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Prof. Fuad Fares University of Haifa

Developing long acting agonists and antagonists of glycoprotein hormones using gene fusion and gene transfer: from bench to clinics. Prof. Fuad Fares University of Haifa. 2 nd International conference on Endocrinology Chicago October 2014. Structure-Function studies

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Prof. Fuad Fares University of Haifa

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  1. Developing long acting agonists and antagonists of glycoprotein hormones using gene fusion and gene transfer: from bench to clinics Prof. Fuad Fares University of Haifa 2nd International conference on Endocrinology Chicago October 2014

  2. Structure-Function studies Using site-directed mutagenesis and gene transfer Development of new analogs

  3. Therapeutical Recombinant proteins 1978 Human Growth Hormone 1979 Human Insulin

  4. The Problem Most therapeutic proteins are <30 kD and hence: • Are filtered out quickly by the kidneys • Are taken up by the liver and cleaved enzymatically • Have to be injected frequently for optimal therapy • Cause adverse effects due to peak dose injection

  5. Success of Long-Lasting Proteins • PEGylation - Interferon  (SGP/Roche) • PEGIntron/Pegasys • $3.2 billion in sales in 2006 • PEGylation - GCSF (Amgen) • Neulasta • $2.5 billion in sales in 2006 • Hyper Glycosylation- EPO (Amgen) • Aranesp (DNA Modifications) • $3.9 billion in sales in 2006

  6. Structure-Function of Glycoprotein Hormones FSH - Human Stimulating Hormone LH - Luteinizing Hormone hCG - Human Chorionic Gonadotropin TSH - Thyrotropin Hormone

  7. hCG hTSH

  8. Glycoprotein Hormone Subunits N N  N N hFSH N hLH N hTSH O-linked N N hCG CTP

  9. The Technology was Created By Nature During Evolution - the CTP “cassette” CTP A Two fertility hormones B hCG – maintains pregnancy and requires long T1/2 hCG C hLH – stimulates ovulation on a pulse mode requiring a short T1/2 D Amino acid sequence of hCG & hLH is almost identical hLH E The 28 amino acid C-terminal peptide (CTP) of hCG with its 4 O-glycans does not exist in hLH T1/2 of hCG is ~5 times longer than T1/2 of LH

  10. SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeu Pro GlyProSerAspThrProIleLeuProGln O O O O CTP

  11. Prediction of Folded and Unfolded Region of human chorionic gonadotropin (HCG) - chain B

  12. Crystal Structure of hGCβ showing long C-term lacking CTP N-terminal CTP not seen in structure

  13. N N  N N hCG TGA Stop Codon N N Mutated hCG N N The Role of CTP

  14. Deletion of CTP from hCG • No effect on the assembly of subunits • No effect on receptor binding • No effect on in vitro bioactivity - Significantly decreased the bioactivity in vivo

  15. + + Protein CTP Half- Life

  16. Designing New FSH Analog

  17. hCG Gene CTP hFSH Gene CTP Designing New FSH Analog hFSH Gene

  18. hFSH - CTP a Exon 2 a Exon 3 a Exon 4 NH2 COOH CTP FSHb Exon3 FSHb Exon 1 FSHb Exon 2 COOH NH2 1- Assembly of the subunits 2- Binding to the receptor 3- In vitro Bioactivity 4- In vivo Bioactivity 5- Immunogenecity

  19. Gene Expression  FSH -CTP Transfection CHO Stable Clone

  20. Assembly of Subunits hFSH-WT hFSH-CTP hFSH-(CTP)2 L M L M L M

  21. in vitro Studies

  22. 10 IU / 24h x 48h (IP) Estrogen (pg/ml) Control FSH-CTP FSH-WT

  23. 4/4 3/3 50 40 4/4 30 Number of ovulated oocytes 20 0/4 0/4 0/4 10 0 WT 1X 10 IU WT 4X 2.5IU C 1 3 10

  24. Half - Life

  25. SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeu Pro GlyProSerAspThrProIleLeuProGln O O O O CTP

  26. FSH -CTP Transfection into LDLD Cells

  27. Biological Activity E2Production(ng/ml) CHO Idl-D 50 40 30 20 10 0 FSH-CTP FSH-WT FSH-CTP

  28. SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeu Pro GlyProSerAspThrProIleLeuProGln O CTP O O O

  29. HumanStudies Organon - Merck • FSH – CTP is effective in follicular stimulation • FSH – CTP is safe • FSH – CTP is not immunogenic

  30. February 2, 2010 — The European Commission (EC) has approved ELONVA (FSH-CTP) Merck Receives Positive Regulatory Opinion for European Marketing of Long-Acting CTP-Modified Fertility Treatment ELONVA

  31. FSH – CTP(ELONVA) World – Wide Use

  32. Start Up Company CTP “Enhancing the potency and longevity of highly valuable proteins”

  33. Start Up Company Public Company • NASDAQ, Stock Exchange, NY, USA. • Tel-Aviv Stock Exchange, Tel-Aviv, Israel.

  34. OPKO Health, Inc. a multinational biopharmaceutical and diagnostics company

  35. Designing Long Acting Proteins Erythropoietin Growth Hormone Interferon Factors, XI & VII Short Peptides

  36. Erythropoietin (EPO) The most common use is in people with anemia (low blood count) related to kidney dysfunction.

  37. C-term N-term 3 - D Structure Analysis Human Erythropoietin  (blue) with its Receptors (cyan) Conclusion: Strands of both termini are fairly long and accessible.

  38. Xba I Not I 5’ - - 3’ CTP EPO - cDNA CTP CTP ATGGGGGTG….GGGGACAGA Met Gly Val….Gly Asp Arg 1 2 3 …. 190 191 192 TCCTCTTCC…..CTCCCACAATAA Ser Ser Ser …. Leu Pro Gln Term 118 119 120 …. 143 144 145 EPO-cDNA hCG-CTP Human EPO-CTP3

  39. 57 EPO (CTP)3 EPO - WT 37 28 Human EPO-CTP3

  40. 40 Control rhEPO 3 x week 30 EPO-(CTP)3 rhEPO 1 x week 20 Haematocrite (change from baseline, %) 10 Days Human EPO-CTP3

  41. rhEPO EPO – (CTP)3 AARANESP Human EPO-CTP3 Erythropoietin (mU/ml) Time (hours)

  42. Human Growth Hormone

  43. Pituitary

  44. Pharmaceutical and Biotechnological Uses of Growth Hormone To treat children of pathologically short stature

  45. C-term N-term 3-D Structure Analysis Human Growth Hormone (blue) with its Receptors (cyan) Conclusion: Both termini pointing away from the receptors and are accessible

  46. A. B. C. D. E. 4 1 2 TCCTCTTCC…..CTCCCACAATAA Ser Ser Ser …. Leu Pro Gln Term 118 119 120 …. 143 144 145 5' ATGGGGGTG….GGGGACAGA Met Gly Val….Gly Asp Arg 1 2 3 …. 190 191 192 '3 3 AgeI BamHI GH CTP GH-cDNA CG-CTP GH CTP CTP GH CTP CTP CTP CTP GH CTP GH CTP Fig.1.

  47. Secretion of GH Analogs from CHO cells

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