The Aptamer Therapeutics Company™ Confidential Preclinical Safety Assessment of Aptamer...

The Aptamer Therapeutics Company™

Confidential

Preclinical Safety Assessment of Aptamer Therapeutics

Scott A. Barros, PhD, DABTSr. Scientist, Toxicology

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apto: “to fit”

mer: “smallest unit of repeating structure”

What is an Aptamer?

Aptamers are single stranded folded oligonucleotides that bind to molecular (protein) targets with high affinity and specificity

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Aptamer Structure

Nature Structural Biology, 7(1):53-57

• 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

• SELEX (Systematic Evolution of Ligands by Exponential Enrichment)Tuerk and Gold (1990) Science 249, p505-510

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Medicinal Chemistry Process

2’-deoxy 2’-OMe

O

OMe

BO

O

O

-OP

H3C

P=O P-Me2’-OMe 2’-deoxy

O

H

BO

O

O

P

P=O P=S

O

OCH3

BO

O

S

-OP

0

0.2

0.4

0.6

0.8

1

0.001 0.01 0.1 1 10 100

Binding affinity measurement

Fract

ion a

pta

mer

bound

[target] nM

beneficial

tolerated

• Increased plasma stability• Increased affinity• Increased potency

• Proprietary processes• Multiple chemistries employed

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●

●●●●●●●●●●

● ● ● ● ● ●●

●●

●●●●●●●●

●

1 2 N

singlesubstitutions,nucleotide A

composites

●●●●●●●●●●

● ● ● ● ● ●●

●●

●●●●●●●●

●

3

singlesubstitutions,

nucleotide B, etc composites

early lead optimized lead

● ● ● ●

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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.

Considerations in Safety Assessment of Aptamers

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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)

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In vitro Complement Activation

Assay method:• Add aptamer or control oligo to

human serum or blood anti-coagulated with direct thrombin inhibitor

• Incubate 37°C, 30 min• Quench complement reaction with

EDTA• Assay for generation of C3a or C5a

split products

Oligonucleotides, especially phosphorothioate oligos, can stimulate complement activation via Factor H or other mechanisms

0

25

50

75

0 10 20 30

PEG-aptameraptamer, no PEGthio-aptamerantisense oligo

[C5

a]

ng/m

L

[oligonucleotide] M

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In vitro Anticoagulation

Assay method:• Add aptamer or control oligo to

citrated human plasma• Add aPTT reagent and calcium, and

measure time to clot

Oligonucleotides, especially phosphorothiate oligos, inhibit coagulation, likely via intrinsic tenase complex (factors IXa and VIIIa, phospholipids, calcium)

0

60

120

180

240

300

0 10 20 30

PEG-aptameraptamer, no PEGthio-aptamerantisense oligo

aPTT,

seco

nds

[oligonucleotide] M

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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

ARCxxx

IL-6 release from PBMCs

0

200

400

600

800

1000

1200

1400

1600

1800

2000

1 10 100 1000

nM ODN

pg

/mL

IL

-6 ARCxxx

ARCyyy

ARCzzz

CpG-B

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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, anti-

coagulation, immune stimulation)―Receptor/enzyme panel screens―In vivo safety pharmacology and general toxicology

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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

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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

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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 off-

target 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

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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

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Exaggerated pharmacology

No spontaneous bleeding despite <3% vWF activity and prolonged cutaneous bleeding times, even at 25X projected human effective dose

Single-dose, 4-hour Infusion

0.1

1

10

100

1000

0 1 4

4.2

5

4.5 5 8

12

16

28

52

76

Hours Post-dose

AR

C1

77

9 (

ug

oli

go

/mL

)v

WF

Ac

tiv

ity

(%

)

0

4

8

12

16

CB

T (

min

)

Dosing

Conc., Control

Conc., 25 mg/kg

Activity, Control

Activity, 25 mg/kg

CBT, Control

CBT, 25 mg/kg

Cynomolgus Monkey

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Anticoagulation

Concentration-dependent prolongation of aPTT


0.1

1

10

100

1000

0 1 4

4.2

5

4.5 5 8

12

16

28

52

76

10

0

16

8

34

0Hours Post-dose

20

K P

EG

-ap

tam

er

(ug

oli

go

/mL

)

25

35

45

55

65

75

aP

TT

(s

ec

)

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)

Cynomolgus Monkey

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Complement Activation Dose-, Rate-, Concentration-Dependent

Threshold for Bb elevation:~50 µg P=S ASO/mL, ~300 µg DNA aptamer/mL

Henry, JPET 1997, 281:810-816


0

1

2

3

4

5

6

7

0.01 0.1 1 10 100 1000 10000

DNA Aptamer Conc. (ug/mL)

Bb

Co

nc

(ug

/mL

)

Control 0.5 mg/kg/min (120 mg/kg)

2.5 mg/kg/min (600 mg/kg) 10 mg/kg/min (2400 mg/kg)

7-day Repeated-dose, 6-hour Infusion

0

1

2

3

4

5

6

7

0.01 0.1 1 10 100 1000 10000

DNA Aptamer Conc (ug/mL)

Bb

Co

nc

(ug

/mL

)

Control 0.5 mg/kg/min (180 mg/kg/day)

1 mg/kg/min (360 mg/kg/day) 2.5 mg/kg/min (900 mg/kg/day)

Repeated-dose, Dose-escalating, 4-hour Infusion

0

1

2

3

4

5

6

7

0.01 0.1 1 10 100 1000 10000

DNA Aptamer Conc (ug/mL)

Bb

Co

nc

(ug

/mL

)

Control 0.5 mg/kg/min (120 mg/kg)

1.5 mg/kg/min (360 mg/kg) 5 mg/kg/min (600 mg/kg)

10 mg/kg/min (2400 mg/kg)

Cynomolgus Monkeys

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Bone marrow suppression

Lower hemoglobin and reticulocyte counts after 14-day repeated-dose in rats

11

11.5

12

12.5

13

13.5

Hgb (g/dL)

0

50

100

150

200

250

aRetic (x10^9/L)

42K PEG Control, 750 mg PEG/kg

40K PEG-aptamer, 5 mg oligo/kg




Sprague-Dawley Rat; Subcutaneous bolus, 3x/week for two weeks

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7-day Continuous Infusion

0.1

1

10

100

1000

10000

00

.5 24

48

96

16

81

68

16

91

69

17

21

76

18

01

92

21

62

40

26

42

88

31

23

36

50

4

Hours Post-dose

40

K P

EG

-ap

tam

er

(ug

oli

go

/mL

)

0

2

4

6

8

10

To

tal

Pro

tein

(g

/dL

)

Dosing

13 ug/kg/min (141 mg/kg)



TP, 0 mg/kg

TP, 141 mg/kg

TP, 423 mg/kg

TP, 1410 mg/kg

Hemodilution; PEG-Associated Plasma Volume Expansion

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

Cynomolgus Monkey

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Basophilic granulation and/or vacuolization, mostly in mononuclear phagocytes

Liver; Kupffer cell vacuolizationSpleen; PAMS vacuolization

Kidney; Basophilic granules in proximal tubulular epithelium

• 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.

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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.)

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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

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The Aptamer Therapeutics Company™ Confidential Preclinical Safety Assessment of Aptamer...

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