©1998 timothy g. standish tissue plasminogen activator: a case study in pharming timothy g....
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©1998 Timothy G. Standish
Tissue Plasminogen Activator:Tissue Plasminogen Activator:A Case Study InA Case Study In
PharmingPharmingTimothy G. Standish, Ph. D.
©1998 Timothy G. Standish
t-PA
BloodClot
What is Tissue Plasminogen What is Tissue Plasminogen Activator (t-PA)?Activator (t-PA)?
t-PA is an enzyme that serves in the cascade of events leading to dissolution of blood clots
PlasminogenActivation
Plasmin
Fibrin Breakdown
Clot DissolutionDamagedTissues t-PA
StreptokinaseFrom Bacteria
UrokinaseFrom the Kidneys
©1998 Timothy G. Standish
t-PA Has Been Developed As t-PA Has Been Developed As A Drug By GenentechA 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-PAProduction 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 CellsGrowing t-PA In CHO Cells
Chinese Hamster Ovary Cell Culture
Human Myeloma Cell Culture
Collect CellsFrom CellCulture
RNAExtraction
mRNA
Insertion IntoChinese Hamster Ovary Cells
cDNA
ReverseTranscription
Grow CHO cells in culture Collect
culturemedium
t-PA
ExtractT-PA
©1998 Timothy G. Standish
PharmingPharming 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?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:Making Recombinant Goats 1:Vector ConstructionVector Construction
Murine Way Acid Protein Promoter
LAtPA cDNA
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
Zygote
Nucleus
Suction
Suction Pipit
Vector
Micro-injector
Making Recombinant Goats 2:Making Recombinant Goats 2:Vector InsertionVector Insertion
©1998 Timothy G. Standish
Making Recombinant Goats 2:Making Recombinant Goats 2:Vector InsertionVector Insertion
Nucleus
Zygote Vector
©1998 Timothy G. Standish
Making Recombinant Goats 2:Making Recombinant Goats 2:Vector InsertionVector Insertion
Nucleus
ZygoteMost copies of the vector are broken down
A few may enter the nucleus
©1998 Timothy G. Standish
Making Recombinant Goats 2:Making Recombinant Goats 2:Vector InsertionVector Insertion
Chromosome
Nucleus Vector
©1998 Timothy G. Standish
Making Recombinant Goats 2:Making Recombinant Goats 2:Vector InsertionVector Insertion
Vector - Inserted into chromosome
©1998 Timothy G. Standish
Making Recombinant Goats 3:Making Recombinant Goats 3:Making KidsMaking Kids
Recombinant zygote or embryo
Milk collection
t-PA Purification
©1998 Timothy G. Standish
The 1987 Trials Of GenentechThe 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 GenentechThe 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
DecNov
OctSept
AprilMar
FebJan
AugJuly
JuneMay
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100110120130
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Dollars
Genentech Stock Prices 1987Genentech Stock Prices 1987
Loss of patentInfringmentsuit
Stock split
FDA approvest-PA license
FDA refusest-PA license
©1998 Timothy G. Standish
The 1988 Trials Of GenentechThe 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 GenentechThe 1988 Trials Of Genentech April
– Ontario Medical Association recommends using streptokinase instead of t-PA because of the expense of t-PA
– 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 from Integrated Genetics in Japan
©1998 Timothy G. Standish
The 1988 Trials Of GenentechThe 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
March– An Italian study shows streptokinase to be equally
effective as t-PA for long-term survival of heart attacks
19901990
©1998 Timothy G. Standish
Genentech Stock Prices 1988Genentech Stock Prices 1988
DecNov
OctSept
AprilMar
FebJan
AugJuly
JuneMay
0
20
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Dollars
©1998 Timothy G. Standish
Current Genentech Stock PricesCurrent Genentech Stock Prices
©1998 Timothy G. Standish
Ethical IssuesEthical 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.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