves T-DNA between cells
GOI - humanised monoclonal antibody
TMV UTR - untranslated region
Strong terminator
If you infect a few cells (dipping) it will spread, all cells will express the transgene and produce antibodies (x3) then mix them. Protein yield can be very high.

Question 17

What are the pros/cons of magnifection?

Answer 17

Pros:
V high protein levels can be achieved
Quick and easy to perform preliminary experiments
Transgenic material can be easily contained
No transgenic seed or pollen, reducing env risk
Cons:
Difficult to control protein levels
Cannot be used for field grown traits. Probably contained in greenhouse.

Question 18

What is another example of a plantibody?

Answer 18

Carox - being developed by Planet Biotechnology. Monoclonal antibody against dental caries causing bacteria Strep mutans. Expressed in tobacco. Prevents colonisation of bacteria for up to 2 years. Painted onto teeth, lots of antibody needed.

Question 19

What are some other uses of antibodies in plants?

Answer 19

Target plant non-proteins eg auxin
Plant protection eg resistance to plant viruses
Creation of novel crops with eg biofilters - they could bind to mercury, clean up pesticides/heavy metals from water

Question 20

What else could plants be used for in terms of immunity?

Answer 20

Expression of the ANTIGEN in plants - not antibodies. Essentially a plant vaccine.

Question 21

Pros/cons of plant vaccines?

Answer 21

Pros:
No refrigeration or injections
V cheap. 30 tomato plants=1000 vaccines. 25cents/dose.
Good for oral vaccines as they reach the lower gut.
No viral contaminants
Cons:
Down-stream processing is challenging

Question 22

Give example of a vaccine from a plant

Answer 22

2006 - Newcastle disease virus causes avian respiratory disease.
Hemaglutinin-neuraminidase protein antigen expressed in tobacco plant cell culture. Provided disease protection in chicks.

Question 23

Another example:

Answer 23

Hep B vaccine. GM potatoes expressing Hep B antigen fed to mice. Immune response similar to commercial vaccine. Mice produced antibodies against Hep B.

Question 24

What oral vaccinations do we already use?

Answer 24

Used against enteric diseases eg cholera, which kills >5milpa.
Recombinant plant vaccines against cholera toxin and enterotoxigenic E.coli both give a degree of protection against disease in mice.

Question 25

What is the benefit of a heat stable oral protein?

Answer 25

Cooking potatoes destroys the protein - not ideal. Heat stable wouldn’t be destroyed.