Preclinical Safety Assessment of Aptamer Therapeutics

1. Preclinical Safety Assessment of Aptamer Therapeutics Scott A. Barros, PhD, DABT Sr. Scientist, Toxicology

2. 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

3. Nature Structural Biology, 7(1):53-57 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 • SELEX (Systematic Evolution of Ligands by Exponential Enrichment) • Tuerk and Gold (1990) Science 249, p505-510

4. optimized lead early lead ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● single substitutions, nucleotide B, etc single substitutions, nucleotide A ● ● ● ● ● ● ● ● ● ● ● ● composites composites ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 1 2 3 N ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● S O O B B B ● ● ● ● ● ● ● ● P P P O O O -O -O H3C O O O beneficial tolerated OMe O O O H OCH3 P=O  P-Me 2’-OMe  2’-deoxy P=O  P=S 2’-deoxy  2’-OMe Medicinal Chemistry Process • Proprietary processes • Multiple chemistries employed • Increased plasma stability • Increased affinity • Increased potency

5. 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.

6. 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)

7. 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

8. 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)

9. IL-6 release from PBMCs 2000 1800 1600 1400 ARCxxx 1200 ARCyyy pg/mL IL-6 1000 ARCzzz 800 CpG-B 600 400 200 0 1 10 100 1000 nM ODN 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

10. 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

11. 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

12. 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

13. 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

14. Typical Findings in General Toxicology Studies • Exaggerated pharmacology • Expected based on target biology • Anticoagulation • Generally a modest effect with good safety margins • C’ activation • Rarely seen and only at very high concentrations with aptamers tested to date • Bone marrow suppression • Seen in repeated-dose toxicity studies, modest effect with good safety margins • Hemodilution (PEGylated oligos only) • Osmotic properties of PEG at high plasma concentrations • Basophilic granulation and/or vacuolization • Mononuclear phagocytes and kidney tubule epithelial cells • Presence of drug-related material in these specific cells

15. Exaggerated pharmacology Cynomolgus Monkey No spontaneous bleeding despite <3% vWF activity and prolonged cutaneous bleeding times, even at 25X projected human effective dose

16. Anticoagulation Cynomolgus Monkey Concentration-dependent prolongation of aPTT

17. Henry, JPET 1997, 281:810-816 Complement Activation Dose-, Rate-, Concentration-Dependent Cynomolgus Monkeys Threshold for Bb elevation: ~50 µg P=S ASO/mL, ~300 µg DNA aptamer/mL

18. Bone marrow suppression Sprague-Dawley Rat; Subcutaneous bolus, 3x/week for two weeks Lower hemoglobin and reticulocyte counts after 14-day repeated-dose in rats

19. Hemodilution; PEG-Associated Plasma Volume Expansion Cynomolgus Monkey 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

20. Basophilic granulation and/or vacuolization, mostly in mononuclear phagocytes Liver; Kupffer cell vacuolization Spleen; 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.

21. 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.)

22. 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

23. The Archemix Gang