Developing long acting agonists and antagonists of glycoprotein hormones using gene fusion and gene transfer:

from bench to clinics

Prof. Fuad FaresProf. Fuad FaresUniversity of HaifaUniversity of Haifa

2nd International conference on EndocrinologyChicago

October 2014

Structure-Function studies Using site-directed

mutagenesis and gene transfer

Development of new analogs

Therapeutical Therapeutical Recombinant proteinsRecombinant proteins

1978 Human Growth 1978 Human Growth HormoneHormone

1979 Human Insulin1979 Human Insulin

The ProblemThe Problem

Cause adverse effects due to Cause adverse effects due to peak dose injectionpeak dose injection

Most therapeutic proteins are <30 Most therapeutic proteins are <30 kD and hencekD and hence::

• Are filtered out quickly by the kidneysAre filtered out quickly by the kidneys Are taken up by the liver and cleaved enzymaticallyAre taken up by the liver and cleaved enzymatically

Have to be injected frequently for optimal therapyHave to be injected frequently for optimal therapy

Success of Long-Lasting Success of Long-Lasting ProteinsProteins

PEGylationPEGylation - Interferon - Interferon (SGP/Roche)(SGP/Roche) PEGIntron/PegasysPEGIntron/Pegasys $3.2 billion in sales in 2006$3.2 billion in sales in 2006

PEGylationPEGylation - GCSF (Amgen) - GCSF (Amgen) NeulastaNeulasta $2.5 billion in sales in 2006 $2.5 billion in sales in 2006

Hyper Glycosylation Hyper Glycosylation - EPO - EPO (Amgen)(Amgen) Aranesp (DNA Modifications) Aranesp (DNA Modifications) $3.9 billion in sales in 2006 $3.9 billion in sales in 2006

Structure-Function of Structure-Function of Glycoprotein HormonesGlycoprotein Hormones

FSH FSH - Human Stimulating Hormone- Human Stimulating Hormone LHLH - Luteinizing Hormone - Luteinizing Hormone

hCGhCG - Human Chorionic Gonadotropin - Human Chorionic Gonadotropin

TSHTSH - Thyrotropin Hormone - Thyrotropin Hormone

hCG hTSH

Glycoprotein Hormone Subunits

hFSH

hLH

hTSH

hCG

N N

NN

N

N

N N

O-linked

CTP

DD

BB

CC

AATwo fertility hormones

hCG – maintains pregnancy and requires long T1/2

hLH – stimulates ovulation on a pulse mode requiring a short T1/2

Amino acid sequence of hCG & hLH is almost identical

The Technology was Created The Technology was Created By Nature During Evolution - By Nature During Evolution - the CTP the CTP ““cassettecassette””

EEThe 28 amino acid C-terminal peptide (CTP) of hCG with its 4 O-glycans does not exist in hLH

hCG

hLH

CTP

T1/2 of hCG is ~5 times longer than T1/2 of LH

SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeu

Pro GlyProSerAspThrProIleLeuProGln

O O

O

O

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

N-terminal

CTP not seen in structure

Crystal Structure of hGCβ showing longC-term lacking CTP

The Role of CTP

Mutated

hCG

N

N N

N

hCG

N N

NN

Stop Codon

TGA

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

+

+

Protein CTP

Half- Life

Designing New FSH AnalogDesigning New FSH Analog

hFSH Gene hCG Gene CTP

CTP hFSH Gene

Designing New FSH Analog

NH2

NH2

Exon 3 Exon 4 Exon 2 COOH

CTPFSH Exon 2FSH Exon3FSH Exon 1 COOH

hFSH - CTP

1 -Assembly of the subunits

2- Binding to the receptor

3 -In vitro Bioactivity

4 -In vivo Bioactivity5 -Immunogenecity

Gene Expression

CHO Transfection

FSH -CTP

Stable Clone

hFSH-WT hFSH-CTP hFSH-(CTP)2

L M L M L M

Assembly of Subunits

in vitro Studiesin vitro Studies

Control

FSH-WT

FSH-CTP

Estr

og

en

(p

g/m

l) 10 IU / 24h x 48h (IP)

1 3 10 WT4X

2.5IU

WT1X

10 IU

C

0/40/4 0/4

3/34/4

4/4

Nu

mb

er

of

ovu

late

d o

ocyt

es

0

10

30

20

40

50

Half - Life

SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeu

Pro GlyProSerAspThrProIleLeuProGln

O O

O

O

Transfection intoLDLD Cells

FSH -CTP

Biological Biological ActivityActivity

0

10

20

30

40

50

FSH

-WT

FSH-C

TP

CHO Idl-D

E2

Pro

du

cti

on

(n

g/m

l)

FSH-C

TP

SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeu

Pro GlyProSerAspThrProIleLeuProGln

O

O O

O

• FSH – CTP is effective in follicular stimulation

• FSH – CTP is safe

• FSH – CTP is not immunogenic

Organon - Merck

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

FSH – CTPFSH – CTP

(ELONVA)(ELONVA)

World – Wide UseWorld – Wide Use

Start Up Start Up CompanyCompany

                                 

“Enhancing the potency and

longevity of highly valuable

proteins”

CTP

Start Up CompanyStart 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 Designing Long Acting ProteinsProteins

ErythropoietinErythropoietin Growth HormoneGrowth Hormone InterferonInterferon Factors, XI & VII Factors, XI & VII Short PeptidesShort Peptides

Erythropoietin (EPO)Erythropoietin (EPO)

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

N-term

C-term

3 - D Structure Analysis

Conclusion: Strands of both termini are fairly long and accessible.

Human Erythropoietin (blue) with its Receptors (cyan)

CTP EPO - cDNA CTP CTP

5’ - - 3’

TCCTCTTCC…..CTCCCACAATAASer Ser Ser …. Leu Pro Gln Term118 119 120 …. 143 144 145

hCG-CTP

ATGGGGGTG….GGGGACAGAMet Gly Val….Gly Asp Arg 1 2 3 …. 190 191 192

EPO-cDNA

Xba I Not I

Human EPO-CTP3

57

37

28

EPO

- W

T

EPO

(CTP) 3

Human EPO-CTP3

Haem

ato

cri

te

)ch

an

ge f

rom

base

lin

e(%

,

40

30

20

10

Days

ControlrhEPO 3 x

weekEPO-(CTP)3

rhEPO 1 x week

Human EPO-CTP3

rhEPO

EPO – (CTP)3

AARANESP

Time (hours)

Ery

thro

poie

tin

(m

U/m

l)

Human EPO-CTP3

Human Growth Hormone

Pituitary

Pharmaceutical and Pharmaceutical and Biotechnological Uses of Biotechnological Uses of

Growth HormoneGrowth Hormone

To treat children of pathologicallyshort stature

C-term

N-term

Conclusion: Both termini pointing away from the receptors and are accessible

3-D Structure Analysis

Human Growth Hormone (blue) with its Receptors (cyan)

GH CTP

1 234

AgeI BamHI

5' ' 3

TCCTCTTCC…..CTCCCACAATAASer Ser Ser …. Leu Pro Gln Term118 119 120 …. 143 144 145

ATGGGGGTG….GGGGACAGAMet Gly Val….Gly Asp Arg 1 2 3 …. 190 191 192

GH-cDNA CG-CTP

GH CTP CTP

CTP GH CTP CTP

GHCTP

GHCTP CTP

A.

B.

C.

D.

E.

Fig.1.

Secretion of GH Analogs from CHO cells

GH – (CTP)3GH – (CTP)3

CTP GH CTP CTP

Biotropin 0.6mg/kg

Biotropin 1.8mg/kg

GH-LA 0.6mg/kg

GH-LA 1.8mg/kg

Time (hours)

IGF

– I

(ng

/ml)

Biological Activity in vivo

Half-life

GH – (CTP)3GH – (CTP)3

Expeiments in Rehsus Monkeys Expeiments in Rehsus Monkeys and human clinical trials phase and human clinical trials phase I that GH-Long- acting is safe I that GH-Long- acting is safe and and not immunogenicnot immunogenic

GH-(CTP)3 is in human GH-(CTP)3 is in human clinical trials phase IIIclinical trials phase III

ConclusionsConclusions

Ligation of the CTP cassette geneLigation of the CTP cassette gene bearing 4 O-linked bearing 4 O-linked Oligosaccharised chains to Oligosaccharised chains to different proteins is an interesting different proteins is an interesting strategy for increasing the strategy for increasing the in vivoin vivo half-life and half-life and in vivoin vivo bioactivity bioactivity

This may allow reducing: A) Drug dose

B) Number of injections

TSH StudiesTSH Studies

hCG hTSH

Thyroid

Peripheral tissue

Pituitary

TSH

T3T4

HypothalamusTRH

TSH Subunits

N N

hTSH N

- WT NH2 ....... Asn -X-Thr...... Asn-X-Thr...

CHO CHO

52 78

- 1 NH2 ....... Asp -X-Thr...... Asn-X-Thr...

CHO

52 78

-2 NH2 ....... Asn -X-Thr...... Asp-X-Thr...

CHO

-1+2 NH2 ....... Asp-X-Thr...... Asp-X-Thr...

52 78

TSH - WT NH2 ...... Asn-X-Thr......

CHO

52 78

23

hTSH Variants

hTSH Single ChainhTSH Single Chain

hTSH Gene Gene

N N N

hTSH

hTSH Gene Gene CTP

N o o o o N N

hTSHCTP

Expressed in CHO cells

Binds to TSH Receptor in high affinity as will as the TSH-WT

Biologically active

hTSH Single Chain

hTSH hTSH –– Single Chain Single Chain VariantsVariants

hTSH Gene Gene CTP

hTSHCTPdeg

hTSH Gene Gene CTP

N o o o o

hTSHCTP1+2

o o o o

N N

hTSH

N

Secretion of TSH variantsSecretion of TSH variants

Dimer WT

CTP(deg)

CTPWT

WT

CTP+2

48K38K

25K

Receptor Binding TSH (Mutants)Receptor Binding TSH (Mutants)

TSH variants (u/ml)

125 I

bT

SH

Bou

nd )

%(

0

20

40

60

80

100

120

0.1 1 10 100 1000 10000

dimer

bCTPa1+2

bCTPa)deg(

dimerCTP

1+2 (sc)

CTP)deg((sc)

variantvariantvaluevalue

hTSH dimer hTSH dimer WTWT

200200

hTSHhTSHCTPCTP1818

hTSHhTSHCTPCTPddegeg

100100

IC50(U/ml)

In vitro Bioactivity

0

20

40

60

80

100

0.1 1 10 100

סידרה1

סידרה2

סידרה3

CTP

CTP1+2

CTP)deg(

cAM

P(p

mol

/we l

l)cA

MP

(pm

ol/w

ell)

hTSH (hTSH (U/ml)U/ml)

     

TSH AntagonistT

3

(fm

o/w

ell)

hTSH(50u/ml)+hTSH variants (u/ml)

0

400

800

1200

1600

2000

0.1 1 10 100 1000

דרה2 סי

דרה3 סי

hTSH+CTP1+2

hTSH+CTP (deg)

0

40

80

120

0.1 1 10 100 1000

דרה2 סי

דרה3 סי

hTSH+CTP1+2

hTSH+CTP (deg)

cAM

P(p

mol

/wel

l)

hTSH(50u/ml)+hTSH variants (U/ml)

T3 (

fmol/

well)

hTSH(50mu/ml)+hTSH variants (U/ml)

Thyroid Stimulating Immunoglobolins

)TSI(

Graves’ Disease

TSH Receptor

Hyperthyroidism

TSI AntagonistTSI Antagonist

0

500

1000

1500

2000

2500

0.1 1 10 100 1000

דרה1 סי

דרה2 סי

hTSI+CTP1+2

T3 (fm

ol/

well)

hTSI (0.75u/ml)+hTSH variants (u/ml)

hTSI+CTPdeg(

hTSI (0.75u/ml)+hTSH variants (u/ml)

0

20

40

60

80

100

0.1 1 10 100 1000

דרה1 סי

דרה2 סי

cAM

P(p

mol

/wel

l)

hTSI+CTP1+2

hTSI+CTPdeg(

Cell membrane

ConclusionsConclusions Deletion of the N-linked oligosaccharides from TSH resulted in partial antagonists of TSH and TSI at the level of the receptor binding site.

TSH variants may offer a novel TSH variants may offer a novel

therapeutic strategy in the treatment oftherapeutic strategy in the treatment of

hyperthyroidism and Graves’ disease.hyperthyroidism and Graves’ disease.

Prof. Zaki Kraiem Prof. Irving Boime

Prof. Aaron Hsueh

Dr. Naeil Azam Dr. Avri Havron Orit Sadeh Dr. Eyal FimaFlonia LeviRinat Alenberg Mr. Shai Novik

Dr. Sharif GanimDr.Taleb Hajoj

Clinical Advisory PanelsClinical Advisory PanelsWorld Known Opinion World Known Opinion

LeadersLeaders hGHhGH

Ron Rosenfeld, MD, StanfordRon Rosenfeld, MD, Stanford Barry Sherman, MD, Genentech, BiParBarry Sherman, MD, Genentech, BiPar Zvi Zadik, MD, Hebrew UniversityZvi Zadik, MD, Hebrew University

EPOEPO Allen Nissenson, MD, UCLAAllen Nissenson, MD, UCLA Anatole Besarab, MD, Henry Ford, DetroitAnatole Besarab, MD, Henry Ford, Detroit

Interferon-betaInterferon-beta William Mobley, MD, StanfordWilliam Mobley, MD, Stanford Hillel Panitch, MD, VermontHillel Panitch, MD, Vermont Ron Milo, MD, IsraelRon 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

Hai

fa