Darbepoetin alfa (Aranesp®) molecular characteristics and

basic research

Presentation overview

The evolution of protein therapeutics

Structure and function of recombinant human erythropoietin

Importance of sialic acid content

Discovery and development of darbepoetin alfa

– molecular characteristics

– implications for clinical use

The evolution of protein therapeutics

Recognition that proteins can be useful ‘hormone-like’ therapeutics eg, insulin (1920s)

First purified from animal and human tissues eg, insulin, growth hormone, factor VIII

Recombinant protein therapeutics – Amgen scientists were among the leaders cloning erythropoietin in 1983

The evolution of protein therapeutics

First purified from animal and human tissues eg, insulin, growth hormone, factor VIII

Recombinant protein therapeutics – Amgen scientists were among the leaders cloning erythropoietin in 1983 Now a new era where protein therapeutics are modified to enhance their properties as therapeutics eg, darbepoetin alfa (Aranesp®)

Recognition that proteins can be useful ‘hormone-like’ therapeutics eg, insulin (1920s)

Darbepoetin alfa

Darbepoetin alfa is a biochemically distinct recombinant erythropoietic protein that stimulates the production of red blood cells

The discovery of darbepoetin alfa resulted from basic research into the structure and function of rHuEPO and its attached carbohydrate

The longer serum residence time and greater biological activity of darbepoetin alfa results from the addition of two extra sialic acid-containing carbohydrate side chains

Structure of EPO bound to an EPO receptor

rHuEPO has four carbohydrate side chains

rHuEPO has a theoretical maximum of 14 sialic acidsReceptor 1

EPO

Receptor 2

Carbohydrate side chains

Typical tetra-antennary carbohydrate

Sialic acid N-linked carbohydrate

The number of branches in carbohydratesand therefore the number of sialic acids is variable

In vivo activity in mice increases with greater sialic acid content

Adapted from Egrie JC, et al.Br J Cancer. 2001;84(suppl 1):3-10.

Isoform

0

5

10

15

20

25

30

8 9 10 11 12 13 14

Incr

ease

in H

ct a

t d

ay 3

0 (p

oin

ts)

Longer serum half-life

Higher receptor binding

Greater in vivo activity

Darbepoetin alfa development strategy

Introduce N-linked glycosylation consensus sequences (Asn-Xxx-Thr/Ser) into rHuEPO

Identify individual variants that have the desired properties

Test optimal combinations of variants

Amino acid sequence of rHuEPO

N-glycosylation sites

Disulphide linkages

O-glycosylation siteALA1

PRO3

PRO2

ARG4

LEU5

CYS7

ASP8

SER9 ARG

10

ILE6

VAL11

LEU12

GLU13

ARG14

TYR15

LEU16

LEU17 GLU

18ALA19

LYS20

GLU21ALA

22GLU23

ASN24

ILE25

THR26

GLY28CYS

29

ASN30

THR27

GLU31

THR32

CYS33 SER

34LEU35

ASN36

GLU37

ASN38

ILE39

THR40

VAL41

PRO42

ASP43

THR44

LYS45VAL

46ASN47

PHE48

TYR49

ALA50TRP

51LYS52

ARG53

MET54

GLU55

VAL56GLY

57

GLN58

GLN59 ALA

60VAL61

GLU62

VAL53

TRP64

GLN65

GLY66

LEU67

ALA68

LEU69 LEU

70SER71

GLU72

ALA73

VAL74

LEU75

ARG76

GLY77

GLN78ALA

79

LEU80

LEU81

VAL82

ASN83

SER84

SER85

GLN86

VAL87ASN

88GLU89

THR90

LEU91

GLN92LEU

93

VAL95

ASP96

LYS97ALA

98VAL99

SER100

GLY101

LEU102

ARG103

SER104

LEU105

THR106

THR107

LEU108

LEU109

ARG110

ALA111

LEU112

GlY113

ALA114

GLN115

LYS116

GLU117

ALA118

ILE119

SER120

PRO121

PRO122

ASP123

ALA124

ALA125

SER126

ALA127

ALA128

PRO129

LEU130

ARG131

THR132 ILE

133THR134

ALA135

ASP136

THR137

PHE138

CYS161

ALA160

GLU159

GLY158

THR157

TYR156

LEU155

LYS154

LEU153

LYS152

GLY151

ARG150

ARG139

LYS140

LEU141

PHE142

ARG143

VAL144

TYR145

SER146

ASN147

PHE148

LEU149

ARG162

THR163

GLY164

ASP165

NH2

COOH

HIS94

rHuEPO has two receptorbinding sites

Site 2Site 1 Site 1

Site 2

The effect of mutations on in vitro activity is indicated: red <2% active, orange <20% active, yellow <70% active

The following needed to be addressed in order to make darbepoetin alfa

Would the glycan addition be efficient?

Would the molecule be properly folded/stable?

Would the ability to stimulate erythropoiesis be retained?

Would in vivo activity be increased?

Discovery of new glycosylationsites in rHuEPO

N-linked carbohydrate consensus sequences were introduced into rHuEPO at positions indicated by vertical lines

Each molecule was tested to see if it had the desired properties

Two positions, Ala30 and Trp88, were selected for further development work

Good bioactivity

Poor bioactivity

Glycosylated

Partial glycosylation

Unglycosylated

a1 a2

N24T26 N38T40 N83S85

B1 COOHNH2

S126

a3 B2 a4

Optimisation of glycosylation sites

A two-fold increase in 9G8A immunoreactivity is suggestive of an altered conformation. Val87 substitutions allow carbohydrate addition at position 88 and normalisation of the conformation

Name

Amino acid/mutations

No. of N-linked chains

RIA-9G8A % of control

rHuEPO Ala30 His32 Pro87 Trp88 Pro90 3 100

rHuEPO denatured Leu91 3 6,600

N13 Asn88 Thr90 3 390

N14 Ser87 Asn88 Thr90 4 780

N18 Val87 Asn88 Thr90 4 120

Darbepoetin alfa Asn30 Thr32 Val87 Asn88 Thr90 5 80

Ala = alanine; Asn = asparagine; His = histidine; Leu = leucine; Pro = proline; Ser = serine; Thr = threonine; Trp = tryptophan; Val = valine

Comparison of rHuEPO and darbepoetin alfa

Three N-linked carbohydrate chains

Maximum 14 sialic acids MW ~ 30,400 daltons 40% carbohydrate

Five N-linked carbohydrate chains

Maximum 22 sialic acids MW ~ 37,100 daltons 51% carbohydrate

New carbohydrate side chains

Receptor 1

Darbepoetin alfa

Receptor 2 Receptor 1

rHuEPO

Receptor 2

Carbohydrate side chains

1Heatherington A, et al. Proc Am Soc Clin Oncol.2002;21:256b. Abstract and poster; 2Macdougall I, et al.

J Am Soc Nephrol. 1999;10:2392-2395.

Darbepoetin alfa has a longer half-life than rHuEPO: single-dose PK of IV administration

100

10

1

0.1

0.010 25 50 75 100

Time post-IV injection (hours)

Mea

n (

SD

) b

asel

ine-

corr

ecte

dse

rum

co

nce

ntr

atio

n (

ng

/mL

)

Darbepoetin alfa (oncology; 0.5 µg/kg, n = 20)*1

Darbepoetin alfa (dialysis; 0.5 µg/kg, n = 11)2

rHuEPO (dialysis; 100 IU/kg, n = 10)2

t1/2 = 25.3 hours

t1/2 = 8.5 hours

t1/2 = 38.8 hours

*Oncology patients received 2.25 µg/kgData shown are normalised for 0.5 µg/kgSD = standard deviation; IV = intravenous

0

20

40

60

80

10 100 1,000 10,000 100,000 1,000,000

ng/mL sample

rHuEPO (three chains)

NM279 (four chains)

Darbepoetin alfa (five chains)

In vivo activity in mice increases with increasing number of glycans

59

Fe

inco

rpo

rate

d

(% o

f m

axim

um

)

The anti-EPO monoclonal Ab F12 does not neutralise EPO bioactivity

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10 100

Amount of Ab added (µg/mL)

Effect of antibodies on EPO in vitro bioactivity

Non-neutralising anti-EPO monoclonal Ab (F12)

Neutralising anti-EPOmonoclonal Ab (D11)

Neutralising anti-EPOpolyclonal Ab (862)

In v

itro

act

ivit

y (%

)*

In vitro activity assay measures formation of erythroid colonies from human bone marrow in soft agar

*

EPO = erythropoietin; Ab - antibody

Development of darbepoetin alfa

A new erythropoietic protein, biochemically distinct from rHuEPO

Increased sialic acid content, resulting in

– a longer circulating half-life (2–3-fold greater than rHuEPO)

– less frequent dosing requirements

– increased biological activity

Pharmacokinetics offer potential for higher response rates and faster onset of action

Darbepoetin alfa: conclusions

Darbepoetin alfa has a similar conformation to rHuEPO

Darbepoetin alfa binds to and activates the same receptor as rHuEPO

Darbepoetin alfa has increased sialic acid-containing carbohydrate resulting in increased in vivo activity and a prolonged half-life1

Provides the opportunity to dose less frequently: QW, Q2W, Q3W or Q4W2–4

Clinical benefits have been demonstrated (high and rapid haematological responses at convenient dosing schedules)5

QW = once every week; Q2W = once every 2 weeks;Q3W = once every 3 weeks; Q4W = once every 4 weeks

1Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10.2Glaspy JA, et al. Br J Cancer. 2002;87:268-276.

3Kotasek D, et al. Eur J Cancer. 2003. In press. 4Kotasek D, et al. Proc Am Soc Clin Oncol. 2002;21:356a. Abstract 1421.

5Glaspy JA, et al. Cancer. 2003;97:1312-1320.