a case study of developing highly hydrophilic...
TRANSCRIPT
Innovation in Linker Technologies A Case Study of Developing Highly
Hydrophilic Drug-Linkers
Robert P. Lyon, Ph.D. Sr. Director, Department of Chemistry
2
Overview: areas where novel ADC chemistry offers potential advancement over current technology
• Pharmacokinetics
o High drug loading accelerates clearance, diminishes activity
Limits drug loading to 3-4 per antibody
• Heterogeneity
o Average of 3-4 drugs per antibody generated by partial reduction of
interchain disulfides or lysine conjugation
• Chemical stability
o Maleimide elimination results in loss of drug-linker
3
Generating ADCs through native disulfides
full
reduction
0 2 4 4 6 8
thiols per antibody
8
thiols per antibody
IgG1
Conjugation with maleimido-drug linkers
8
drugs per antibody
W2996 Extraction at 280 nm
min
2.00 4.00 6.00 8.00 10.00 12.00
W2996 Extraction at 280 nm
min
2.00 4.00 6.00 8.00 10.00 12.00
time (min) time (min)
(0)
(2) (4)
(6)
(8)
(8)
Hydrophobic
Interaction
Chromatography
partial
reduction
(excess
TCEP) (limiting
TCEP)
0 2 4 4 6 8
drugs per antibody
4
High drug loading can result in faster clearance
Hamblett,K et al., Clinical Cancer Res. 2004, 10, 7063-7070
0 7 14 21
1
10
100
1000 0 2 4 8
time (days)
c o
n c e n t r
a t i
o n (
m g / m
L )
mc-vc-MMAE loading
1 reduced
disulfide
4 reduced
disulfides
Conjugation with mc-vc-MMAE:
Antibody / ADC Pharmacokinetics
5
High drug loading can result in faster clearance
Hamblett,K et al., Clinical Cancer Res. 2004, 10, 7063-7070
0 7 14 21
1
10
100
1000 0 2 4 8
time (days)
c o
n c e n t r
a t i
o n (
m g / m
L )
mc-vc-MMAE loading
0 1 0 2 0 3 0 4 0 5 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
d a y s p o s t tu m o r im p la n t
tum
or v
olu
me
(m
m3) u n tr e a te d
d o s e : 0 .5 m g /k g
Karpas-299 xenograft
Conjugation with mc-vc-MMAE:
Antibody / ADC Pharmacokinetics
6
Disulfide reduction does not adversely impact antibody pharmacokinetics
capping agent
native IgG1 capped IgG1
1) disulfide reduction
2) thiol capping
0 5 1 0 1 5
1
1 0
1 0 0
tim e (d a y s )
pla
sm
a c
on
ce
ntr
ati
on
(mg
/mL
)
c a p p e d
n a tiv e
2 8 0 n m H I C
W 2 9 9 6 E x t r a c t i o n a t 2 8 0 n m 9 1 5 0 4 7 E ; ; 2 / 1 0 / 2 0 1 2 1 : 1 5 : 5 8 P M P S T
W 2 9 9 6 E x t r a c t i o n a t 2 8 0 n m h 1 F 6 ; ; 3 / 1 3 / 2 0 1 2 4 : 5 2 : 3 8 P M P D T
m in
3 . 0 0 4 . 0 0 5 . 0 0 6 . 0 0 7 . 0 0 8 . 0 0 9 . 0 0 1 0 . 0 0
280 nm HIC
W2996 Extraction at 280 nm 915047E; ; 2/10/2012 1:15:58 PM PST
W2996 Extraction at 280 nm h1F6; ; 3/13/2012 4:52:38 PM PDT
min
3.004.005.006.007.008.009.0010.00
retention time (minutes)
native
capped
Hydrophobic Interaction Chromatography Pharmacokinetics
7
Evaluating the role of drug-linker hydrophobicity
• Auristatin drug-linker engineered to eliminate any unnecessary hydrophobic elements and introduce hydrophilic moieties:
‘Auristatin T’
phenylalanine of MMAF
replaced with threonine
valine-citrulline cleavage site
replaced with hydrophilic sequence
self-immolative PAB group
eliminated
8
Apparent hydrophobicity correlates with plasma clearance
• Auristatin ADCs with 8 drugs per antibody evaluated for apparent
hydrophobicity and pharmacokinetics
280 nm HIC
W2996 Extraction at 280 nm h1F6; ; 3/13/2012 7:13:04 PM PDT
W2996 Extraction at 280 nm h1F6-1251; ; 3/13/2012 7:52:57 PM PDT
W2996 Extraction at 280 nm h1F6-4808; ; 3/13/2012 8:12:54 PM PDT
W2996 Extraction at 280 nm h1F6-1269; ; 3/13/2012 8:32:51 PM PDT
min
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
retention time (minutes)
Hydrophobicity
Mouse Pharmacokinetics
(antibody concentration in plasma)
0 2 4 6 8 1 0
0 .1
1
1 0
1 0 0
tim e (d a y s )
[Ab
] ( m
g/m
l)
native h1F6
Auristatin T
mcMMAF
mc-vc-MMAF
ADC ADC:
Lyon, R. P. et al. Nature Biotechnology 33, 733-735 (2015).
• HIC retention time predicts pharmacokinetics reasonably well
9
Cells of mononuclear phagocytic system may mediate the accelerated clearance of hydrophobic ADCs
Kupffer cells hepatic sinusoids
• No observable MPS uptake of unmodified antibody or hydrophilic Auristatin T ADC
• Recognition of hydrophobic ADCs by the MPS may underlie the increased clearance from plasma
perfused rat livers stained with anti-human Fc antibody:
h1F6 h1F6:mc-vc-MMAF8 h1F6:Auristatin T8
(1 hour post-dose)
10
In vivo activity of Auristatin T ADCs increase with drug loading
0 2 0 4 0 6 0 8 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
d a y s p o s t tu m o r im p la n t
Me
an
Tu
mo
r V
olu
me
(m
m3
)
m c -v c -M M A F 4
m c -v c -M M A F 8
U n tr e a te d A u r is ta t in T 4
A u r is ta t in T 8
2 mg/kg 0.5 mg/kg 786-O
(RCC model)
Anti-CD70 ADCs
Hydrophilic drug-linkers can overcome the 4 drug loading limit
• Enable homogeneous conjugates with 8 drugs / antibody
Reverses the situation observed with the hydrophobic mc-vc-MMAF:
4
4 8
8
11
Can PEG can be used to ‘mask’ the hydrophobicity of an MMAE drug-linker?
• Glucuronide cleavable linker less hydrophobic than val-cit
• Prepared with discrete PEG24 in two configurations:
PEG24
‘stretcher’
PEG24
‘side chain’
12
Pharmacokinetics, in vivo activity depend upon PEG configuration
L540cy Hodgkin Lymphoma model
Single dose
cAC10 (anti-CD30) ADCs
8 drugs / antibody
0 2 0 4 0 6 0 8 0 1 0 0
0
5 0 0
1 0 0 0u n tr e a te d
1 m g /k g
1 m g /k g
d a y s p o s t im p la n t
me
an
tu
mo
r v
olu
me
(m
m3)
2 m g /k g
ADC pharmacokinetics
3 mg/kg iv dose
0 7 1 4
0 .1
1
1 0
1 0 0
c A C 1 0 -1
c A C 1 0 -2
c A C 1 0 -3
c A C 1 0
day
tota
l a
nti
bo
dy
(m
g/m
l)
P E G 2 4 s id e c h a in
n o P E G
P E G 2 4 s tr e tc h e r
13
perfused rat livers stained with anti-human Fc antibody:
Appropriate PEG ‘masking’ greatly diminishes hepatic MPS uptake
14
Instability of ADCs generated with maleimide drug-linkers
• Slow elimination via retro-Michael reaction regenerates reactive
drug-linker over several days
• Drug-linker can subsequently react with biological thiols
o e.g. serum albumin
Alley, S.C. et al, Bioconjugate Chemistry 19 (3), 2008, pp 759-765
serum albumin
15
How can drug-linkers conjugated to cysteines be stabilized? • Hydrolysis: a second process in maleimide ADCs:
very slow
X stabilized ADC
16
A potential mechanism to accelerate ring hydrolysis in ADC manufacturing • Intramolecular catalysis:
X
very fast?
stabilized ADC
17
Hydrolysis rates at pH 7.4, 22°C, as a function of amine group spacing
0 6 1 2 1 8 2 4
0
2 0
4 0
6 0
8 0
1 0 0
t im e (h o u rs )
% h
yd
roly
sis
(li
gh
t c
ha
in)
1
t1 /2
(h rs )
0 .4
1 .8
5 .2
1 3 .54
3
2
x
t1/2
(hours)
0.4
1.8
5.2
13.5
x
1
2
3
4
Monitoring hydrolysis by mass spectrometry
vc-MMAE x=1 ‘mDPR’
maleimide
diaminopropionyl
Lyon R.P. et al. Nature Biotechnology 32, 1059-1062 (2014)
18
Stability against drug-linker elimination
H3 L1 H3 L1
H3 H2 H1 H0
L0 L1 H3
H2 L0
L1
retention time (min) retention time (min)
t=0
t=14d Drug loading determined by
reversed-phase HPLC
L1
H3
pH 8.0 buffer, 37 °C, +10 mM NAC
0 1 2 3 4 5 6 7
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
t im e (d a y s )
% d
ru
g r
em
ain
ing
in vitro rat plasma R = val-cit-MMAE
19
Combining stability, homogeneity, and slower clearance
• Inclusion of mDPR into PEGylated MMAE to provide stability
• How long does the PEG chain need to be to overcome
pharmacokinetic liabilities of homogeneous 8-load ADCs?
20
ADC pharmacokinetics profoundly impacted by PEG chain length
• 8 units sufficient to slow clearance to levels
similar to unconjugated antibody
• Incremental pharmacokinetic benefit from
extending PEG chain to 24 units
0 4 8 1 2 1 6 2 0 2 4 2 8
0
1 0
2 0
3 0
4 0
5 0
A D C c le a ra n c e a s a fu n c t io n o f P E G s iz e
P E G le n g th
Cle
ara
nc
e (
mL
·kg
-1·d
ay
-1)
ADC clearance
vs
PEG length
0 5 1 0
0 .1
1
1 0
A D C ra t p h a rm a c o k in e tic s ,
3 m p k , iv d o s e , D A R 8 c o n ju g a te s
T im e (d a y s )
To
tal
Ab
(m
g/m
l)
Ig G -1 0 (n o P E G )
Ig G -9 (a c e ty l)
Ig G -8 (P E G 2 )
Ig G -7 (P E G 4 )
Ig G -6 (P E G 8 )
Ig G -5 (P E G 1 2 )
Ig G -4 (P E G 2 4 )
Ig G n a k e d a n tib o d y
unconjugated antibody
24
12
8
4
2
0 (acetyl)
0 (no lysine spacer)
PEG subunits
0 5 1 0
0 .1
1
1 0
A D C ra t p h a rm a c o k in e tic s ,
3 m p k , iv d o s e , D A R 8 c o n ju g a te s
T im e (d a y s )
To
ta
l A
b (
mg
/ml)
Ig G -1 0 (n o P E G )
Ig G -9 (a c e ty l)
Ig G -8 (P E G 2 )
Ig G -7 (P E G 4 )
Ig G -6 (P E G 8 )
Ig G -5 (P E G 1 2 )
Ig G -4 (P E G 2 4 )
Ig G n a k e d a n tib o d y
21
Tolerability inversely correlated with clearance
animals sac’d in PEG 0, 2, and 4
groups due to weight loss
0 5 1 0
-2 5
-2 0
-1 5
-1 0
-5
0
5
1 0
S in g le d o s e m o u s e to le ra b ility
5 0 m p k , ip d o s e , D A R 8 c o n ju g a te s
T im e (d a y s )
% w
eig
ht
ch
an
ge
Ig G -1 0 (n o P E G )
Ig G -9 (a c e ty l)
Ig G -8 (P E G 2 )
Ig G -7 (P E G 4 )
Ig G -6 (P E G 8 )
Ig G -5 (P E G 1 2 )
Ig G -4 (P E G 2 4 )
u n tre a te dTolerability in mice, 50 mg/kg
dose tolerated in PEG 8, 12, and 24
groups
0 5 1 0
0 .1
1
1 0
A D C ra t p h a rm a c o k in e tic s ,
3 m p k , iv d o s e , D A R 8 c o n ju g a te s
T im e (d a y s )
To
tal
Ab
(m
g/m
l)
Ig G -1 0 (n o P E G )
Ig G -9 (a c e ty l)
Ig G -8 (P E G 2 )
Ig G -7 (P E G 4 )
Ig G -6 (P E G 8 )
Ig G -5 (P E G 1 2 )
Ig G -4 (P E G 2 4 )
Ig G n a k e d a n tib o d y
unconjugated antibody
24
12
8
4
2
0 (acetyl)
0 (no lysine spacer)
PEG subunits
0 5 1 0
0 .1
1
1 0
A D C ra t p h a rm a c o k in e tic s ,
3 m p k , iv d o s e , D A R 8 c o n ju g a te s
T im e (d a y s )
To
ta
l A
b (
mg
/ml)
Ig G -1 0 (n o P E G )
Ig G -9 (a c e ty l)
Ig G -8 (P E G 2 )
Ig G -7 (P E G 4 )
Ig G -6 (P E G 8 )
Ig G -5 (P E G 1 2 )
Ig G -4 (P E G 2 4 )
Ig G n a k e d a n tib o d y
22
Antitumor activity parallels ADC PK
0
4
4
5
2
0
untreated
24
12
8
4
0
PEG
subunits
anti-CD19 ADCs at
3 mg/kg single dose
RL model of DLBCL
mostly cures with
PEG 8, 12, and 24
no cures in absence of PEG
0 5 1 0
0 .1
1
1 0
A D C ra t p h a rm a c o k in e tic s ,
3 m p k , iv d o s e , D A R 8 c o n ju g a te s
T im e (d a y s )
To
tal
Ab
(m
g/m
l)
Ig G -1 0 (n o P E G )
Ig G -9 (a c e ty l)
Ig G -8 (P E G 2 )
Ig G -7 (P E G 4 )
Ig G -6 (P E G 8 )
Ig G -5 (P E G 1 2 )
Ig G -4 (P E G 2 4 )
Ig G n a k e d a n tib o d y
unconjugated antibody
24
12
8
4
2
0 (acetyl)
0 (no lysine spacer)
PEG subunits
0 5 1 0
0 .1
1
1 0
A D C ra t p h a rm a c o k in e tic s ,
3 m p k , iv d o s e , D A R 8 c o n ju g a te s
T im e (d a y s )
To
ta
l A
b (
mg
/ml)
Ig G -1 0 (n o P E G )
Ig G -9 (a c e ty l)
Ig G -8 (P E G 2 )
Ig G -7 (P E G 4 )
Ig G -6 (P E G 8 )
Ig G -5 (P E G 1 2 )
Ig G -4 (P E G 2 4 )
Ig G n a k e d a n tib o d y
cures
(5 mice/group)
0 2 0 4 0 6 0 8 0
0
3 0 0
6 0 0
9 0 0
1 2 0 0
d a y s p o s t im p la n t
me
an
tu
mo
r v
olu
me
(mm
3)
0 2 0 4 0 6 0 8 0
0
3 0 0
6 0 0
9 0 0
1 2 0 0
d a y s p o s t im p la n t
me
an
tu
mo
r v
olu
me
(mm
3)
23
Summary of PEG length findings
• PEG chain of 8-24 units resulted in ADCs with pharmacokinetics similar to
unconjugated antibody
o These ADCs exhibited greater tolerability in a rodent model
o Greater exposure of these ADCs also improved activity in xenografts
• Appropriately configured PEG chain can improve the apparent therapeutic
index on both sides of the activity / tolerability ratio
24
Overview: areas where novel ADC chemistry offers potential advancement over current technology
• Pharmacokinetics
o High drug loading accelerates clearance, diminishes activity
Limits drug loading to 3-4 per antibody
o Hydrophilic and polymer-masked drug-linkers overcome PK limitations
Lyon, R. P. et al. Nature Biotechnology 33, 733-735 (2015).
• Heterogeneity
o Average of 3-4 drugs per antibody generated by partial reduction of interchain disulfides or lysine
conjugation
o Drug-linkers designed for hydrophilicity allow for uniform 8-load ADCs
• Chemical stability
o Maleimide elimination results in loss of drug-linker
o Self-stabilizing maleimide through rapid ring hydrolysis
Lyon R.P. et al. Nature Biotechnology 32, 1059-1062 (2014)
• These advances can be combined and applied to new drug classes