Preclinical Safety Assessment of Aptamer Therapeutics

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Preclinical Safety Assessment of Aptamer
Therapeutics

• Scott A. Barros, PhD, DABT
• Sr. Scientist, Toxicology

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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
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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

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Medicinal Chemistry Process

• Increased plasma stability
• Increased affinity
• Increased potency

• Proprietary processes
• Multiple chemistries employed

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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.

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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
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 anti-coagulated with direct thrombin
  inhibitor
• Incubate 37C, 30 min
• Quench complement reaction with EDTA
• Assay for generation of C3a or C5a split products

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

• Assay method
• Add aptamer or control oligo to citrated human
  plasma
• Add aPTT reagent and calcium, and measure time to
  clot

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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
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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

• 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

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Exaggerated pharmacology
Cynomolgus Monkey
No spontaneous bleeding despite lt3 vWF activity
and prolonged cutaneous bleeding times, even at
25X projected human effective dose
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Anticoagulation
Cynomolgus Monkey
Concentration-dependent prolongation of aPTT
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Complement Activation Dose-, Rate-,
Concentration-Dependent
Cynomolgus Monkeys
Threshold for Bb elevation 50 µg PS ASO/mL,
300 µg DNA aptamer/mL
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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
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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
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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.

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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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