Toxicology for Industrial and Regulatory ScientistsToxicology for Industrial and Regulatory Scientists

Nonclinical Safety Evaluation of Biotechnology-Derived Therapeutics

Melanie Hartsough  Ph DMelanie Hartsough, Ph.D.Senior Consultant

Biologics Consulting Group, Inc.

April 30, 2015

History of biotechnology derived products

Presentation Overview History of biotechnology-derived products Fundamental differences between small and

large molecule pharmaceuticals g p Challenges specific to biologics Species specificity

Immunogenicity Immunogenicity Alternative approaches/models Reproductive/developmental studiesReproductive/developmental studies Studies in Nonclinical Programs Summary

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Pharmaceutical Products

Majority are small molecule (traditional) pharmaceuticals Extensive experience and large historical database Industry Regulatory agencies (e g FDA EMA)Regulatory agencies (e.g.,FDA, EMA)

Scientific and regulatory expectations for nonclinical toxicology studies are well defined

3

Pharmaceutical Products

Biotechnology-derived products (Biologics) Increased production over the last 25 years for use in

various clinical indicationsvarious clinical indications Relatively new (compared to small molecules) Less experience (industry; regulatory agencies) Less experience (industry; regulatory agencies) Smaller historical database (clinical and nonclinical)

4

Biologics: DefinitionS b t d i d f li i i Substances derived from living organisms Humans Animals Animals Plants

Substances produced by biotechnology methodsSubstances produced by biotechnology methods

5

Biopharmaceuticals The definition of biologics encompasses: Vaccines Blood and blood-derived products Anti-toxins

All i Allergenics Cell, gene and tissue products Monoclonal antibodies Therapeutic proteins Monoclonal antibodies Recombinant proteins Fusion proteins

Therapeutic proteins,biopharmaceuticals, biotechnology‐derived productsp

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BiopharmaceuticalsBiologics (using recombinant DNA technology) Biologics (using recombinant DNA technology) initially developed in early 1980’s Revolutionized the treatment of human disease by:

Mimicking or supplementing a human endogenous protein (growth hormone, erythropoietin)e y opo e )

Activating (agonistic) or blocking (antagonistic) a signaling pathway through specific receptor or ligand binding (monoclonal antibodyligand binding (monoclonal antibody, fusion protein)

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Bi h ti l E lBiopharmaceuticals: Examples Humulin® first approved recombinant therapeutic Humulin® - first approved recombinant therapeutic

protein was recombinant human insulin Produced by genetically modified bacteria Approved in 1982 Diabetes

Intron® - interferon--2b Approved in 1986 Hairy cell leukemiaOrthoclone OKT 3 ® (muromonab) Orthoclone OKT-3 ® (muromonab) Anti-CD3 murine monoclonal antibody (IgG2a) Approved in 1986

R l t l t

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Renal transplant

Bi h ti l E lBiopharmaceuticals: Examples Enbrel® Fc (IgG ) fusion protein Enbrel® - Fc (IgG1) fusion protein

Approved in 1998 Specific to human tumor necrosis factor

Rh t id th iti Rheumatoid arthritis Avastin® (Bevacizumab)

Anti-vascular endothelial growth factor antibody (IgG1)g y ( g ) Approved in 2004 Colorectal cancer

Vectibix® (Panitumumab) Vectibix® (Panitumumab) Anti-epidermal growth factor receptor monoclonal antibody

(IgG2)Approved in 2006

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Approved in 2006 Colorectal cancer

Bi h ti l VBiopharmaceuticals Vs. Small Molecules

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Biopharmaceuticals Vs. Small Molecules

Fundamental differences exist between small molecules and biopharmaceuticalsp Differences influence the types of nonclinical

toxicology studies to support the safety assessment Guidance documents support different

approach for the nonclinical toxicology studies (ICH S6(R1): Preclinical Safety Evaluation of Biotechnology derived Pharmaceuticals)Biotechnology-derived Pharmaceuticals)

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Biopharmaceuticals Vs Small MoleculesBiopharmaceuticals Vs. Small Molecules Small Molecules

M f t d b h i l Biopharmaceuticals

D i d f ti Manufactured by chemical processes

Derived from genetic manipulation of DNA

Manufactured by living cells

Low molecular weight1000 d lt

High molecular weight ≥ 1000 daltons

< 1000 daltons Potential for extensive

distribution within the b d

≥ 1000 daltons High molecular weight

proteins are largely confined initially to thebody confined initially to the vascular space

More limited distribution within the bodywithin the body

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S Bi h i l VStructure: Biopharmaceuticals Vs. Small Molecules

Fab = 1/3 mAb 1 structure Higher order

Fab = ~1/3 mAb

Statinstructure Post translational

modification

Statin

modification microheterogeneity

Statin MW ~400 DaMonoclonal Antibody MW ~ 150 000 Da

87 AMonoclonal Antibody MW ~ 150,000 Da

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C f M f i fConsequence of Manufacturing from Living Cells– Comparability Complexity of protein structure results in

microheterogeneity of final product Glycosylation deamidation oxidation disulfide bonds free thiols Glycosylation, deamidation, oxidation, disulfide bonds, free thiols,

clipping, aggregates

Manufacturing of biologics evolves with product d l t (i f t h l t )development (i.e., frequent changes, scale-ups, etc)

Since manufacturing changes may alter product, comparability assessments are frequently performedcomparability assessments are frequently performed

Comparability assessment ensures that the manufacturing changes have not affected the safety,

ff fidentity, purity or efficacy, including immunogenicity, of the product

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C f M f i fConsequence of Manufacturing from Living Cells– Comparability Analytical and functional assays performed (quality

attributes/ CMC issues) If product comparability cannot be established with If product comparability cannot be established with

quality assessments, preclinical studies may be required PK assessments PD assessments Toxicology studies Toxicology studies

Product used in nonclinical studies not required to be “identical” to that going into the clinic, but does have to be “comparable” in order to extrapolate the preclinical safety data to the clinical scenario

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Bi h ti l V S ll M l lBiopharmaceuticals Vs. Small Molecules Small Molecules

Metabolized by Liver Biopharmaceuticals

Catabolized by body Metabolized by Liver Biotransformation studies Metabolites identified

Catabolized by body (proteolytic degradation) Amino acids Small peptides

Potential for active or toxic metabolites

S a pep des Active or toxic breakdown

protein products generally not a concern

Biotransformation studies generally not performed

Biopharmaceuticals Vs. Small Molecules

Small Molecules Biopharmaceuticals Small Molecules Drug-drug interactions

potential concern Half-life relatively short

Biopharmaceuticals Drug-drug interactions are

less of a concern Half-life can be long Half-life relatively short

Minutes Hours Few days

Half life can be long mAb-Days to weeks

y Impurities generally consist

of: Organic residuals

Impurities generally consist of host cell molecules: DNA

Solvents Toxicity studies for

impurities may be

Protein Toxicity studies for

impurities usually not d dnecessary needed

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Biopharmaceuticals Vs. Small Molecules

Small Molecules Biopharmaceuticals Small Molecules Less target specificity

(compared to biologics)

Biopharmaceuticals High target specificity Toxicity often related to ( g )

Toxicities often non-specific/not related to the intended target

o c y o e e a ed ointended target/ pharmacological action of the drugthe intended target

“Off-target toxicity”of the drug “On-target toxicity” Exaggerated

pharmacology

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Biopharmaceuticals Vs. Small Molecules

Small Molecules Immunogenicity

generall not a concern

Biopharmaceuticals Immunogenicity is often

a challengegenerally not a concern

Species limitations

a challenge

Species limitations may Species limitations generally not a concern 2 species usually used for

toxicology studies

Species limitations may be a challenge Only 1 relevant animal

species may be availabletoxicology studies (1 rodent; 1 non-rodent)

species may be available

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Properties of Biologics that Can Influence Nonclinical Toxicology Programs High degree of target and species specificity High degree of target and species specificity Immunogenicity

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Species Specificity

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Properties of Biologics:Properties of Biologics:Species Specificity

Most biopharmaceuticals are: Human proteins that are highly targeted to a human receptorHuman proteins that are highly targeted to a human receptor Antibodies specific for a human protein or receptor

Due to high specificity to the human target many Due to high specificity to the human target, many biopharmaceuticals do not recognize the target in all animal species commonly used in nonclinical t i l t ditoxicology studies. Mouse, rat, rabbit, dog, minipig, monkey

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Species Specificity Toxicology studies for biopharmaceuticals should beToxicology studies for biopharmaceuticals should be

conducted in a pharmacologically relevant species (ICHS6(R1)).

Relevant species is one in which the biopharmaceutical is pharmacologically active due to the expression of the intended target (e.g., receptor or epitope).g ( g , p p p )

Toxicology studies in non-relevant animal species (one that does not express the target) can be misleading and

di d (ICHS6(R1))are discouraged (ICHS6(R1)). Unreliable safety data Not predictive of potential human toxicity

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Species Specificity How to demonstrate relevant species: How to demonstrate relevant species: Sequence identity of target Target expression and distributionTarget expression and distribution Binding assays Biacore, flow cytometry, ELISA

Functional bioassays Cell proliferation, signal transduction modulation, enzyme

changeschanges In vivo pharmacology

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S i S ifi itSpecies Specificity

There are cases when only one relevant animal species can be identified

M t ft h i t ( l Most often nonhuman primates (e.g., cynomolgus monkeys)

One species is acceptable with sufficient justificationOne species is acceptable with sufficient justification

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Species Specificity Nonhuman PrimatesSpecies Specificity – Nonhuman Primates NHP often the only pharmacologically relevant animal species

for toxicology studies gy Old world NHPs Cynomolgus monkey

Most common NHP used (larger historical database)( g ) Rhesus monkey

New world NHPs (marmosets) Limited historical database for toxicity studiesLimited historical database for toxicity studies Very sensitive to disruptions in environment More often used for PK studies

Great apes Great apes Chimpanzees are NEVER appropriate for toxicology!

Limited data obtained (endangered species) Small animal number and large variability Small animal number and large variability Difficult data interpretation Experimentally naïve animals not available

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Ch ll U i N h P i t Various challenges using NHP as the animal model in

t i l t di

Challenges Using Nonhuman Primates

toxicology studies Limited availability (compared to rodents and canines) Contact CROs early to place study

Limited number of animals/group (compared to rodents/canines) Can make data interpretation difficult

Cost is high (compared to rodents/canines) Cost is high (compared to rodents/canines) particularly sexually mature animals!

Background and/or sub-clinical disease ( i b t i l it )(viruses, bacterial, parasites) Hepatitis, malaria, shigella Impact data interpretation

Particularly if the molecule is an immunosuppressive Particularly if the molecule is an immunosuppressive Are observed changes due to pre-existing infection or molecule?

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Challenges Using Nonhuman Primates Cont. NHPs of different originsg Indonesian vs. Chinese origin

NHPs are heterogenousC d t i b d i t Compared to inbred mice or rats

Increases variability in data

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Species Specificity There are cases when NO relevant species can be There are cases when NO relevant species can be

identified Specific for human or human and chimpanzeep p Alternative approaches can be used to identify

potential safety risks Alternative approaches: Surrogate/analogous proteins

T i i l Transgenic animals Knockout animals Animal models of disease Animal models of disease

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Immunogenicity

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Immunogenicity

Immunogenicity = immune response against the product, resulting in production of anti-product antibodiesantibodies Biopharmaceuticals are often immunogenic in

animalsanimals Abbreviations that you may see: ADA = anti-drug antibodies RAHA= rat anti-human antibodies PAHA = primate anti-human antibodies MAHA = monkey anti-human antibodies MAHA = monkey anti-human antibodies

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Immunogenicity Types of anti-product antibodiesyp p Binding Minimal to no impact on product exposure

Clearing Increase clearance of product which decrease systemic

exposure of productexposure of product Can alter distribution of product to tissues

Sustaining (rare) Decrease clearance of product which prolong half-

life/exposure of product Potentially lead to or increase toxicityy y

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Immunogenicity

Types of anti-product antibodies Neutralizing Interfere with product binding to target Neutralize pharmacological activity of product

Cross-reactive with endogenous proteinsCross reactive with endogenous proteins Neutralize the pharmacological activity of the product and

the corresponding endogenous proteinC i f t li ti f ti it f d d t Concern is for neutralization of activity of nonredundant system

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Immunogenicity Anti product antibodies can affect the outcome of a Anti-product antibodies can affect the outcome of a

toxicology study No effect Decrease exposure by increasing clearance of the

product or neutralizing the product’s activity Sustain exposure by decreasing clearance (rare) Cause adverse effects not directly related to the

product (anaphylaxis immune complex deposition)product (anaphylaxis, immune complex deposition) Cross-react with the endogenous protein

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Immunogenicity Included as an endpoint in biopharmaceutical Included as an endpoint in biopharmaceutical

toxicology studies Aid in the interpretation of the study p y

Primary concern is for the development of clearing and/or neutralizing anti-product antibodies Lower exposure of the target organs to

biopharmaceutical Generation of misleading toxicity data Generation of misleading toxicity data

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Immunogenicity

Mere presence of anti-product antibodies is not a reason to terminate a study (does not invalidate the study)study) Sufficient number of animals may be exposure to

active productp Studies terminated when complete loss of exposure of

activity of product and/or severe adverse reactions l d dditi l d ipreclude additional dosing

36

Immunogencity Various factors can influence the immunogenic Various factors can influence the immunogenic

potential of biopharmaceuticals Route of administration

Inhalation> SC> IP> IM >IV > topical* Duration of dosing

Typically repeated dosing is more immunogenic than a single dose

Product Protein structure Manufacturing processes (glycosylated vs. nonglycosylated) Impurities (DNA, host cell proteins) Aggregates (high order)

*Braun A et al (1997) Pharm Res. 14(10) 1472-14878

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Immunogenicity Anti-product antibodies may make long term toxicity

studies and reproductive and developmental studies difficultdifficult

Immunogenicity in animals is not necessarily predictive of human response (ICHS6(R1))predictive of human response (ICHS6(R1))

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Immunogenicity Case Studyg y y

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I i itImmunogenicity

St d D i Study Design: Human monoclonal antibody

Administered to cynomolgus monkeys Administered to cynomolgus monkeys Intravenous injection

Once weekly for 4 weeksy Development of clearing ADAs resulted in:

Increase drug clearance Decreased exposure over time

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Clearing Antibody: Impact on Exposure

Day 1 Day 281000 1000

100 100

n (

g/m

l)

n (

g/m

l)

Antibody negative

1

10

1

10

ncen

trat

ion

ncen

trat

ion g

0.1

1

0.1

1

rug

X C

on

rug

X C

on

0.01 0.01Seru

m D

r

Seru

m D

r

Antibody positive

41Time (days)

0 2 4 6 8 10 12 14

Time (days)

0 2 4 6 8 10 12 14

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Alternative Approaches and Models

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Alt ti A h /M d l In some cases, alternative approaches for the

Alternative Approaches/Modelspp

nonclinical toxicology studies for biologics are necessary Product pharmacologically active in humans only Product pharmacologically active in humans only Anti-product antibodies imposed limitations on the ability

to conduct comprehensive nonclinical safety studies Clearing and/or neutralizing antibodies Unable to maintain exposure/pharmacological activity

throughout studyg y ICH S6(R1) – allows for flexibility so alternative

approaches can potentially be used for the nonclinical safety assessment

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Alternative Approaches/Models Homologous (Surrogate/Analogous)

proteinsp Transgenic animals Knockout animals Knockout animals Animal models of disease

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H l P t i Homologous proteins recognize the ortholog

Homologous Proteing p g g

of the original human target Animal models

Mouse (most common)Mouse (most common) Rat Cynomolgus monkey

Advantage Advantage Good model for molecules that cross-react only with

human target Most commonly used alternative methodMost commonly used alternative method

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H l (S t ) P t i

ICH S6(R1) h l t i h ld

Homologous (Surrogate) Protein

ICH S6(R1)- homologous proteins should resemble the clinical candidate as much as possible with regard to: p g Production process Range of impurity and/or contaminant profileN t h t i th h l Necessary to characterize the homologous protein to ensure it is a suitable surrogate for the clinical candidateclinical candidate

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H l (S t ) P t i Demonstrate surrogate has pharmacological

Homologous (Surrogate) Protein Demonstrate surrogate has pharmacological mechanism similar to clinical candidate Binding studies

A /bi k f h l i l ti it Assays/biomarker of pharmacological activity Downmodulation/upmodulation of receptor/target or expression of cytokine(s)

or proliferation of certain cell populations Necessary to characterize PK and ADA responses of

surrogate in animal species used in toxicologysurrogate in animal species used in toxicology studies

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H l (S t ) P t i

N t d t i th d f th h l

Homologous (Surrogate) Protein

Necessary to determine the dose of the homologous protein in your animal species that is pharmacologically equivalent to the human dose g ywith the clinical candidate Based on pharmacology and PK data Data used to determine doses used for toxicologyData used to determine doses used for toxicology

studies with the homologous protein

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Homologous (Surrogate) Protein Challenges using an homologous protein:

Homologous (Surrogate) Protein g g g p

It will never be your clinical candidate! Differences from clinical candidate

Manufacturing process Manufacturing process Different impurity profile PK and ADA profile

Example: lower exposure with surrogate due Example: lower exposure with surrogate due to high rate of ADA responses

Pharmacological activity Potency equal to clinical candidate?Potency equal to clinical candidate? Mechanism of action similar?

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Homologous (surrogate) Protein

Challenges using an homologous protein: No established criteria or regulatory guidance for

h l t i l t dipharmacology or toxicology studies How rigorous should the assessment of the

pharmacological/toxicological activities of the homologue be? Aggregates Host-cell proteins

How rigorous should assessment of the homologous material be?

Company/regulatory concerns Suitability of homologue to characterize safety of clinical y g y

candidate when compared to human data

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Homologous (Surrogate) ProteinHomologous (Surrogate) Protein Challenges using an homologous protein:g g g p Resource intensive! Development & manufacturing resources needed

(essentially developing a 2nd molecule!!) Generation of material Characterization of material Characterization of material

Impurities Aggregates Formulation excipients differ from clinical candidate

Studies to demonstrate comparability to the clinical candidate

Assay development for PK and ADA 51

Alt ti A h /M d l Transgenic animals (mice)

Alternative Approaches/Models

Animal model expressing human target Often used in pharmacology studies

Advantageg Allows for evaluation of clinical candidate (unlike

surrogate) Limitations

Expensive to develop and maintain colony Lack of historical data; data interpretation can be difficult Limited lifespan; limited number of animals Spontaneous pathologies can be associated with having

the gene expressed

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g p May be difficult to produce mice with consistent

expression and tissue distribution of the human target

Alt ti A h /M d l Knockout models

Alternative Approaches/Models

Evaluate the pathologies associated with not having the target expressed

Limitations Expensive to develop and maintain colony

Lack of historical data Lack of historical data Limited lifespan Limited animal availabilityy Other spontaneous pathologies associated with not

having gene expressedD l i di t l i l’ lif

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Develop immediately or over animal’s life span Does not mimic therapeutic setting

Alt ti A h /M d l Animal models of disease

Alternative Approaches/Models

Allows for evaluation of general toxicity and undesirable promotion of disease progression during treatment with drugdrug

Limitations Lack of historical data Limited lifespan or number of animals Confounding effects of disease

May interfere with interpretation of the toxicological data GLP compliance may be difficult due to the complexity

of these studies

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Often available in research facilities that do not have GLP capabilities

Reproduction and Development

Toxicity StudiesToxicity Studies

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Reproduction/DevelopmentReproduction/Development Studies ICHS6(R1)- Studies performed according to

ICHS5(R2); however, study design and dosing schedule may be modified based on speciesschedule may be modified based on species specificity, the nature of the product and mechanism of action, immunogenicity and/or PK behavior and , g yembryo-fetal exposure Studies should only use pharmacologically relevant

species, ideally with clinical product

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Reproduction/DevelopmentReproduction/Development Studies Studies should be performed in rat and rabbit, if

pharmacologically relevant species When no relevant species, alternative models

may be explored If weight of evidence (e.g., mechanism of action,

phenotypic data from genetically modified animals class effects etc) suggests adverseanimals, class effects, etc) suggests adverse effect on fertility or pregnancy outcome may not need to perform studiesneed to perform studies

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Reproduction/DevelopmentReproduction/Development Studies If NHP only relevant species: Only developmental studies are necessaryy p y May use alternative model with scientific justification Study may be conducted during Phase 3 and

submitted at time of licensure, as long as appropriate clinical precautions are taken

ICHS6(R1) provides a design consideration for ICHS6(R1) provides a design consideration for combined embryo-fetal and pre/post-natal development (PPND) study (referred to as ePPND)

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Reproduction/DevelopmentReproduction/Development Studies

Effects on fertility can be assessed in repeat-dose toxicity studies of at least 3 months

Sexually mature NHPs Sexually mature NHPs evaluation of reproductive tract (organ weights and

histopathological evaluation) If concern, menstrual cyclicity, sperm count, sperm

morphology/motility, and reproductive hormone levels can be evaluated

Not recommended to develop homologous protein or transgenic model to conduct mating studies in rodents

Studies are used for hazard identificationStudies are used for hazard identification

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Nonclinical Studies to Support th S f t f Bi h ti lthe Safety of Biopharmaceuticals

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Biopharmaceutical NonclinicalBiopharmaceutical Nonclinical Programs Pharmacodynamic studies Pharmacodynamic studies

Mechanism of action, proof of concept

Safety Pharmacology Studies Many times can be incorporated into the general toxicology

studies (ICHS7a) hERG assay not performed y p

Pharmaco-/toxicokinetics Absorption studies

Ti di t ib ti t di t t i ll f d d t Tissue distribution studies not typically performed due to limitations with data interpretation

Metabolism/ excretion studies not typically performed ( t b li )(catabolism)

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Biopharmaceutical NonclinicalBiopharmaceutical Nonclinical Programs General toxicology studies Program designed based on properties of the

bi h ti lbiopharmaceutical Human Tissue Cross-reactivity study (generally

mAbs only)mAbs only) Local tolerance studies Incorporated into the general toxicology studiesIncorporated into the general toxicology studies

Genotoxicity assay Not typically performed, inability of product to enter y y y

living cells

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Biopharmaceutical NonclinicalBiopharmaceutical Nonclinical Programs Reproductive and developmental toxicology studies

Study designs dependent upon relevant species, immunogenicity, mechanism of action embryo-fetal exposuremechanism of action, embryo fetal exposure

If NHP the only relevant species- studies are more complicated ICHS6(R1) provides design considerations

C i i it t di Carcinogenicity studies Weight of evidence approach Published data, class effects, product biology and mechanism of p gy

action, etc Studies may not be required and/or may not be feasible

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Summary There are fundamental differences between There are fundamental differences between

biopharmaceuticals and small molecules Properties of biologics can influence the approachProperties of biologics can influence the approach

and design for the nonclinical toxicology studies Critical to conduct the appropriate study based on

the properties of the product pharmacologically relevant species

I i it fil Immunogenicity profile

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Summary

Consider using alternative models for the program when appropriate

E Ab th t t l ith h t t Ex: mAb that cross-reacts only with human target Develop surrogate antibody rather than conducting toxicity

studies in non-relevant animal species

There is “no one program fits all” approach for biopharmaceuticals

65

Summary Approach for the nonclinical safety assessment Approach for the nonclinical safety assessment

strategy for a biopharmaceutical should be based on: Scientific rationale Relevant species, mechanism of action, study feasibility,

other product propertiesother product properties Indication/patient population Ex: Oncology vs psoriasis Ex: Women of child-bearing potential vs. post-menopausal

women Relevance of the studies for determining and Relevance of the studies for determining and

understanding human risk66

ReferencesReferences ICH S6 (R1): Preclinical Safety Evaluation of Biotechnology-Derived Products (1997), with addendum (2011)Derived Products (1997), with addendum (2011) ICHS5 (R2):Detection of Toxicity of Reproduction for Medicinal Products and Toxicity to Male Fertility (2000) Points to Consider in the Manufacture and Testing of Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (CBER, 1997) Raptiva®, FDA Review, Application Number 125075/0, approval 10/27/03 Clarke, J. et al (2004). Reg. Tox. Pharm., 219-226 Braun, A. et al (1997). Pharm. Res, 14(10), 1472-1478Braun, A. et al (1997). Pharm. Res, 14(10), 1472 1478 Mounho, B. et al (2003). The Toxicologist #1880 pg 387 Clarke, J. et al (2003). The Toxicologist #361 pg 74 www bio org

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www.bio.org www.fda.gov

Extra Slides

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Homologous Antibody: Case ExampleHomologous Antibody: Case Example

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C E l Efalizumab (Raptiva®)

Case ExampleEfalizumab (Raptiva ) Humanized IgG1 monoclonal antibody to human

CD11a Cross-reacts only with human and chimpanzee CD11aCross reacts only with human and chimpanzee CD11a

Indication: moderate-severe plaque psoriasis Men & women of childbearing age

Administered chronically Administered chronically Chronic and reproductive toxicity studies necessary

Developed surrogate antibody Chimeric rat/mouse anti-mouse CD11a antibody

muM17 Toxicology studies to support registration were conducted

i CD 1 i

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in CD-1 mice

Clarke, J. et al (2004). Reg. Tox. Pharm., 219-226Raptiva, FDA Review, Application Number 125075/0, approval 10/27/03

C E l C t Evaluation of muM17 as suitable surrogate antibody

Case Example Cont. Evaluation of muM17 as suitable surrogate antibody for efalizumab Binding affinity to CD11a

efalizumab to human CD11a ~ 3.0 nM muM17 to murine CD11a ~ 2.7 nM

Similar tissue binding propertiesg p p Efalizumab: human leukocytes in blood and spleen muM17: mouse leukocytes in blood and spleen

Characterize PK/antibody responses of muM17 in mice Characterize PK/antibody responses of muM17 in mice Demonstrate pharmacological activity of muM17 in mice

similar to efalizumab in humans

71Clarke, J. et al (2004). Reg. Tox. Pharm., 219-226Raptiva, FDA Review, Application Number 125075/0, approval 10/27/03

decrease CD11a expression on T lymphocytes

C E l C t Determined dose of muM17 in mice that was

Case Example Cont.

pharmacologically equivalent to the efalizumab human dose Based on CD11a downmodulation on T lymphocytes Based on CD11a downmodulation on T lymphocytes

Very sensitive biomarker on pharmacodynamic activity for efalizumab in humans and muM17 in mice

3 mg/kg/week identified as minimal muM17 dose that maintained maximal CD11a downmodulation Approximately equivalent to efalizumab human dose pp y q

of 1 mg/kg/week muM17 doses for toxicology studies

3, 10, and 30 mg/kg (10-fold safety factor based on dose)

72Clarke, J. et al (2004). Reg. Tox. Pharm., 219-226Raptiva, FDA Review, Application Number 125075/0, approval 10/27/03

C E l C t Toxicology program with muM17

Case Example Cont.

Repeated-dose toxicity (up to 6 months duration) Reproductive toxicity

F tilit Fertility Embryo/fetal development Peri/postnatalp

Immunotoxicology Adult mice

Off i b t d d i i t d M17 d i Offspring born to dams administered muM17 during gestation and lactation

73Raptiva, FDA Review, Application Number 125075/0, approval 10/27/03

Transgenic Animal Model: Case ExampleTransgenic Animal Model: Case Example

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T i A i l M d l C St d Keliximab

H l k hi i Ab

Transgenic Animal Model: Case Study

Human-cynomolgus monkey chimeric mAb directed against human CD4

Cross-reactivity limited to human CD4y Indication

Rheumatoid arthritisM l d f l f hildb i Males and females of childbearing age

Chronic treatment

Developed human CD4 transgenic mouseDeveloped human CD4 transgenic mouse Model (HuCD4/Tg mice)

Murine CD4 knockout, human CD4 knock-in

75Bugelski, PJ et al. (2000). Hum. Exp. Toxicol. 19: 230 – 243

Case Study Cont Necessary to characterize HuCD4/Tg mice to ensure

suitable animal model for toxicology studies

Case Study Cont.

suitable animal model for toxicology studies Compare distribution of cells expressing human CD4

transgene in comparison to murine CD4 Demonstrate pharmacological activity of keliximab in

HuCD4/Tg miceDepletion of HuCD4+ lymphocytes Depletion of HuCD4+ lymphocytes

Determine suitability of mice for chronic studies with regard to background pathologies and survivalregard to background pathologies and survival

Ensure immune system of mice was competent Immunotoxicology studies

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Developing/maintaining animal model resource intense!

C St d C t Toxicology studies using HuCD4/Tg mice

Case Study Cont.

Short term and chronic (6-month) toxicity studies Male/female fertility Embryo-fetal development Immune function in F1 mice

Host resistance to infection Host resistance to infection Immune function

Host resistance to infection

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