1. ©1998 Timothy G. Standish Tissue Plasminogen Activator: A Case Study In Pharming Timothy G. Standish, Ph. D.

2. ©1998 Timothy G. Standish t-PA Blood Clot 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 Plasminogen Activation Plasmin Fibrin Breakdown Clot Dissolution Damaged Tissues t-PA Streptokinase From Bacteria Urokinase From the Kidneys

3. ©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

4. ©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

5. ©1998 Timothy G. Standish Growing t-PA In CHO Cells Chinese Hamster Ovary Cell Culture Human Myeloma Cell Culture Collect Cells From Cell Culture RNA Extraction mRNA Insertion Into Chinese Hamster Ovary Cells cDNA Reverse Transcription Grow CHO cells in culture Collect culture medium t-PA Extract T-PA

6. ©1998 Timothy G. StandishPharming 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

7. ©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.

8. ©1998 Timothy G. Standish Making Recombinant Goats 1: Vector 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

9. ©1998 Timothy G. Standish Zygote Nucleus Suction Suction Pipit Vector Micro-injector Making Recombinant Goats 2: Vector Insertion

10. ©1998 Timothy G. Standish Making Recombinant Goats 2: Vector Insertion Nucleus Zygote Vector

11. ©1998 Timothy G. Standish Making Recombinant Goats 2: Vector Insertion Nucleus Zygote Most copies of the vector are broken down A few may enter the nucleus

12. ©1998 Timothy G. Standish Making Recombinant Goats 2: Vector Insertion Chromosome Nucleus Vector

13. ©1998 Timothy G. Standish Making Recombinant Goats 2: Vector Insertion Vector - Inserted into chromosome

14. ©1998 Timothy G. Standish Making Recombinant Goats 3: Making Kids Recombinant zygote or embryo Milk collection t-PA Purification

15. ©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

16. ©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

17. ©1998 Timothy G. Standish Dec Nov Oct Sept April Mar Feb Jan Aug July June May 0 20 30 40 100 110 120 130 50 60 80 90 10 70 Dollars Genentech Stock Prices 1987 Loss of patent Infringment suit Stock split FDA approves t-PA license FDA refuses t-PA license

18. ©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

19. ©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 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

20. ©1998 Timothy G. Standish 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 March –An Italian study shows streptokinase to be equally effective as t-PA for long-term survival of heart attacks 1990

21. ©1998 Timothy G. Standish Genentech Stock Prices 1988 Dec Nov Oct Sept April Mar Feb Jan Aug July June May 0 20 30 40 50 10 Dollars

22. ©1998 Timothy G. Standish Current Genentech Stock Prices

23. ©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?

24. ©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?

25. ©1998 Timothy G. Standish