tissue plasminogen activator: a case study in pharming

©1998 Timothy G. Standish

Tissue Plasminogen Activator:Tissue Plasminogen Activator:A Case Study InA Case Study In

PharmingPharmingTimothy G. Standish, Ph. D.


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


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


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


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


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


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.


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


Zygote

Nucleus

Suction

Suction Pipit

Vector

Micro-injector

Making Recombinant Goats 2:Making Recombinant Goats 2:Vector InsertionVector Insertion



Nucleus

Zygote Vector



Nucleus

ZygoteMost copies of the vector are broken down

A few may enter the nucleus



Chromosome

Nucleus Vector



Vector - Inserted into chromosome


Making Recombinant Goats 3:Making Recombinant Goats 3:Making KidsMaking Kids

Recombinant zygote or embryo

Milk collection

t-PA Purification


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


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


DecNov

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AprilMar

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Genentech Stock Prices 1987Genentech Stock Prices 1987

Loss of patentInfringmentsuit

Stock split

FDA approvest-PA license

FDA refusest-PA license


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


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


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


Genentech Stock Prices 1988Genentech Stock Prices 1988

DecNov

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Dollars


Current Genentech Stock PricesCurrent Genentech Stock Prices


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?


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?

tissue plasminogen activator: a case study in pharming

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