Developing long acting agonists and antagonists of glycoprotein

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 Using site-directed mutagenesis and gene transfer Development of new analogs

Therapeutical Recombinant proteins n 1978 Human Growth Hormone n 1979 Human Insulin

The Problem Most therapeutic proteins are <30 k. D 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

Success of Long-Lasting Proteins § PEGylation - Interferon (SGP/Roche) § § § PEGylation - GCSF (Amgen) § § § PEGIntron/Pegasys $3. 2 billion in sales in 2006 Neulasta $2. 5 billion in sales in 2006 Hyper Glycosylation - EPO (Amgen) § § Aranesp (DNA Modifications) $3. 9 billion in sales in 2006

Structure-Function of Glycoprotein Hormones n FSH - Human Stimulating Hormone n LH - Luteinizing Hormone n h. CG - Human Chorionic Gonadotropin n TSH - Thyrotropin Hormone

h. CG h. TSH

Glycoprotein Hormone Subunits N N h. FSH N N N h. LH h. TSH N O-linked N h. CG N CTP

The Technology was Created By Nature During Evolution - the CTP “cassette” Two fertility hormones h. CG – maintains pregnancy and requires long T 1/2 CTP A B h. LH – stimulates ovulation on a pulse mode requiring a short T 1/2 Amino acid sequence of h. CG & h. LH is almost identical h. CG C D The 28 amino acid C-terminal peptide (CTP) of h. CG with its 4 O-glycans does not exist in h. LH E h. LH T 1/2 of h. CG is ~5 times longer than T 1/2 of LH

O O O Ser. Lys. Ala. Pro. Ser. Leu. Pro. Ser. Arg. Leu O Pro Gly. Pro. Ser. Asp. Thr. Pro. Ile. Leu. Pro. Gln

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

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

The Role of CTP N N h. CG Mutated h. CG N N TGA N N Stop Codon

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

+ Protein +CTP Half- Life

Designing New FSH Analog

Designing New FSH Analog h. FSH Gene h. CG h. FSH Gene CTP

h. FSH - CTP NH 2 Exon 2 FSH Exon 1 Exon 3 Exon 4 FSH Exon 2 FSH Exon 3 1 - Assembly of the subunits 2 - Binding to the receptor 3 - In vitro Bioactivity 4 - In vivo Bioactivity 5 - Immunogenecity COOH CTP COOH

Gene Expression FSH -CTP CHO Stable Clone Transfection

Assembly of Subunits h. FSH-WT h. FSH-CTP L L M M h. FSH-(CTP)2 L M

in vitro Studies

Estrogen (pg/ml) 10 IU / 24 h x 48 h (IP) C on tr ol FS FS H H -W T -C TP

4/ 4 3/3 Number of ovulated oocytes 50 40 4/4 30 20 10 0/4 0/4 0 C WT 1 X 10 IU WT 4 X 2. 5 IU 1 3 10

Half - Life

O O O Ser. Lys. Ala. Pro. Ser. Leu. Pro. Ser. Arg. Leu O Pro Gly. Pro. Ser. Asp. Thr. Pro. Ile. Leu. Pro. Gln

FSH -CTP Transfection into LDLD Cells

Biological Activity E 2 Production (ng/ml) 50 40 30 20 10 0 P CT HFS P CT H- FS WT HFS CHO Idl-D

O O O Ser. Lys. Ala. Pro. Ser. Leu. Pro. Ser. Arg. Leu O Pro Gly. Pro. Ser. Asp. Thr. Pro. Ile. Leu. Pro. Gln

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

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

FSH – CTP (ELONVA) World – Wide Use

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

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

OPKO Health, Inc. a multinational biopharmaceutical and diagnostics company

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

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

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

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

57 37 28 P) (CT O EP EP O -W T 3 Human EPO-CTP 3

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

Erythropoietin (m. U/ml) Human EPO-CTP 3 rh. EPO – (CTP)3 AARANESP Time (hours)

Human Growth Hormone

Pituitary

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

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

A. '5 Age. I 4 3 1 GH ATGGGGGTG…. GGGGACAGA Met Gly Val…. Gly Asp Arg 1 2 3 …. 190 191 192 2 CTP GH CTP C. CTP GH D. CTP GH E. CTP GH Fig. 1. Bam. HI TCCTCTTCC…. . CTCCCACAATAA Ser …. Leu Pro Gln Term 118 119 120 …. 143 144 145 GH-c. DNA CG -CTP B. 3' CTP CTP

Secretion of GH Analogs from CHO cells

GH – (CTP)3 CTP GH CTP

Biological Activity in vivo Biotropin 0. 6 mg/kg IGF – I (ng/ml) Biotropin 1. 8 mg/kg GH-LA 0. 6 mg/kg GH-LA 1. 8 mg/kg Time (hours)

Half-life

GH – (CTP)3 n Expeiments in Rehsus Monkeys and human clinical trials phase I that GHLong- acting is safe and not immunogenic n GH-(CTP)3 is in human clinical trials phase III

Conclusions n Ligation of the CTP cassette gene bearing 4 O-linked Oligosaccharised chains to different proteins is an interesting strategy for increasing the in vivohalf-life and in vivo bioactivity This may allow reducing : A) Drug dose B) Number of injections

TSH Studies

h. CG h. TSH

Hypothalamus TRH Pituitary TSH Thyroid T 3 T 4 Peripheral tissue

TSH Subunits N h. TSH N N

h. TSH Variants

h. TSH Single Chain N N h. TSH Gene h. TSH N h. TSH Gene N Gene o o N CTP Gene h. TSH CTP N

h. TSH Single Chain u. Expressed in CHO cells u. Binds to TSH Receptor in high affinity as will as the TSH-WT u. Biologically active

h. TSH– Single Chain Variants N N o o h. TSH Gene CTP h. TSH CTP 1+2 o oo o h. TSH Gene CTP Gene h. TSH CTP deg

Secretion of TSH variants 48 K 38 K 25 K Di me r. W C T TP C ( de g TP ) W T C TP 1+ 2

125 I b. TSH Bound (%) Receptor Binding TSH (Mutants) IC 50( U/ml) variant value h. TSH dimer WT 200 h. TSH CTP 1+2 18 h. TSH CTP (deg) 100 TSH variants ( u/ml)

c. AMP(pmol/well) In vitro Bioactivity h. TSH ( U/ml)

c. AMP(pmol/well) TSH Antagonist T 3 (fmol/well) T 3 (fmo/well) h. TSH(50 u/ml)+h. TSH variants ( U/ml) h. TSH(50 mu/ml)+h. TSH variants ( u/ml) h. TSH(50 u/ml)+h. TSH variants ( U/ml)

Graves’ Disease Thyroid Stimulating Immunoglobolins (TSI) TSH Receptor Hyperthyroidism

c. AMP(pmol/well) TSI Antagonist h. TSI+ CTP 1+2 h. TSI+ CTP (deg) h. TSI (0. 75 u/ml)+h. TSH variants ( u/ml) T 3 (fmol/well) h. TSI+ CTP 1+2 h. TSI+ CTP (deg) h. TSI (0. 75 u/ml)+h. TSH variants ( u/ml)

Cell membrane

Conclusions Deletion of the N-linked oligosaccharides from TSH resulted in partial antagonists of TSH and TSI at the level of the receptor binding site. n n TSH variants may offer a novel therapeutic strategy in the treatment of hyperthyroidism and Graves’ disease.

Prof. Zaki Kraiem Dr. Naeil Azam Orit Sadeh Flonia Levi Rinat Alenberg Dr. Sharif Ganim Dr. Taleb Hajoj Prof. Irving Boime Prof. Aaron Hsueh Dr. Avri Havron Dr. Eyal Fima Mr. Shai Novik

Clinical Advisory Panels World Known Opinion Leaders n n n h. GH n Rosenfeld, MD, Stanford n Barry Sherman, MD, Genentech, Bi. Par n Zvi Zadik, MD, Hebrew University EPO n Allen Nissenson, MD, UCLA n Anatole Besarab, MD, Henry Ford, Detroit Interferon-beta n William Mobley, MD, Stanford n Hillel Panitch, MD, Vermont n Ron Milo, MD, Israel

• National Institutes of Health (NIH) • The Rockefeller Foundation • United States - Israel Binational Science Foundation (BSF) • Israel Science Foundation (ISF) • The Israel Ministry of Science • The Israel Ministry of Industry and Trade • Private Investors

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