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Copyright © 2014 Covance. All Rights Reserved

NO TWO ARE THE SAME: APPROACHES TO THE NONCLINICAL SAFETY ASSESSMENT OF BIOLOGICS

General Considerations for Study DesignJanice Lansita, PhD, DABT, Senior Managing Scientist, ToxStrategies, Inc.

The Value of Understanding the Biologic for Non-GLP Candidate Optimization and Group Size Considerations for FIH GLP StudiesShawn Heidel, DVM, PhD, Executive Director, Global Lead Optimization and Program ManagementCovance

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General Considerations for Study Design

Janice Lansita PhD, DABT

jlansita@toxstrategies.com

General Considerations for Study Design

Overview

Introduction to Biological Products (biologics)• Regulatory guidance ICH S6(R1), Preclinical Safety Evaluation of

Biotechnology-Derived Pharmaceuticals

Toxicology Study Design• Dose• Dose Selection• Recovery Duration

Species Selection

Assessment of Immunogenicity

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Definition of Biologics

Biologics – protein pharmaceuticals derived from living organisms including:

Humans, animals, plants, microorganisms

Biotechnology methods (recombinant DNA/cell culture technology)

• Recombinant human proteins (enzymes, cytokines, growth factors)

• Monoclonal antibodies (mAb)• Fusion proteins• Antibody-drug conjugates (mAb + cytotoxic)• Bispecifics• Biosimilars

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Small Molecules vs. Biologics

Property Small Molecules Biologics

Molecular weight

Small• < 1000 daltons

Large• ≥ 1000 daltons, e.g. mAb ≈ 150 kDA

Target specificity

Less (compared to biologics)• Toxicities generally non-specific/not related to target (“off-target toxicity”)

High target specificity• Toxicity generally related to target/pharmacology or “on-target toxicity”; expected

Species Specificity

Generally not a concern• 2 species (rodent; non-rodent)

May be a challenge• Only 1 animal species may be relevant

Half-life Short (compared to antibodies)• Minutes - hours - days

Long – especially molecules with Fc of IgG FcRn receptor, protects IgG from catabolism• Avastin® ≈ 20 days

Distribution Potential for extensive distribution within the body

More limited distribution within body • Initially, largely confined to vascular space

Immunogenicity Generally not a concern Common challenge in animals and humans

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Biologics Regulatory Guidance: ICH S6 Addendum/ICH S6(R1) Key Points

ICH S6(R1) (2011), Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals

Guidance addresses biologics-specific issues.

For today’s discussion, we will focus on:

Study design • Dose selection• Duration • Recovery

Species selection

Immunogenicity

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Toxicology Studies: Study Design Objectives

Dose Selection• High-dose: generally a dose that produces some toxicity. However, with

biologics, often no overt toxicity is observed.– From ICH S6(R1): (1) a dose that provides the maximum intended

pharmacological effect in the preclinical species and (2) a dose that provides an approximately 10-fold exposure multiple over the maximum exposure to be achieved in the clinic

• Mid-dose: generally an even multiple between the low and high dose.• Low-dose: generally a dose that results in a similar exposure as the

anticipated clinically efficacious dose.

Clinical Start Dose• Define a NOAEL (STD 10 or HNSTD for oncology products)• NOAEL used to define the starting dose for clinical trials• Clinical trial starting dose depends on overall risk vs. benefit

NOAEL = No observable adverse effect levelSTD 10 (rodents) = severely toxic dose in 10% of animals HNSTD (non-rodents) = highest non-severely toxic dose

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Toxicology Studies: Study Design Objectives

Duration• Duration should support clinical trial. For chronic toxicity testing, 6-month

duration generally adequate

Dose Schedule/Frequency • Mimic clinical trial • Example: if biologic is to be administered twice weekly in patients, it

should be administered twice weekly in animals• Recovery and Duration of Recovery Period: – Address reversibility not delayed toxicity

– Complete recovery not essential

– Recovery duration should consider T1/2 of biologic

– Biologic product may interfere with detection of ADAs

Key objective of toxicology study is to define

a safe clinical dose

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Toxicology Studies: Key Objectives

Identify target organ(s) of toxicity: • Related to pharmacological action of drug? • Dose-response and time-course • Biomarkers or clinical parameters

• Monitorable, manageable and/or predictable?• Mechanism of toxicity• Reversibility

Clinical Risk vs. Benefit Assessment • Patient population and indication • Mitigate risks with clinical monitoring, inclusion/exclusion criteria, smaller

N, more conservative dose escalation, e.g., waiting period between patients and dose cohorts

• Risk and benefit become more defined; however, rare events difficult to predict until post-marketing

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Species Specificity

Species specificity - specific activity in a pharmacologically relevant species

• Species in which biologic is pharmacologically active due to expression of the intended target (e.g., receptor or epitope)

• Rats and dogs standard for small molecules

• Biologics – specific to human target• Nonhuman primate may be only relevant species• Cynomolgus monkey most common

ICH S6(R1) – “Toxicity studies in nonrelevant species may be misleading and are discouraged.”

• Animal species that does not express the target• Unreliable safety data • Not predictive of potential human toxicity• Example: early biologics drug development of interferons in rodents

(nonrelevant species) vs. non-human primates (pharmacologically relevant species).

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Determination of a Pharmacologically Relevant Species

How to determine a pharmacologically relevant species?

Comparison of animal species vs. human:• Sequence homology

• Expression and distribution of target • Binding affinity • In vitro functional bioassay – e.g., neutralization/activation of target

– Cell proliferation, tyrosine kinase phosphorylation

• In vivo assay (if available)– E.g., serum marker of pharmacological activity

– Up-regulation/down-regulation of cytokine or

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Example of Data Used to Determine Pharmacologically Relevant Species

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Species Sequence Homology

Binding Affinity (pM)

In vitro Functional Activity IC50 (μM)

Human --- 160 10

Cynomolgus

Monkey98% 80 5

Rat 95% 140 15

Hamster 89% 1700 550

Mouse 87% 2000 980

Monkey & rat are pharmacologically relevant

Data set consists of: target sequence homology, binding affinity of mAb to target, and in vitro functional activity.

Immunogenicity

Immunogenicity is a common challenge for biologics and is a normal and expected response

• Animal’s immune system recognizes the biologic as foreign • Develops anti-drug antibodies (ADA) to eliminate from body

Measuring animal ADA responses in toxicology studies is necessary to aid in study interpretation

Primary concern for development of clearing and/or neutralizing ADA in toxicology studies:

• Lower exposure of target organs to the drug

• Fewer treatment-related toxicities or false-negative results

ADA responses in animals are not relevant to predict human ADA responses

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Immunogenicity: Various Types of ADA Responses Can Occur

Binding: minimal to no impact

Clearing: increase drug clearance (decrease exposure)

Sustaining: decrease drug clearance (increase exposure)

Neutralizing: interfere with biologic binding to the target

Cross-reactive: binds to biologic and endogenous protein• Neutralizes pharmacological activity of biologic and endogenous protein• Endogenous protein mediates a unique biological action – Toxicity may be a concern if function is neutralized

Erythropoietin (pure red cell aplasia)

Thrombopoietin (thrombocytopenia)

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Day 1 Day 28

Time (days)

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Antibody positive

Clearing Antibody: Impact on Exposure

Antibody negative

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Immunogenicity: Take-home Messages

ADA responses can make long-term studies difficult

ADA responses do not necessarily invalidate a study• Sufficient number of animals exposed to active drug

Potential solutions: • Higher doses or more frequent dosing– “Saturate” or “dose through” the ADA response

– Sufficient drug “on board” to maintain systemic exposure

– Do not include low dose group if unlikely to maintain exposure throughout toxicology study

• Other approaches

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THE VALUE OF UNDERSTANDING THE BIOLOGIC FOR NON-GLP CANDIDATE OPTIMIZATION AND GROUP SIZE CONSIDERATIONS FOR FIH GLP STUDIES

Shawn Heidel, DVM, PhD Executive Director, Global Lead Optimization and Program ManagementCovance

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Goal for Candidate Optimization Studies (Non-GLP)

► Eliminate molecules with an unacceptable margin of safety

► Provide data to set doses for GLP toxicology studies

► Establish a dose response relationshipTarget saturation plays a key role

in dose selection for biologics

► Maximum saturation = maximum effect at the intended target

Toxicity associated with biopharmaceuticals is usually due to sustained, saturating pharmacology

Exceeding maximum saturation of the intended target can result in activating related targets

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Low Dose

Mid Dose

High Dose

Simple Pharmacodynmic (PD) Curve with Recommended Doses For GLP Studies

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Dose Selection

“…PK-PD…can assist in high dose selection by identifying 1) a dose which provides the maximum intended pharmacological effect in the preclinical species; and 2) a dose which provides an approximately 10-fold exposure multiple over the maximum exposure to be achieved in the clinic. The higher of these two doses should be chosen for the high dose group ....”

Note: It’s also important to ensure the low-dose isn’t overtly toxic and that the mid-dose identifies a dose response between the low and high doses.

FROM ICHS6(R1) SECTION 3.1 DOSE SELECTION AND APPLICATION OF PK/PD PRINCIPLES

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What Candidate Optimization Studies Should be Done?

Case #1 – only active in NHPs

No in vivo pharmacology data, because NHP are the only appropriate species and no pharmacology models exist in NHPs

► Sequence homology and in vitro binding completed

► Affinity for NHP target < 10-fold and rodent target > 30-fold compared to the human target

► No in vivo pharmacodynamic (PD) marker available

► No related targets that the biologic could bind to and cause toxicity

IT DEPENDS ON THE BIOLOGIC AND WHAT IS KNOWN

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What Candidate Optimization Studies Should be Done?

Case #1 continued

Reactive only in NHPs and target is not anticipated to have any toxicology findings, then conduct only a single dose Pharmacokinetic (PK) study► A good example is a anti-cytokine antibody to a cytokine that’s

expressed mostly during disease; anti-IL6 mAb

► Typically don’t need more than 3 NHPs/dose, since this is range finding

► Dose selection needs to start low to observe any target mediated clearance

► Usually a single dose, because of material requirements

► Collect clinical pathology and hematology to ensure no unanticipated toxicity

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Determining Target Saturation with PK Study

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Cynomolgus Monkey PK curve

Target-mediated CL

Normal antibody CL pathways after target is saturated

Target Saturation ~ 0.5 mg/kg

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NHPs the Only Appropriate Species

Reactive Only in NHPs and Target Activation Could Result in Toxicity, then NHP PK + Toxicology Study

► EGFR is also known as Erb-1 and belongs to a family of receptors that includes Erb-2, -3 and -4

► The family members can form homodimers or heterodimers with other members of the family

► Multiple ligands bind different receptors

► Binding to other receptors in the family may be intended for the desired pharmacological effect or unintended due to structural similarity across receptors in the family

► Very complex biology

ANTI-EGFR AS AN EXAMPLE

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Case #2

NHP = Non-Human Primate

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Complexity of EGF Receptor Biology

Ligand Receptor

ErbB-1 ErbB-2 ErbB-3 ErbB-4

EGF + - - -TGF-α + - - -HB-EGF + - - +

amphiregulin + - - -

betacellulin + - - +

epigen + - - -epiregulin + - - +

neuregulin 1 - - + +

neuregulin 2 - - + +

neuregulin 3 - - - +

neuregulin 4 - - - +

24 Table from Wikipedia

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NHPs the Only Appropriate Species

► EGFR is expressed on multiple normal tissues

Skin, adrenal, cecum, colon, heart, etc.

► EGFR is over-expressed on multiple tumor types

► Mouse xenograft models with different human tumors is a common pharmacology model

Each tumor responds to a different dose of anti-EGFR

Tumor responsiveness does not always correlate with EGFR expression level

ANTI-EGFR AS AN EXAMPLE

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It is difficult/impossible to use mouse xenograft data to model doses that may cause toxicity in monkeys

Case #2 – continued

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NHPs the Only Appropriate Species

► What doses should be selected for monkey toxicology studies?

► Does the monkey PK screen identify a dose that approximates target saturation?

► How much target occupancy is needed for toxicology?

► Given the complexity of the target biology, is it possible to estimate doses that have partial pharmacology, but not toxicity?

ANTI-EGFR AS AN EXAMPLE

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Case #2 – continued

Given the complexity of targets like EGFR, having a pharmacodynamic marker of target inhibition is very useful and perhaps critical

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What Can Happen if You Don’t Have Enough Data to Establish a Dose-Response?

► No dose-response!

► If target saturation is associated with toxicity, then the result is toxicity at all doses!

► What is a safe starting clinical dose?

► Clinical hold?

POSSIBLE TO SELECT ALL DOSES THAT SATURATE THE TARGET

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Low Dose

Mid DoseHigh Dose

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How to Utilize a Pharmacodynamic Marker to Set Doses ?► Concentrations of an anti-EGFR antibody in cynomolgus monkeys following a

single dose of 1, 10, or 100 mg/kg (not actual data)

► The effective concentrations that provide 100% or 50% target inhibition (TI) are shown (not actual data)

Time after dosing (hr)

0 10 20 30 40 50 60 70

Co

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Estimated TI 50

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NHPs and Rodents, Toxicity Anticipated

Reactive in NHPs and Rodents, and is anticipated to have toxicity, then 14-day toxicity study in the rodent, 7 days in the non-rodent

►Establish target saturation in both species as in Case Study #1

►Pharmacology model(s), if available

Dose selection based on PK modeling and target saturation

Spread out the doses to cover the anticipated PKPD curve

►Use data from PK studies and pharmacology study(ies) to model doses for the non-GLP 14-day rodent and 7-day non-rodent toxicology studies

►Common to run 2 NHP/sex/gp for a “range finding” toxicology study

Typically no reversibility in Candidate Optimization studies unless a decision to discontinue development could be made

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Case #3

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Group Size Considerations for GLP Studies

► “It depends”

► Golimumab (humanized anti-TNF)Approved more than a decade after the first anti-TNF

mAb and therefore, not anticipating any serious findings

Only 2 dose groups in mouse and NHP studies

3 NHP/sex/group + 2/sex/group recovery

► When a robust data set exists, the study design can be minimal

► No absolute regulatory requirements for group size >3/sex/group

Most companies use 3-4/sex/group, a control and 3 dose groups

HOW MANY ANIMALS IS ENOUGH?

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Recovery Group Considerations

“Recovery from pharmacological and toxicological effects with potential adverse clinical impact should be understood when they occur at clinically relevant exposures. This information can be obtained by an understanding that the particular effect observed is generally reversible/non-reversible or by including a non-dosing period in at least one study, at least one dose level, to be justified by the sponsor. The purpose of the non-dosing period is to examine reversibility of these effects, not to assess delayed toxicity. The demonstration of complete recovery is not considered essential. The addition of a recovery period just to assess potential for immunogenicity is not required.”

RECOVERY: ICHS6(R1)

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Do not include recovery animals at this stage::

•Consider excluding recovery animals from initial studies•Only include recovery animals in later studies if there is an indication of need•Re-asseaa the need for recovery based on the outcome of the other studies i.e. once the toxicity profile has been determined How to include recovery animals:

Recovery Group ConsiderationsWHEN TO INCLUDE RECOVERY GROUPS AND HOW MANY ANIMALS?

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Sewell et al_Reg PharmTox_2014

When to include recovery animals:Are recovery animals necessary at this stage? i.e. does recovery need to be

assessed now, rather than in later studies, once more information is available?

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How Does this Compare to Actual Practice with FIH Studies for Biologics?

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Sewell et al_Reg PharmTox_2014

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Why Are Recovery Groups Usually Included for Biologics?

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Biologicals

Ranking Rationale for inclusion No. studies No. compounds No. companies

1234567891011

Company PracticePerceived Regulatory ExpectationBiotherapeuticLiterature/Known Class EffectsPrevious Internal Data on the CompoundPerceived First in ClassSignal from Prior In Vivo StudyOncology DrugClinical Request (internal or external)OtherFormal Regulatory Feedback

41363418131197221

(59%)(52%)(49%)(26%)(19%)(16%)(13%)(10%)(3%)(3%)(1%)

3125241511765211

(66%)(53%)(51%)(32%)(23%)(15%)(13%)(11%)(4%)(2%)(2%)

1161067554211

Sewell et al_Reg PharmTox_2014

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What are the Reasons for Not Including Recovery in Any Dose Group and the Impact?

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Biologicals

Ranking Rationale for exclusion No. studies No. compounds No. companies

123456

Company practice Signal (or lack thereof) from prior in vivo study Previous internal data on the compound No indication of need 5 Literature/known class effects (MOA) Other

11109981

(85%)(77%)(70%)(70%)(62%)(8%)

443331

(67%)(67%)(50%)(50%)(50%)(17%)

332211

Ranking Impact of exclusion No. studies No. compounds No. companies

1 None 13 (100%) 6 (100%) 4

Sewell et al_Reg PharmTox_2014

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Reversibility in All Dose Groups was Most Common. Consistent with “at clinically relevant exposures” from ICHS6(R1)?

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Summary

► Consider each biologic and design the Candidate Optimization package of studies appropriately

► Establishing a pharmacodynamic curve is critical for selecting the appropriate doses for GLP studies

Dose selection must consider pharmacology and how that might relate to toxicity/adverse events

► Reversibility should be carefully considered at clinically relevant exposures

NO TWO ARE EVER THE SAME

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Let’s Start the Conversation

Shawn Heidel, DVM, PhD

Executive Director, Global Lead Optimization and Program Management

Covance

Shawn.Heidel@covance.com

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