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Tissue Plasminogen Activator: A Case Study In Pharming

Tissue Plasminogen Activator: A Case Study In Pharming. Timothy G. Standish, Ph. D. Clot Dissolution. Blood Clot. t-PA. Fibrin Breakdown. t-PA. Activation. Plasmin. Urokinase. Streptokinase. From the Kidneys. From Bacteria. What is Tissue Plasminogen Activator (t-PA)?.

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Tissue Plasminogen Activator: A Case Study In Pharming

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  1. Tissue Plasminogen Activator:A Case Study InPharming Timothy G. Standish, Ph. D.

  2. Clot Dissolution Blood Clot t-PA Fibrin Breakdown t-PA Activation Plasmin Urokinase Streptokinase From the Kidneys From Bacteria 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 Plasminogen

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

  6. 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

  7. 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. LAtPA cDNA Murine Way Acid Protein Promoter SV40 Poly A Signal Making Recombinant Goats 1:Vector Construction • 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. Micro-injector Vector Nucleus Suction Pipit Suction Making Recombinant Goats 2:Vector Insertion Zygote

  10. Vector Making Recombinant Goats 2:Vector Insertion Zygote Nucleus

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

  12. Vector Chromosome Making Recombinant Goats 2:Vector Insertion Nucleus

  13. Vector -Inserted into chromosome Making Recombinant Goats 2:Vector Insertion

  14. Recombinant zygote or embryo t-PA Purification Milk collection Making Recombinant Goats 3:Making Kids

  15. 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. 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. 130 120 110 Stock split 100 90 80 FDA refuses t-PA license FDA approves t-PA license Dollars 70 60 50 40 30 20 10 0 Jan Mar May July Sept Nov Feb April June Aug Oct Dec Genentech Stock Prices 1987 Loss of patent Infringment suit

  18. 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. 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. 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 attacks

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

  22. Current Genentech Stock Prices

  23. 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. 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. The End

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