Designing new agonists and antagonists of Designing new agonists and - - PowerPoint PPT Presentation

Designing new agonists and antagonists of Designing new agonists and antagonists of glycoprotein hormones using site glycoprotein hormones using site-

• directed

directed mutagenesis and gene transfer mutagenesis and gene transfer

• Dr. Fuad Fares

November 8, 2004

Recombinant Proteins Recombinant Proteins

Gene Cloning Transfection into Eukaryotic or

Prokaryotic cells

Purification

Gene Modifications Gene Modifications

Site - Directed Mutagenesis Gene Fusion

Development of new agonists & antagonists

• Site – Directed Mutagenesis

& gene fusion

Structure – Function Glycoprotein Hormones

Development of Agonists & Antagonists

TSH LH FSH

Glycoprotein Hormone Family

hCG

CTP

Pituitary Placenta

Glycoprotein hormones mechanism of action

3

Glycoprotein Hormone Subunits

 hFSH hLH hTSH hCG

N N N N N N N N

SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeu Pro GlyProSerAspThrProIleLeuProGln

O O O O

3

Glycoprotein Hormone Subunits

 hCG Mutated hCG

N N N N N N

Stop Codon

N N

TAA TAG 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

Designing New Analogs Designing New Analogs

Follicle-Stimulating Hormone

• Used for stimulation of follicular

development in women

• Injected daily

hFSH Gene hCG Gene CTP CTP hFSH Gene

Designing New FSH Analog

d d

Secretions

Biological Activity Biological Activity in vitro in vitro

Receptor binding Biological Activity

Control FSH-WT F S H

• C

T P

Estrogen (pg/ml)

SerSerSerSerLysAlaProProProSerLeuProSerProSerArgLeu Pro GlyProSerAspThrProIleLeuProGln

O O O O

Biological Activity Biological Activity

10 20 30 40 50 FSH-WT FSH-CTP

CHO Idl-D

E2 Production (ng/ml) FSH-CTP

Half Life, in vivo Half Life, in vivo

Designing Long Acting Designing Long Acting Erythropoietin (EPO) Erythropoietin (EPO)

EPO is Produced in the kidney Glycoprotein hormone involved with

growth and development of mature red blood cells from erythrocyte precursor cells.

Therapeutic use of EPO Therapeutic use of EPO

• EPO has a wide clinical use;
• Several forms of anemia, including

those associated with renal failure, HIV infection, blood loss, and chronic disease.

• EPO injected three times/week

EPO Market EPO Market

Treatment for chronic dialysis patients

estimated between $8,000 and $10,000 per patient per year

Market volume of its usage worldwide

estimated at around $6 billion per year

hEPO Gene hCG Gene CTP CTP hEPO Gene

Designing New EPO Analog

Human EPO Human EPO

Amino Acid Sequence Amino Acid Sequence

EPO EPO-

• CTP Construction

CTP Construction

N N N O O O O O | | | | | | | | NH2...Asn-X-Thr…Asn-X-Thr...Asn-X-Ser…...Ser…..Ser...Ser...Ser...Ser.....COOH 24 38 83 126 2 4 1 3

AgeI BamHI

EPO cDNA CTP

Human EPO Human EPO-

• CTP

CTP cDNA cDNA Sequence Sequence

ACCAGATCTACCGGTCATCATGGGGGTGCACGAATGTCCTGCCTGGCTGTGGCTTCT CCTGTCCCTTCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCACGCCT CATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAA TATCACGACGGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGA CATCAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGA AGTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTT GGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCAG TGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCAT CTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTT CCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACAC AGGGGAGGCCTGCAGGACAGGGGACAGATCCTCTTCCTCAAAGGCCCCTCCCCCGAG CCTTCCAAGTCCATCCCGACTCCCGGGGCCCTCGGACACCCCGATCCTCCCACAATA AAGGTCTTCTGGATCCGCACTCTGGAGGTTAA

Cloning Cloning Plasmid Plasmid

invivogen Cloning site

Secretion

Receptor Binding

EPO U/ml

0.1 2.5 5 10 20

In vitro Bioactivity

In vivo Bioactivity

In vivo Bioactivity

In vivo Bioactivity

EPO EPO – – long acting long acting

Bind with high affinity to hEPO receptor The half – life is 2-3 times higher than

EPO-WT

In vivo half-life

Summary Summary

O- linked oligosaccharides have no role in

receptor binding and in vitro bioactivity

• f the hormone

Ligation of the CTP to different proteins,

resulted in increasing of the in vivo half- life and bioactivity.

3

Structure-Function of hTSH

 Subunit hTSH Sununit

N N N

Carbohydrate Recognition Carbohydrate Recognition Signal Signal

N-linked Oligosacharides

Asn – X – Ser/Thr

O-linked oligosacharides

Ser/Thr

 - W T N H 2 ....... A s n -X -T h r ...... A s n - X -T h r ... C H O C H O 5 2 7 8  - 1 N H 2 ....... A s p -X -T h r ...... A s n - X -T h r ... C H O 5 2 7 8  - 2 N H 2 ....... A s n - X - T h r ...... A s p - X - T h r ... C H O  -1 + 2 N H 2 ....... A s p - X -T h r ...... A s p - X -T h r ... 5 2 7 8 T S H  - W T N H 2 ...... A s n - X -T h r ...... C H O 5 2 7 8 2 3

hTSH Variants

hTSH hTSH Mutants Mutants

Expressed in CHO Cells Bioactivity was detected by measuring the

ability of the hTSH to stimulate cAMP formation and T3 secretion from cultured human thyroid follicles.

hTSH hTSH Mutants Mutants

Reduction in the protein secretion No effect on receptor binding Reduction in biological activity

hTSH hTSH Single Chain Single Chain

hTSH Gene  Gene hTSH Gene  Gene CTP

N N N N

• o o o

N N

hTSH  hTSHCTP

. .

.

BamHI TTT TCT GTC GCT CCT GAT Phe Ser Val Ala Pro Asp 136 137 138 1 2 3 TSH Exon 3  Exon 2

• A. TSH

TSHxon 2 TSH Exon 3  Exon 2  Exon 3  Exson 4

• B. TSHCTP

TTT TCT GTC TCC TCT TCC --- CTC CCA CAA GCT CCT GAT Phe Ser Val Ser Ser Ser --- Leu Pro Gln Ala Pro Asp 136 137 138 118 119 120

• -- 143

144 145 1 2 3

TSH Exon 3 CGCTP  Exon 2

BamHI 2 3 1 Sal I Sal I

4

TSH Exon 2 TSH Exon 3 CGCTP  Exon 2 Exon 3 Exon 4

5 4 1 6

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 Variants Single Chain Variants

.

hTSH Gene  Gene CTP hTSHCTPdeg hTSH Gene  Gene CTP

N

• o o o

hTSHCTP1+2

• o o o

Secretion of TSH variants Secretion of TSH variants

Dimer WT CTP(deg) CTPWT   W T CTP+2

48K 38K 25K

Receptor Binding TSH (WT) Receptor Binding TSH (WT)

hTSH variants (u/ml)

125I bTSH Bound (%)

20 40 60 80 100 120 0.1 1 10 100 1000 10000

dimer bCTP,a dimer bCTPa WT baWT

βCTP,α dimer

dimer β CTPαWT(sc) β αWT(sc)

10 hTSH 100 hTSHCTP 200 hTSH CTP, dimer 200 hTSH dimer WT

value variant

IC50 (U/ml)

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

TSH variants (u/ml)

125I bTSH Bound (%) 20 40 60 80 100 120 0.1 1 10 100 1000 10000

dimer bCTPa1+2 bCTPa(deg)

dimer

βCTPα1+2 (sc) βCTPα(deg)(sc)

value variant

100 hTSHCTPdeg 18 hTSHCTP 200 hTSH dimer WT

IC50(U/ml)

In vitro Bioactivity

20 40 60 80 100 0.1 1 10 100 1סידרה 2סידרה 3סידרה βCTPα βCTPα1+2 βCTPα(deg)

cAMP cAMP( (pmol pmol/well) /well) hTSH hTSH ( ( U U/ml) /ml)

In vitro Bioactivity

20 40 60 80 100 0.1 1 10 100 1סידרה 2סידרה 3סידרה βCTPα βCTPα1+2 βCTPα(deg)

cAMP cAMP( (pmol pmol/well) /well) hTSH hTSH ( ( u u/ml) /ml)

TSH Antagonist

value variant 158

hTSHCTPa(deg)

70

hTSHCTPa1+2

IC50 (U/ml)

value variant 135

hTSHCTPa(deg)

33

hTSHCTPa1+2 400 800 1200 1600 2000 0.1 1 10 100 1000

2סידרה 3סידרה

hTSH+βCTPα1+2 hTSH+βCTPα(deg)

T3 (fmo/well)

hTSH(50u/ml)+hTSH variants (u/ml)

40 80 120

0.1 1 10 100 1000

2סידרה 3סידרה

hTSH+βCTPα1+2 hTSH+βCTPα(deg)

cAMP(pmol/well)

hTSH(50u/ml)+hTSH variants (u/ml)

TSH Antagonist TSH Antagonist

500 1000 1500 2000 2500 0.1 1 10 100 1000

1סידרה 2סידרה

T3 (fmol/well)

hTSI (0.75u/ml)+hTSH variants (u/ml) hTSI+CTP1+2 hTSI+CTPdeg)

20 40 60 80 100 0.1 1 10 100 1000

1סידרה 2סידרה

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

cAMP(pmol/well)

hTSI+CTP1+2 hTSI+CTPdeg)

G G-

• protein

protein cellular responses: cellular responses:

hTSH/

hTSI

Biological activity of Biological activity of hTSH hTSH variants in variants in the presence and absence of the presence and absence of forskolin forskolin

Fsk 1+210 

1 + 2

1 + F s k

hTSH variant (u/ml)

F s k 1+250 1+250+Fsk 

1 + 2

1 + F s k Fsk 1+2100

cAMP (pmol/well)

20 40 60 80 100 120

*

G G-

• protein

protein cellular responses: cellular responses:

hTSH

Agonist/ Antagonist hTSI forskolin

G G-

• protein

protein cellular responses: cellular responses:

hTSH

Antagonist

hTSH TSI

In Vivo (8h after injection) In Vivo (8h after injection)

T4 (g/dl)

2 4 6 8 10 12

8

bTSH WT (sc) CTPdeg (sc) CTP1+2 (sc)

** **

In Vivo In Vivo -

• Competition (

Competition ( 1+2

1+2)

)

Time (h) T4 (g/dl)

1 2 3 4 5 6 7 8 9 4 8

bTSH(2punch) a1+2(2punch) A1+2+bTSH(2punch) bb

1+2+bTSH

CTP1+2

bTSH

** ** ** **

In Vivo In Vivo – – Competition ( Competition (deg deg) )

Time (h) T4 (g/dl)

1 2 3 4 5 6 7 8 9 4 8

bTSH(2punch) deg(2punch) bTSH+deg(2punch)

bTSH CTP(deg) deg+bTSH

** **

** **

• hTSH  and  subunits, encoded as single

chain, retain a biologically active conformation similar to that

• f

the heterodimer

• The N-linked oligosaccharide units are not

important for hTSH receptor binding.

• However, N-linked oligosaccharide play a

dominant role in signal transduction of hTSH

Summary

Conc nclusions Conc nclusions

Ligation of the CTP cassete gene to

different proteins, could be an interesting strategy for increasing the in vivo half-life and in vivo bioactivity This will reduce the amount of drug, and the number of injections

Conclu lusi sion

• ns

Conclu lusi sion

• ns

Deletion

• f

the N-linked

• ligosaccharides

from TSH resulted in partial antagonists of TSH and TSI at the level

• f the

receptor binding site.

 TSH variants may offer a novel

TSH variants may offer a novel therapeutic strategy in the treatment of therapeutic strategy in the treatment of hyperthyroidism and Graves hyperthyroidism and Graves’ ’ disease. disease.

Science & Biotechnology

Technion-Incubator, Haifa Focused

• n

enhancing the potency and longevity of short half-life proteins with strong business potential. Ltd.

• Dr. Fuad Fares
• Prof. Irving Boime
• Prof. Zaki Kraiem
• Dr. Naeil Azam
• Dr. Sharif Ganim

Dr.Taleb Hajoj Orit Sadeh Flonia Levi Rinat Alenberg

Clinical Trials

3 4 5 4 5 6 7 EPO-CTP EPO kDa 57 46.5 28

Appendix: Shareholders

100% Total 13% Employees (options) 6% Mor Research Implementation Ltd. 2.5% Washington University 24% Technion Incubator 54.5%

• Dr. Fuad Fares

% Shareholders

ModigeneTech Ltd. Technion-Nesher Science Park POB 212, Nesher 36601, Israel

• Tel. +972-4-830-8357
• Fax. +972-4-821-0531

E-mail: sherifg@modigenetech.co.il Web: www.modigenetech.co.il

Enhancing the potency and longevity of highly valuable proteins