Transcript The Aptamer Therapeutics Company™ Preclinical Safety Assessment of Aptamer Therapeutics Scott A. Barros, PhD, DABT Sr.

The Aptamer Therapeutics Company™
Preclinical Safety Assessment of
Aptamer Therapeutics
Scott A. Barros, PhD, DABT
Sr. Scientist, Toxicology
Confidential
What is an Aptamer?
apto:
“to fit”
mer:
“smallest unit of repeating structure”
Aptamers are single stranded
folded oligonucleotides that
bind to molecular (protein)
targets with high affinity and
specificity
Confidential
2
Aptamer Structure
• Unique tertiary structures allow
aptamers to fold into stable scaffolds
for carrying out molecular recognition
• van der Waals, hydrogen bonding,
and electrostatic interactions drive
high affinity target binding
• Designed to block protein-protein
interactions
• Share properties of both small
molecules and biologics
Nature Structural Biology,
7(1):53-57
• SELEX (Systematic Evolution of Ligands by Exponential Enrichment)
Tuerk and Gold (1990) Science 249, p505-510
Confidential
3
Medicinal Chemistry Process
• Increased plasma stability
• Increased affinity
• Increased potency
• Proprietary processes
• Multiple chemistries employed
1
S
Fraction aptamer bound
3
composites
N
Binding affinity measurement
0.8
0.6
single
substitutions,
nucleotide B, etc
composites
2
●●
●
●
● ● ●●●●●●●
●
●
●● ●●●●●●●●
●
●● ●
1
●●
●
●
● ●●●●●●●●
●
●
●● ●●●●●●●●
●
●● ●
●● ●●
●
● ● ●●●●●●
●
●
●● ●●●●●●●●
●● ●●
●● ●●
●
● ● ●●●●●●
●
●
● ●●●●●●●●
●● ●●
single
substitutions,
nucleotide A
●● ●●
●
● ● ●●●●●●
●
●
● ● ●●●●●●●●
●● ●●
optimized lead
early lead
-O
beneficial
P
O
O
O
B
-O
P
O
O
O
B
H 3C
P
B
O
O
tolerated
0.4
O
OCH3
O
OMe
O
H
0.2
0
0.001
0.01
Confidential
0.1
1
[target] nM
10
100
P=O  P=S
2’-deoxy  2’-OMe
P=O  P-Me
2’-OMe  2’-deoxy
4
Considerations in Safety Assessment of
Aptamers
In general, aptamers have toxicological properties similar
to other oligonucleotide therapeutics, but with a few
modality-specific considerations:
• The diversity and combinations of chemical compositions employed
distinguish aptamers from other oligonucleotide therapeutic modalities
― 15-40 mer, with variety of stabilizing 2’ ribose modifications and 3’-idT
― Often with large molecular weight PEG conjugate
• Species restriction and pharmacological activity
― Species restriction is often observed; similar to mAbs
― Two species toxicology testing, typically rat (off-target species) and
monkey (on-target species)
• Our goal is to keep aptamer at the site of action in the plasma and
interstitial tissue compartments, outside of cells
― Plasma concentration and plasma exposure is more of a focal point than
tissue concentrations
• Dose regimens vary widely depending on the aptamer compositions
and the intended use
― IV bolus, infusion, repeated bolus, SC bolus, etc.
Confidential
5
Discovery Toxicology
Purpose of Discovery Toxicology:
• To detect potential development-limiting toxicological liabilities as early as
possible and avoid or engineer them out
Discovery Toxicology for Aptamers:
• Thus far, the general toxicological properties of aptamer therapeutics have
been mostly predictable, class-based, and with good safety margins for the
intended uses
― Therefore, we do not consider in vivo discovery toxicology important since
we would only expect to find the predictable outcomes (discussed later)
• But, we do not fully understand what attributes modulate the occurrence or
potency of the known class-based effects (not yet fully predictable)
― Therefore, we screen in vitro for oligo class-based toxicities during lead
optimization in order to detect early and engineer if necessary
• These in vitro screening assays include:
― Anti-coagulation – Polyanion effect, sequence independent, influenced by
composition
― Complement activation – Polyanion effect, sequence independent,
influenced by composition
― Immune Stimulation – Sequence dependent, influenced by composition
(TLRs)
Confidential
6
In vitro Complement Activation
[C5a] ng/mL
75
PEG-aptamer
aptamer, no PEG
thio-aptamer
antisense oligo
50
25
0
0
10
20
30
[oligonucleotide] M
Confidential
Oligonucleotides, especially
phosphorothioate oligos, can
stimulate complement activation via
Factor H or other mechanisms
Assay method:
• Add aptamer or control oligo to
human serum or blood anticoagulated with direct thrombin
inhibitor
• Incubate 37°C, 30 min
• Quench complement reaction with
EDTA
• Assay for generation of C3a or C5a
split products
7
In vitro Anticoagulation
aPTT, seconds
300
PEG-aptamer
aptamer, no PEG
thio-aptamer
antisense oligo
240
180
Assay method:
• Add aptamer or control oligo to
citrated human plasma
• Add aPTT reagent and calcium, and
measure time to clot
120
60
0
Confidential
Oligonucleotides, especially
phosphorothiate oligos, inhibit
coagulation, likely via intrinsic tenase
complex (factors IXa and VIIIa,
phospholipids, calcium)
0
10
20
30
[oligonucleotide] M
8
In vitro Immune Stimulation Screens
• Cytokine release and proliferation assays measure TLR 3,7/8,9 activation
• CpG oligonucleotides and transfected immunostimulatory RNAs induce
PBMC/mouse splenocytes to produce IL-6 and interferon alpha
• Class A and C type CpGs induce PBMCs and mouse splenocytes to proliferate
IL-6 release from PBMCs
ARCxxx
2000
1800
1600
pg/mL IL-6
1400
ARCxxx
1200
ARCyyy
1000
ARCzzz
800
CpG-B
600
400
200
0
1
10
100
1000
nM ODN
Confidential
9
Secondary Pharmacology
• “Off-target” protein interactions with ASOs have been referred to as
“aptamer effects”
• All oligonucleotides can have relatively low affinity interactions with
unintended target proteins (polyanion effects)
• This is to be distinguished from a therapeutic aptamer which has been
selected and optimized for high potency interactions with a target
protein
• These “off-target” effects can manifest as secondary pharmacology, at
some concentration
• How do we test for secondary pharmacology?
―Directed specificity testing depending on the target protein
―Discovery in vitro toxicology screens (C’ activation, anticoagulation, immune stimulation)
―Receptor/enzyme panel screens
―In vivo safety pharmacology and general toxicology
Confidential
10
Safety Pharmacology
• We adhere to the principles of ICH S7a
―CNS:
 Standard CNS study in rats
―CV
 hERG patch clamp
 Telemetered cynomolgus monkey in vivo study
―Respiratory:
 Respiratory endpoints incorporated into cynomolgus monkey
CV study
• We have seen no significant test article related effects in
these studies to date
Confidential
11
Genetic Toxicology
• We have conducted standard ICH battery of genetic toxicity studies
―Ames assay
―Human HPBL chromosomal aberrations
―In vivo micronucleus (rat)
• We have tested the final development compound in these assays
(e.g., PEGylated) using standard practice for dose selections
• All results have been negative for genotoxic effects
Confidential
12
General Toxicology - Principles
• Species selection:
― We conduct two species general toxicology testing
― Rodents often non-pharmacologically responsive “off-target” species
― Monkeys generally pharmacologically responsive “on-target and offtarget species”
• Route and regimens appropriate for the intended clinical use
― Can vary widely (IV bolus, infusion, SC bolus; continuous, daily,
weekly, etc)
― Have successfully used single-dose toxicology to support single dose
in man
― Repeated-dose designs may mimic those for mAbs when PEGylated
aptamer has long half-life (e.g., twice weekly dosing, etc)
• Dose selection
― Clinical equivalent (low), max feasible or chosen multiple of human
(high), and log mean (mid), based on plasma exposure multiples
― Clinically-relevant dose range is generally similar to what is seen with
mAbs
― We generally express dose on basis of aptamer mass, exclusive of
PEG; PEG doses are generally 3-4X aptamer doses
Confidential
13
Typical Findings in General Toxicology Studies
1. Exaggerated pharmacology
• Expected based on target biology
2. Anticoagulation
• Generally a modest effect with good safety margins
3. C’ activation
• Rarely seen and only at very high concentrations with aptamers
tested to date
4. Bone marrow suppression
• Seen in repeated-dose toxicity studies, modest effect with good
safety margins
5. Hemodilution (PEGylated oligos only)
• Osmotic properties of PEG at high plasma concentrations
6. Basophilic granulation and/or vacuolization
• Mononuclear phagocytes and kidney tubule epithelial cells
• Presence of drug-related material in these specific cells
Confidential
14
Exaggerated pharmacology
16
100
12
10
8
Dosing
Conc., Control
Conc., 25 mg/kg
Activity, Control
Activity, 25 mg/kg
CBT, Control
CBT, 25 mg/kg
76
52
28
16
12
8
5
4.5
0
4.25
0.1
4
4
1
1
0
ARC1779 (ug oligo/mL)
vWF Activity (%)
1000
CBT (min)
Cynomolgus Monkey
Single-dose, 4-hour Infusion
Hours Post-dose
No spontaneous bleeding despite <3% vWF activity and prolonged
cutaneous bleeding times, even at 25X projected human effective dose
Confidential
15
Anticoagulation
Cynomolgus Monkey
Single-dose, 4-hour Infusion
75
65
100
55
10
45
1
35
340
168
100
76
52
28
16
12
8
5
4.5
4.25
4
1
25
0
0.1
aPTT (sec)
20K PEG-aptamer (ug oligo/mL)
1000
Dosing
Conc., Control
Conc., 3 ug/kg/min (1 mg/kg)
Conc. 15 ug/kg/min (5 mg/kg)
Conc., 75 ug/kg/min (25 mg/kg)
aPTT, Control
aPTT, 75 ug/kg/min (25 mg/kg)
aPTT, 3 ug/kg/min (1 mg/kg)
aPTT 15 ug/kg/min (5 mg/kg)
Hours Post-dose
Concentration-dependent prolongation of aPTT
Confidential
16
Complement Activation
Dose-, Rate-, Concentration-Dependent
Cynomolgus Monkeys
7-day Repeated-dose, 6-hour Infusion
Henry, JPET 1997, 281:810-816
7
Bb Conc (ug/mL)
6
5
4
3
2
1
0
0.01
0.1
1
10
100
1000
10000
DNA Aptamer Conc (ug/mL)
Control
1 mg/kg/min (360 mg/kg/day)
Repeated-dose, Dose-escalating, 4-hour Infusion
Single-dose, 4-hour Infusion
7
7
6
6
Bb Conc (ug/mL)
Bb Conc (ug/mL)
0.5 mg/kg/min (180 mg/kg/day)
2.5 mg/kg/min (900 mg/kg/day)
5
4
3
2
5
4
3
2
1
1
0
0.01
0
0.01
0.1
1
10
100
1000
10000
DNA Aptamer Conc. (ug/mL)
0.1
1
10
100
1000
10000
DNA Aptamer Conc (ug/mL)
Control
0.5 mg/kg/min (120 mg/kg)
5 mg/kg/min (600 mg/kg)
Control
0.5 mg/kg/min (120 mg/kg)
1.5 mg/kg/min (360 mg/kg)
2.5 mg/kg/min (600 mg/kg)
10 mg/kg/min (2400 mg/kg)
10 mg/kg/min (2400 mg/kg)
Threshold for Bb elevation:
~50 µg P=S ASO/mL, ~300 µg DNA aptamer/mL
Confidential
17
Bone marrow suppression
Sprague-Dawley Rat; Subcutaneous bolus, 3x/week for two weeks
42K PEG Control, 750 mg PEG/kg
250
13.5
40K PEG-aptamer, 5 mg oligo/kg
40K PEG-aptamer, 15 mg oligo/kg
200
13
40K PEG-aptamer, 50 mg oligo/kg
12.5
150
12
100
11.5
50
11
0
Hgb (g/dL)
40K PEG-aptamer, 150 mg oligo/kg
aRetic (x10^9/L)
Lower hemoglobin and reticulocyte counts after
14-day repeated-dose in rats
Confidential
18
Hemodilution; PEG-Associated Plasma Volume
Expansion
10
1000
8
100
6
10
4
2
0.1
0
504
1
0
0.5
24
48
96
168
168
169
169
172
176
180
192
216
240
264
288
312
336
40K PEG-aptamer (ug oligo/mL)
10000
Total Protein (g/dL)
Cynomolgus Monkey
7-day Continuous Infusion
Dosing
13 ug/kg/min (141 mg/kg)
39 ug/kg/min (423 mg/kg)
130 ug/kg/min (1410 mg/kg)
TP, 0 mg/kg
TP, 141 mg/kg
TP, 423 mg/kg
TP, 1410 mg/kg
Hours Post-dose
Other parameters comparably affected included:
alb, glob, ALT, LD, ALP, GGT, chol, trig, RBC, Hgb, Hct, retic, WBC, neut, lymph, plat
PEG doses and concentrations are 4X oligo
Confidential
19
Basophilic granulation and/or vacuolization,
mostly in mononuclear phagocytes
Spleen; PAMS vacuolization
• Presence of test article-related
material in cells has not been
associated with apparent adverse
effects on those cells or tissues.
• Therefore, we have not considered
this finding alone to be an adverse
effect.
Confidential
Liver; Kupffer cell vacuolization
Kidney; Basophilic granules in
proximal tubulular epithelium
20
Additional Toxicology Testing
• We plan to do standard ICH-guided testing for reproductive
toxicology, chronic toxicology and carcinogenicity, when
appropriate
• We desire to test in at least 1 pharmacologically active species
whenever possible
• We do not propose to use surrogate molecules in toxicology
testing (surrogate molecules would always differ appreciably in
sequence, composition, potency, specificity, etc.)
Confidential
21
Conclusions
• Aptamers share many “class- based” properties with other
oligonucleotides
• But aptamers also differ appreciably from other oligonucleotides
in both MOA and chemical compositions
• We have developed a customized toxicology testing strategy for
aptamers
• The toxicities we have seen are class-based, as seen with other
oligonucleotides or with other PEGylated macromolecules
• The aptamers tested to date have shown good safety margins
between efficacious dose and concentrations and NOAELs in
toxicology studies
Confidential
22
The Archemix Gang
Confidential
23