Tissue Plasminogen Activator:
A Case Study In
Pharming
Timothy G. Standish, Ph. D.
©1998 Timothy G. Standish
What is Tissue Plasminogen
Activator (t-PA)?
t-PA is an enzyme that serves in the cascade of
events leading to dissolution of blood clots
Damaged
Tissues
t-PA
Blood
Clot
Clot Dissolution
Fibrin
Breakdown
t-PA
Plasminogen
Activation
Plasmin
Urokinase
Streptokinase
From the Kidneys
From Bacteria
©1998 Timothy G. Standish
t-PA Has Been Developed As
A Drug By Genentech
The biotechnology company Genentech has
cloned human t-PA for use in treating unwanted
or life-threatening blood clots
Activase (Alterplase recombinant) is the trade
name of Genentech’s t-PA
Activase is useful in treating heart attacks and
strokes when administered within 5 hours of
thrombosis formation or embolism lodging in the
heart or brain
The FDA approval in 1987 and medical use of
Activase has a very interesting history
©1998 Timothy G. Standish
Production of t-PA
Scientists at Genentech took human mRNA
coding for t-PA from a human myeloma cell line
The mRNA was converted into cDNA
Human t-PA coding cDNA was inserted it into
Chinese hamster ovary cell lines
When grown in culture the CHO cells excrete
human t-PA into their growth medium
Activase is produced by isolating t-PA from the
growth medium
This is an expensive and difficult process
©1998 Timothy G. Standish
Growing t-PA In CHO Cells
Collect Cells
From Cell
Culture
RNA
Extraction
Reverse
Transcription
mRNA
cDNA
Human Myeloma
Cell Culture
Grow CHO
cells in
culture
Insertion Into
Chinese Hamster Ovary Cells
Collect
culture
medium
Extract
T-PA
t-PA
Chinese Hamster Ovary
Cell Culture
©1998 Timothy G. Standish
Pharming
Because production of t-PA in cell culture is a
difficult and expensive process, t-PA is an
expensive drug
t-PA’s main competition in the thrombolytic (clot
busting) market is streptokinase
Streptokinase costs 1/10th the price of t-PA and
seems to do an equivalent job
A cheaper way to produce t-PA would be beneficial
(unless you are a streptokinase manufacturer)
Pharming offers the promise of cheap production of
a wide variety of proteins including t-PA
t-PA was the first viably produced protein using
pharming
©1998 Timothy G. Standish
What Is Pharming?
Pharming is the use of recombinant domestic
animals to produce proteins and other bioactive
compounds
One of the most common strategies in pharming
involves engineering mammals to produce
desired proteins in their milk
This strategy was first used to produce t-PA in the
milk of goats
It is not coincidental that this work was done at
the Roslin Institute in Scotland, the same institute
that cloned Dolly.
©1998 Timothy G. Standish
Making Recombinant Goats 1:
Vector Construction
LAtPA cDNA
Murine Way Acid Protein Promoter
SV40 Poly A Signal
The murine (mouse) way acid protein promoter
signals for expression of the gene in mammary
cells as part of milk
LAtPA cDNA codes for a point mutated form of
tPA (asn->glu at AA 117) that is glycosylated
differently resulting in longer acting (LA) tPA
SV40 is a virus that provides the poly A signal
telling cells mRNA is ready for expression
©1998 Timothy G. Standish
Making Recombinant Goats 2:
Vector Insertion
Micro-injector
Zygote
Vector
Nucleus
Suction
Suction
Pipit
©1998 Timothy G. Standish
Making Recombinant Goats 2:
Vector Insertion
Zygote
Vector
Nucleus
©1998 Timothy G. Standish
Making Recombinant Goats 2:
Vector Insertion
Zygote
Most copies of
the vector are
broken down
A few may enter
the nucleus
Nucleus
©1998 Timothy G. Standish
Making Recombinant Goats 2:
Vector Insertion
Nucleus
Vector
Chromosome
©1998 Timothy G. Standish
Making Recombinant Goats 2:
Vector Insertion
Vector - Inserted
into chromosome
©1998 Timothy G. Standish
Making Recombinant Goats 3:
Making Kids
Recombinant
zygote or
embryo
t-PA Purification
Milk collection
©1998 Timothy G. Standish
The 1987 Trials Of Genentech
March
– Japanese patent for t-PA awarded to Genentech
– Several companies file objections
June
– FDA refuses to license t-PA - Genentech stock loses $
1,000,000,000 or $ 11.50 per share
– Wellcome challenges Genentech’s UK t-PA patent
©1998 Timothy G. Standish
The 1987 Trials Of Genentech
July
– Genentech loses to Wellcome but appeals - Stock loses
$ 1.375
– t-PA is on the market in New Zealand, The
Philippines, and France
November
– FDA approves t-PA for human use
– T-PA is on the market in Austria, West Germany and
South Korea
December
– In the last 6 weeks of 1987, US sales of t-PA total
58,000,000
©1998 Timothy G. Standish
Genentech Stock Prices 1987
Dollars
130
120
110
100
90
80
70
60
50
40
30
20
10
0
Jan
Stock split
FDA refuses
t-PA license
Loss of patent
Infringment
suit
Feb
Mar
April
May
June
July
Aug
Sept
Oct
FDA approves
t-PA license
Nov
Dec
©1998 Timothy G. Standish
The 1988 Trials Of Genentech
January
– $ 20,000,000 in US t-PA sales
March
– Genentech stock is listed on the NYSE
– A study reveals t-PA reduces mortalitiy two weeks
after acute myocardial infarction
– Sales of t-PA drop to $ 11,000,000 for February and
March
– 2,000 patients a week are treated with t-PA
– April
©1998 Timothy G. Standish
The 1988 Trials Of Genentech
April
– Ontario Medical Association recommends using
streptokinase instead of t-PA because of the expense of tPA
– Medicare refuses to pay for t-PA
– Genentech stock drops 18 % in two weeks
June
– US patent for t-PA is granted to Genentech, but only
covers the purified form and does not exclude others
from filing patents
– In a suing-o-rama, Genentech sues Burroughs Wellcome
and Genetics Institute for their t-PA collaboration,
Abbott sues Genentech for patent infringement,
Genentech fails to win a restraining order to prevent
clinical trials of t-PA produced by Toyobo under license
©1998 Timothy G. Standish
from Integrated Genetics in Japan
The 1988 Trials Of Genentech
September
– Genentech is issued a US patent on its human
recombinant version of t-PA
– On the basis of the new patent Burroughs Wellcome
and Genetics Institute are sued again
November
– Genentech loses its appeal of a lower-court decision
to void its patent for being too broad in scope
1990
March
– An Italian study shows streptokinase to be equally
effective as t-PA for long-term survival of heart
©1998 Timothy G. Standish
attacks
Genentech Stock Prices 1988
50
40
Dollars
30
20
10
0
Jan
Feb
Mar
April
May
June
July
Aug
Sept
Oct
Nov
Dec
©1998 Timothy G. Standish
Current Genentech Stock Prices
©1998 Timothy G. Standish
Ethical Issues
Is production of recombinant organisms ethical?
Do the risks of recombinant organism production
outweigh the benefits of recombinant products?
Is use of organisms as tools to make products
purely for human benefit ethical?
Is introduction of animal products into humans for
therapeutic purposes ethical?
Is the cost of making recombinant products the
most productive use of health-care resources?
Is it ethical to withhold treatment using expensive
recombinant products from the poor so that
investment can be recouped and reinvested?
©1998 Timothy G. Standish
Ethical Issues Cont.
Is it ethical to produce high tech/high cost health
care products that are only marginally better than
lower tech/lower cost products?
Is it possible to own (patent) a naturally occurring
human gene?
Is it product infringement when another company
produces a comparable product that has identical
activity and other properties, but differs
structurally from the original?
©1998 Timothy G. Standish
©1998 Timothy G. Standish