Darbepoetin alfa (Aranesp ® ) molecular characteristics and basic research.
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Transcript of Darbepoetin alfa (Aranesp ® ) molecular characteristics and basic research.
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.