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DOI: 10.1177/1091581809337262 2009; 28; 230 Int J Toxicol

Beyer and Christopher Horvath Jeanine L. Bussiere, Pauline Martin, Michelle Horner, Jessica Couch, Meghan Flaherty, Laura Andrews, Joseph

Alternative Strategies for Toxicity Testing of Species-Specific Biopharmaceuticals

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Alternative Strategies for Toxicity Testingof Species-Specific Biopharmaceuticals

Jeanine L. Bussiere, Pauline Martin, Michelle Horner,Jessica Couch, Meghan Flaherty, Laura Andrews,Joseph Beyer, and Christopher Horvath

Although toxicology studies should always be conductedin pharmacologically relevant species, the specificity ofmany biopharmaceuticals can present challenges inidentification of a relevant species. In certain cases,that is, when the clinical product is active only inhumans or chimpanzees, or if the clinical candidateis active in other species but immunogenicity limitsthe ability to conduct a thorough safety assessment,alternative approaches to evaluating the safety of a bio-pharmaceutical must be considered. Alternativeapproaches, including animal models of disease, gene-tically modified mice, or use of surrogate molecules,

may improve the predictive value of preclinical safetyassessments of species-specific biopharmaceuticals,although many caveats associated with these modelsmust be considered. Because of the many caveats thatare discussed in this article, alternative approachesshould only be used to evaluate safety when the clinicalcandidate cannot be readily tested in at least one rele-vant species to identify potential hazards.

Keywords: alternate strategies; biologics; biopharma-ceuticals; genetically modified mice; homologous pro-teins; surrogate molecules

Although toxicology studies should always beconducted in pharmacologically relevant spe-cies, the high degree of species specificity of

many biopharmaceuticals can present certain chal-lenges in identification of a relevant species. Thisspecificity also implies that the toxicity is generallydue to on-target activity (based on the intended phar-macology) rather than off-target effects (nonspecificbinding) as is often the case for traditional smallmolecule pharmaceuticals. ICH S61 defines a phar-macologically relevant species as ‘‘one in which thetest material is pharmacologically active due toexpression of the receptor or an epitope (in the case

of monoclonal antibodies).’’ In some cases, it maynot be possible to evaluate the toxicity of a clinicalproduct in animals (for example, when the productis active only in humans or chimpanzees, or if immu-nogenicity limits the ability to conduct a thoroughsafety assessment). Alternative approaches to evalu-ating the safety of a biopharmaceutical should beconsidered in these cases. ICH S6 has identifiedthe following as potentially viable alternativeapproaches: animal models of disease, transgenic/knock-out models, and surrogate molecules (Table 1).Each of these has unique advantages and disadvan-tages that must be considered in the developmentof preclinical safety assessment strategies (Table 2).Multiple approaches to understanding the toxicitiesof a development compound may be equally valid,provided that they can be scientifically justified asrelevant to understanding the potential safety ofthe molecule. This article will discuss some of thebenefits and limitations of each approach and willprovide a series of examples from approved productsas well as current development programs on how

From Amgen Inc, Thousand Oaks, California; Centocor Research& Development, Inc, Radnor, Pennsylvania; Genzyme,Framingham, Massachusetts; Genentech Inc, South SanFrancisco, California; and Taligen Therapeutics, Cambridge,Massachusetts.

Please address correspondence to Jeanine L. Bussiere, PhD,DABT, Scientific Executive Director, Toxicology, Amgen Inc,One Amgen Center Dr, MS 29-2-A, Thousand Oaks, CA91320-1799; e-mail: [email protected].

InternationalJournal of ToxicologyVolume 28 Number 3

May/June 2009 230-253# 2009 The Author(s)

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these alternative approaches have been used.Although most of the discussion will focus onprotein biopharmaceuticals (eg, recombinant pro-teins, monoclonal antibodies, and fusion proteins),the principles for alternative testing approaches arealso relevant for nonprotein biopharmaceuticals(eg, peptides, antisense oligonucleotides, small inter-fering RNAs, and aptamers) and could potentially beapplied to some new chemical entities (eg, smallmolecules showing restricted species specificity).

Animal Models of Human Disease

One challenge in identifying a pharmacologicallyrelevant species for biopharmaceutical toxicity

testing is that, although the target may be expressedin different animal species and may even havecomparable function as in humans, the target maynot be recognized (pharmacologically modulated)by the clinical candidate. Alternatively, the therapeu-tic target may be expressed or upregulated only incertain disease settings. Examples include nonhostproteins such as viral or bacterial antigens that arenot normally present in healthy animals, or hostproteins that are expressed only in certain diseasestates such as unique cancer antigens, Rh factor, oraltered (mutated) host proteins. Because such tar-gets are unlikely to be present in normal animals,these represent perhaps the most challenging targetsfor which to assess the pharmacologic and toxicolo-gic effects of the clinical candidate.

Table 2. General Issues With Nonclinical Development Programs for Biopharmaceuticals LackingCross-Reactivity in Traditional Species (ie, Rat and Dog)

Evaluate Human Test Article(Clinical Candidate) in

Cross-Reactive NonhumanPrimate Species

Evaluate Surrogate Rodent Homologous TestArticle in Rodent Species

(Mouse or Rat)

Evaluate Effect of GeneticDeletion of Target in

Knockout Mice

Preference May be preferable because usingactual test article

May be less preferable because not evaluatingactual test article

Relevance Relevance to humans is assumedinitially

Relevance to humans is questioned initially

Acceptance Historically well accepted(becoming ‘‘traditional’’)

Historically of varying acceptance (remainsnontraditional)

Design andtiming

May require nonstandard studydesigns, cost structures andtimelines

Standard study designs modeled after traditionalstudies with standard cost structures ($, Personnel)and standard timelines

Expenses Standard costs for materials,assays, reagents; often serve as‘‘dry run’’ for clinical trials

Requires dual development of clinicalcandidate and homologue; additional costfor surrogate materials, assays, reagents

Additional costs for breeding,genotyping, assays and reagents,licensing fees, etc

Otheroptions

Sometimes the ‘‘relevant’’species may not be primate(eg, pigs)

Sometimes the surrogate homologue may betested in a non-rodent species (eg, primate)

Sometimes the clinical candidate maybe tested in rodent knockout micewith human ‘‘knockin’’ target

Table 1. Alternative Approaches Identified in ICH S6

Approach Advantages Limitations

Animal modelsof disease

More closely mimics effects in humanpatients

Lack of historical data; limited life span; confounding effects ofdisease

Geneticallymodifiedanimals

Can gather data in species where nocross-reactivity exists

Can do additional studies in a morestandard toxicology model

Lack of historical data; limited number of animals; limited life span;technical challenges

Surrogatemolecules

Can gather data in species where nocross-reactivity exists

Can do additional studies in a morestandard toxicology model

Different product (production process, impurities, mechanism);technical challenges, pharmacology differences (ie, potentialdifferences in signaling based on the epitope that the surrogatebinds)

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Animal models of human disease have been usednot only to facilitate compound selection and allowfor an early understanding of mechanism of actionbut also to assess safety. The use of animal modelsof disease to assess in vivo activity as well as toxicitycan provide a better understanding of the therapeuticindex and therefore improve clinical dose selection.2

Animal models of human disease may exist spon-taneously (eg, dogs with factor VIII deficiency)or may be experimentally developed for preclinicaltesting. Common examples of the latter include theuse of virally challenged animals to test the efficacyof a vaccine or a therapeutic directed against viralproteins, mice inoculated with xenogenic (human)tumors expressing the target antigen, micechallenged with infectious agents, or geneticallymodified animals that develop spontaneous disease.As with any model, there are both advantages anddisadvantages to this approach (Table 1). One keyconsideration is that the immune system may be verydifferent between rodents and humans.3 Forexample, animal models of many inflammatory dis-eases such as multiple sclerosis, where complexmulticomponent processes are involved, may differsubstantially between mice and humans.4 In addi-tion, animal models of disease are generally not wellcharacterized to understand what the ‘‘background’’lesions are relative to what may be a compound-related effect.

Genetically Modified Animals

Genetically modified (knock-out, knock-in, trans-genic) mice are rapidly gaining acceptance as toolsfor mechanistic research and validation of targetbiology and have considerable potential as specificmodels of toxicological importance. Gene-targetedor knock-out (KO) animals have been created usingmolecular and cellular genetic engineering tech-niques to produce animals that specifically lack anendogenous gene and therefore fail to express therelated protein(s), whereas transgenic (Tg) mice areengineered to overexpress a target protein.5 ManyKO and Tg mice appear morphologically normalalthough functional abnormalities may be apparent;in other cases, these mice may exhibit a normalphenotype. Pharmacological challenges or otherphysiological stressors may also unmask subtle phe-notypes (functional and/or morphologic changesresulting from the genetic engineering event).6,7 In

addition, alteration of a target that has a significantrole in prenatal growth and development may havesignificant adverse effects (eg, may be embryolethal)that are not relevant when inhibiting the target in anadult. Embryolethal effects can be avoided throughdevelopment of conditional KO animals in which tar-get disruption is limited to a specific developmentalstage and/or tissues of interest.8

Knock-out mice have been used to assess drugspecificity, investigate mechanisms of toxicity, andscreen for mutagenic and carcinogenic activities oftherapeutic candidates. Similarly, the effect of targetblockade by novel therapeutic candidates can beestimated in KO mice; for example, generation ofviable and fertile animals with null mutations for apotential target protein may suggest that pharmaco-logical inhibition of the molecule in vivo is unlikelyto elicit major adverse effects on normal physiologi-cal functions.9 As such, an apparent lack of a deleter-ious phenotype could be used as supportive evidencefor the safety of inhibiting the intended target follow-ing extensive characterization of the KO mouse.

However, because KO mice may have uncharac-terized compensatory mechanisms or redundantpathways that are not readily apparent to replace thefunction of the absent protein(s) or target,10-12 theiruse in assessing safety will likely be supportive ratherthan providing definitive safety data. One possiblealternative to avoid this issue could be directedtoward utilizing conditional KO mice, which permitevaluation of effects resulting from inhibition of aparticular gene product during only the relevantstage of life. Finally, when considering animal mod-els of disease using a complete or partial gene KO, itis important to consider the genetic structure andfunction of the gene, because genetic mutationsunderlying human disease may have a different phy-siological effect in the rodent because of differencesin gene structure and/or function between species.13

‘‘Humanized’’ knock-in (KI) animals, in whichthe human gene is inserted into the mouse genome(either independently or in conjunction with aknock-out of the endogenous mouse gene), are ofparticular utility in evaluating the efficacy andtoxicity of human biopharmaceuticals that are notpharmacologically active in normal rodents.14,15 Onecriticism of this approach is that humanized micemay express one or a few human proteins of interest,but other proteins that interact with the humanmolecules are still of mouse origin. The physiologicaleffects of human-mouse protein interactions may

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differ slightly—or substantially—from that of thenormal human-human association (eg, Toll-likereceptors16,17). It is important to note that in severalcases, KI mice have been used to support appro-priate function of the human KI gene.13,15 Prior touse, comprehensive studies must be conducted todefine the biology of mouse-human protein interac-tions to allow validation of the human gene KI miceas appropriate models. As transgenic insertions maynot be targeted to a specific site in the genome,some of the regulatory sequences around them mayfunction differently than those of the endogenousgene, which may affect both spatiotemporal patternsas well as overall levels of protein expression. Inaddition, administration of the clinical candidate inthese animals may still be immunogenic thus limit-ing long-term studies in KI mice.

With the increasing number of biopharmaceuti-cals on the market, data from studies in geneticallyengineered mice will become important to demon-strate that they have utility as a viable alternative totesting in nonhuman primates and that findings ofefficacy and toxicity obtained using these models arerelevant to humans. This will take time, because theultimate validation will not occur until there areclinical data to compare with relevant preclinicalmodels.

Despite their potential utility, the use of KO/Tgmice for toxicity studies may be technically challen-ging because of the activity of the target (ie, may bedifferent in mice and humans) and/or early embryoniclethality. In cases of embryonic lethality, developmentof alternative models, such as the BRCA1 andBRCA2 conditional KO animals, may enable a moreappropriate evaluation of target inhibition in theadult animal.8

The technical and feasibility challenges associ-ated with using KO/Tg mice may significantly affectprogram time lines, and consequently, the potentialuse of KO/Tg mice in a safety assessment programmust be considered very early in development. Aspreviously discussed, standard KO mice that lack atarget throughout embryogenesis and developmentmay not accurately reflect disease or pharmacologi-cal interventions in the clinical scenario, whereprotein function may be nullified only during adult-hood. Another important consideration when usinga KO/Tg mouse is that they are often generatedon different background strains (which may producedifferent phenotypes) or on strains that havenot typically been used in toxicity assessments

(eg, C57Bl/6 and S129), and therefore less historicaldata may be available. Multigenerational studies orincreased numbers of KO/Tg control animals maytherefore be required for interpretation of safetyresults in the absence of adequate historical data.This may be particularly pertinent in reproductivetoxicity assessments, where fetal variations orabnormalities may be strain dependent. For lesswell-characterized strains of mice, it may be neces-sary to rely on the characterization of the wild-typemice as well as the genetically modified mice. Asimilar situation exists for pharmacology or diseasemodels, which are notoriously specific for certainstrains of mice. Immune function in these strains isgenerally not well characterized and specific straindifferences are known to exist, especially in theimmune response.18 Therefore, the pharmacologicactivity of the biotherapeutic must be evaluated inthe KO/Tg animal prior to committing to its use forsafety assessment. Last, genetically modified miceare often developed in discovery or academicresearch settings, where the incidence of latent oropportunistic pathogenic infections tends to behigher, and may necessitate rederivation of the strainprior to scaling up their production. This couldpotentially result in a 1- to 2-year delay before themodel would be ready to use in a safety assessmentsetting. During the rederivation process andthroughout the use of a KO/Tg model, it may benecessary to genotype each animal to ensure thathetero- or homozygosity and/or copy number areappropriate for the desired genetic modification.There are also certain KO/Tg mice that are sterileor not viable as homozygotes, so the animals mustbe generated from heterozygotes and therefore mustbe genotyped each time. Finally, as all of thesemodels have strengths and limitations, the scientificrationale for their use must be justified andthoroughly discussed (generation of the geneticallymodified animal, backcrossing stage, strain, etc).

Surrogate Molecules

Considerations for Use of a SurrogateMolecule for Safety Assessment

For the purpose of this discussion, a surrogate mole-cule is one that is selected for use in place of thedevelopment compound to evaluate pharmacologyand/or toxicology parameters in a species that is

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believed to be biologically relevant to humans. Thesemolecules have also been referred to as analogous orhomologous compounds. However, until the clinicalcandidate has been evaluated in humans, the extentto which the surrogate molecule is truly homologousor analogous to the clinical candidate cannot becompletely understood. Of course, the same maybe said with respect to the biological relevance of theanimal species selected for testing of the clinicalcandidate. Although all analogous or homologouscompounds are surrogates for the clinical candidate,not all surrogates will be proven to be analogous orhomologous (ie, a monoclonal antibody [mAb] to theanimal target may be a completely different mole-cule). These caveats should be kept in mind wheninterpreting the results of any alternative testingstrategy.

Surrogate molecules may enable an evaluation ofthe consequences of modulating the target-mediatedpharmacologic activity of the drug in cases when anevaluation of the clinical candidate is limited by spe-cies specificity. The surrogate molecule shouldresemble the clinical candidate as much as possiblewith regard to pharmacologic activity (ie, target epi-tope recognized, binding affinity, potency, etc) if itis to provide relevant and useful safety information.Because the formulation, production process, rangeof impurities/contaminants, glycosylation patterns,and many other factors could also influence the find-ings on a study, one should also understand theessential quality characteristics of the molecule. Thismay be challenging and will require significantresources to characterize a molecule that will neverbe a drug candidate.

Although in some cases surrogate molecules haveemerged as scientifically accepted tools for the safetyassessment of biopharmaceuticals, surrogate mole-cules inherently differ from the clinical candidatesthey are designed to represent. As such, several ques-tions regarding the validity of a surrogate moleculefor toxicity assessments, as well as the overalladvantages and disadvantages of using a surrogatemolecule over the clinical candidate, should be takeninto consideration. Both in vivo and in vitro assess-ments should be used to characterize the relativesimilarity of the surrogate molecule to the clinicalcandidate. An important consideration for certainmonoclonal antibodies is use of the appropriateisotype for the surrogate, particularly for those withanticipated ADCC or CDC activity as part of themechanism of toxicity or pharmacologic action.

In vitro assessments should include pharmacolo-gic activity, potency, affinity/avidity, as well asgeneral physicochemical characterization. In addi-tion, consideration must be given to the appropriateisotype to understand the similarity of ADCC orCDC activity of the Fc region of the antibody.Similarities in tissue binding and specificity mayalso be useful in assessing the appropriateness ofthe surrogate molecule. In vivo evaluations of thepharmacokinetics and pharmacodynamics of thesurrogate molecule should also be conducted, withcomparisons drawn to the clinical candidate wherepossible. Investigations into biomarkers of thesurrogate molecule’s activity and/or application ofthe surrogate molecule in models of efficacy may alsoaid in further characterization.

Limitations in the utility of surrogate moleculesare dictated by the nature of the molecule itself. Thesurrogate molecule should be comparable inpharmacologic activity as well as structure, to theclinical candidate. However, in some cases, the sur-rogate molecule may be structurally distinct from theclinical candidate but have similar pharmacody-namic effects in vivo. This is often a requirement ifsignificant interspecies variability exists in the targetmolecule structure, but may also occur when thedevelopment of the surrogate molecule is conductedeither separately from the clinical candidate or usingdifferent manufacturing methods. In cases where ananimal model lacks the human target, a surrogatemolecule may be developed to mimic biologicallyequivalent pharmacologic effects rather than specifictarget recognition. An example of this might be amolecule targeting murine keratinocyte (KC) proteinas a surrogate for a clinical candidate directedagainst human IL-8.19,20

Additional limitations of the surrogate moleculemay exist with respect to the magnitude or specificityof its biological effect, if the intended pharmacologi-cal effect is either amplified or reduced relative to theclinical candidate. It is important to be aware thatsurrogate molecules may elicit different downstreamsignaling cascades that could yield potentiallysignificant, yet irrelevant, effects. Differences ineither pharmacologic effect or molecular structurecan also affect the pharmacokinetics of the surrogatemolecule. Thus, when the pharmacokinetic profile ofthe clinical candidate is known in a relevant animalspecies, relative comparisons between the surrogatemolecule and the clinical candidate should be made,taking into consideration species differences in

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affinity, target distribution, or differences in otherclearance processes (ie, Fc-mediated clearance) thatmay impact pharmacokinetics. When the pharmaco-kinetic profile of the clinical candidate in an animalmodel is unknown due to species specificity, thesimilarity in pharmacologic effects between mole-cules (assessed using in vitro or in vivo data) can beused to justify comparability.

When a surrogate molecule believed to be repre-sentative of the clinical candidate is identified, and adecision to use it to support development of the clin-ical candidate is made, the sponsor has effectivelyassumed the responsibility of codeveloping 2 mole-cules, only one of which will be tested in the clinic.Several factors should be considered when assessingthe feasibility of surrogate molecule codevelopmentwith the clinical candidate, including the require-ments for development, manufacture, and character-ization of the surrogate molecule. The extent towhich the surrogate molecule will be manufacturedto Good Manufacturing Practice (GMP) guidelinesor tested in safety studies compliant with GoodLaboratory Practice (GLP) guidelines will need tobe decided upon, with appropriate justification(s).Development, including validation (or qualification),of different assays to detect the surrogate moleculeand to monitor the effects of immunogenicity (ie,anti-product antibodies) is also necessary. Asproduction processes associated with manufacturinga surrogate molecule can be different from theclinical product, attention should be given to anypotential impact these changes may have on thepharmacologic activity and/or the range of impuri-ties. Manufacture of the surrogate molecule willrequire additional resources to develop the process,formulation, and packaging of the material. In casesof limited manufacturing capabilities or when clinicalprocesses are utilized in the manufacturing of the sur-rogate molecule, additional pressure may be placedon manufacturing infrastructure. Additional assaysare needed for characterization (aggregates, clips ofthe intact protein and impurities such as host cellprotein, endotoxin, etc) and stability testing to sup-port the duration of the toxicology studies.

Similar to the use of KO/Tg animals in preclinicalsafety evaluations, the use of surrogate moleculesshould be undertaken with an understanding of thetime, cost, and effort of surrogate molecule develop-ment, in context with the applicability and relativebenefits toward assessing clinical safety. Ultimately,a clear understanding and characterization of the

pharmacological and biological behavior of thesurrogate molecule is critical not only to the designof a relevant toxicity program, but also to the deter-mination of the overall risk-to-benefit ratio forhumans. As long-term repeated dosing of a humanbiopharmaceutical in rodent species is often not fea-sible due to immunogenicity and development ofneutralizing antibodies that reduce exposure, a mur-ine surrogate molecule may be developed on theassumption that it would be less immunogenic inmice. However, just as a human protein can beimmunogenic in humans, the homologous proteincan also be immunogenic in rodents. Therefore, thepotential for immunogenicity from a surrogate mole-cule should be evaluated as early as feasible. Theintended application of a surrogate molecule in anonclinical safety program should also be considered(ie, for developmental and reproductive toxicology[DART], for chronic toxicology, etc), as it will definethe extent of in vitro and in vivo studies required foradequate surrogate molecule characterization. Forexample, if a surrogate molecule is intended for usein DART assessments, in vitro placental transferstudies should be considered at an early stage ofsurrogate molecule development to characterize therelevance of the surrogate molecule to the clinicalcandidate in this respect and demonstrate that thefetus will be exposed to the drug.21 Finally, determi-nation of clinically relevant starting doses frompreclinical toxicity evaluations using a surrogatemolecule should involve careful consideration ofrelevant biological and pharmacokinetic differencesand their potential impact on extrapolation of asafe dose.

Potential Advantages/Disadvantages ofUsing a Surrogate Molecule

In situations where the biopharmaceutical ispharmacologically active only in humans and/orchimpanzees, the use of a surrogate molecule couldbe helpful for determining potential hazards associ-ated with the use of the product. Although limitednonterminal safety studies can be conducted inchimpanzees, the small group sizes, the heterogene-ity of the animals that are available, previous drugexposure, and lack of histopathological endpointslimit the use of chimpanzees to an assessment ofacute tolerability rather than an assessment of toxi-city. Because of this, many companies now screencandidates and select lead molecules that have

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pharmacologic activity in at least one toxicologicallyrelevant species. However, when a suitable speciescannot be identified, a full single-species toxicologyassessment can be conducted using a surrogatemolecule. To date, the molecular classes that haveshown the greatest degree of species specificity arethe monoclonal antibodies and the interferons.Surrogate monoclonal antibodies and homologousinterferons have been generated for some species-restricted products and have been used in nonclini-cal safety assessments.21-24 For the monoclonalantibodies, surrogate antibodies could potentiallybe developed for any species, but the most commonsurrogate molecules are rat anti-mouse antibodies.These rat antibodies can then be chimerized (ratFab-mouse Fc) or engineered to replace certain ratsequences with murine sequences. One consider-ation for surrogate antibodies or Fc-fusion proteinsis that the Fc region in the murine surrogate mole-cule must match the appropriate effector functionof the Fc in humans (ie, the IgG2a isotype in miceto match the effector function of an IgG1 isotypein humans).25,26

One of the advantages of developing a murinesurrogate for toxicology evaluation is that theoutbred CD-1 mouse is a common toxicological spe-cies and therefore standard, well-validated toxicologyprotocols and endpoints can be applied to these stud-ies. For immunomodulatory biopharmaceuticals,many of the standard immunotoxicity endpoints havebeen well established in mice, and many reagents areavailable for evaluating immunological endpoints.An added advantage of using the mouse is that manywell-established disease models have been created inmice, allowing both pharmacology and toxicology tobe conducted in the same species and providing apotential margin of safety in that species. A disadvan-tage of using mice is the limited blood volume forvarious endpoints and thus the relatively large num-ber of animals needed for each study.

With the availability of an appropriate murinesurrogate molecule, acute, subchronic, and chronictoxicity studies can potentially be conducted,depending on the immunogenicity of the surrogatemolecule. Additional endpoints can be incorporatedinto the studies based on the pharmacology of themolecule, such as immunotoxicity endpoints forimmune modulating biopharmaceuticals as men-tioned previously. It is important to evaluate toxico-kinetic parameters to demonstrate that exposure tothe surrogate molecule is maintained throughout the

dosing period and that antibodies that may developagainst the surrogate molecule do not limit theexposure. However, if mice are used for toxicologyevaluations, the limited blood volume does not allowthe toxicokinetic and immune response to be mea-sured in the main toxicology animals. Consequently,satellite animals must be included for the toxicoki-netic and immune response evaluations, makingdirect correlations between exposure, immuneresponse, and toxicity difficult.

For biopharmaceuticals that require an evaluationof potential effects on reproduction and/or develop-ment, DART studies in rodents using a surrogatemolecule can be an acceptable option. This approachfor DART studies can be used for molecules that donot cross-react with relevant toxicology species, or canbe used to reduce the use of nonhuman primates forthose molecules that show cross-reactivity only tohumans and nonhuman primates. However, becauseof the caveats discussed previously, it may be moreappropriate for developmental studies to evaluate theclinical candidate in nonhuman primates than to usea surrogate molecule that may not be truly representa-tive. With the use of a surrogate molecule in rodents,all aspects of the DART evaluation as outlined in ICHS5 (R2)27 can be evaluated using standard well-established protocols. Numerous testing facilitieshave the experience and capabilities of conductingDART studies in rodents, and large historical data-bases are available for comparison with normal back-ground findings.

Rodent studies may be particularly useful for theevaluation of fertility (ie, the number of successfulpregnancies). Mating behavior and number of suc-cessful pregnancies cannot be evaluated practicallyin nonhuman primates because they have a naturallylow fertility rate and high spontaneous abortion rate.Reproductive potential can, however, be evaluated inprimates by evaluation of hormones, menstrualcycles, semen parameters, and other indicators ofreproductive capability. In contrast, fertility mea-sured by pregnancy success is a well-establishedendpoint in rodents.

Embryofetal development studies can also beconducted in rodents using a surrogate molecule.There are, however, certain caveats concerningthe use of a surrogate molecule for embryofetaldevelopment studies that must be taken into con-sideration. The mechanisms that regulate largemolecular weight protein transport and diffusionacross the placenta are species dependent and

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differ between rodent and primate placenta.28 Forantibodies that are known to be transported acrossthe placenta, the timing of placental transfer andthe efficiency of transfer differs between rodentsand primates.29,30 In rodents fetal exposure toantibodies occurs earlier in development than inprimates. Therefore, adverse effects may beobserved in the rodents that are not relevant tohumans and may therefore overpredict the poten-tial risk. For large molecular weight proteins thatdo not cross the placenta, embryofetal develop-ment studies are likely to be restricted to maternaleffects rather than direct teratogenic effects and astudy in rodents with the surrogate molecule maymodel these effects similarly as a study inprimates.

Pre- and postnatal development studies can alsobe conducted in rodents using the rodent surrogatemolecule. In this case, endpoints for establishingeffects of treatment on postnatal development arewell established in rodents. The rodent postnataldevelopment studies allow for an evaluation of sexualmaturity and second-generation reproductive perfor-mance that cannot be evaluated in nonhuman pri-mates because of the long period of time betweenbirth and sexual maturity. However, because thetoxicity of biopharmaceuticals is generally relatedto exaggerated pharmacology, unless there is ascientific reason why the molecule might affectsexual maturation, the absence of this information isnot considered to be critical to assessing human safety.

In addition to the discussed limitations of usingprimates for reproductive toxicity studies, the otherconsiderations for reproductive and developmentaltoxicity testing include the ethical use of primatesversus rodents, especially for fertility studies, and theduration of the studies. A primate embryofetaldevelopment study can take up to 1 year to completeand a primate pre- and postnatal development studycan take 2 years or more to complete. Reproductivetoxicity studies can be conducted in rodents using asurrogate molecule in a fraction of this time, andcould provide safety information earlier in the devel-opment process than is possible for primate studies.However, this advantage must be balanced with thepreviously mentioned disadvantages associated withdeveloping a surrogate molecule and the possibilitythat the surrogate molecule in the rodent may notbe pharmacologically identical to the clinical candi-date in the primate. Also the resources required todevelop, characterize, and test a surrogate molecule

can exceed that of conducting nonhuman primateDART studies, so there may not be any advantageto the sponsor in developing a surrogate molecule.The most relevant model to assess risk ofreproductive effects in humans should be utilizedand justified to the regulatory agencies. Therefore,because of their limitations, the surrogate moleculeapproach should be considered as an alternate tononhuman primate DART studies, not as an addi-tional species.

There are several examples of approved prod-ucts on the market in the United States for whichthe safety assessment included surrogate moleculesin the regulatory filing. These products includeActimmune (interferon-g; InterMune, Brisbane,CA), Remicade (infliximab; Centocor, Horsham, PA),Raptiva (efalizumab; Genentech, Inc, South SanFrancisco, CA, and Xoma Ltd, Berkeley, CA),Cimzia (Certolizumab pegol; UCB Inc, Smyrna,GA), and Solaris (eculizumab; Alexion Pharmaceu-ticals, Cheshire, CT). For example, to conduct athorough safety assessment of interferon (IFN)-g,which lacks activity in rodents, the sponsor electedto develop a recombinant murine IFN-g and usedthat product to conduct toxicology studies inmice.22,23 Efalizumab and infliximab, monoclonalantibodies recognizing human CD11a and tumornecrosis factor-a (TNF-a), respectively, are activein humans and chimpanzees only. For both prod-ucts, initial toxicology studies that supported thesafety of clinical trials, were conducted in chimpan-zees. To conduct a more thorough safety evaluation,which was necessary for product approval, thesponsors for these products elected to developantibodies that recognized mouse CD11a andTNF-a.21,24 Ecalizumab, an anti-C5 antibody,showed cross-reactivity only to humans. Therefore,a surrogate mouse antibody was developed andchronic and DART studies were conducted withthis surrogate molecule (Solaris approval informa-tion, http://www.fda.gov/). Certolizumab pegol isan anti-TNF antibody Fab0 fragment conjugatedwith PEG to extend the terminal plasma eliminationhalf-life. Inasmuch as certolizumab pegol does notcross-react with mouse or rat TNF-a, reproductionstudies were performed in rats using a rodentanti-murine TNF-a pegylated Fab fragment (cTNFPF), similar to certolizumab pegol. A few of theseexamples will be highlighted in more detail in thefollowing section, along with cases of molecules stillin development.

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Case Study Examples Using AlternateApproaches to Safety Assessment

Approved Products

Efalizumab

Efalizumab is a recombinant humanized monoclonalIgG1 antibody specific for the a subunit (CD11a) ofleukocyte function associated antigen-1 (LFA-1)approved for treatment of chronic plaque psoriasis.Efalizumab binds specifically to human and chimpan-zee CD11a. As a result of the limited species binding,muM17, a murinized rat anti-mouse chimeric IgG2asurrogate antibody specific for murine CD11a wasdeveloped for preclinical safety evaluation. Themurine surrogate antibody was of the IgG2a isotypeto match the potential effector functions of an IgG1isotype in humans. The general preclinical safety pro-gram conducted with muM17 to support registrationof efalizumab included a tissue cross-reactivity study,1- and 6-month repeat dose toxicity studies, maleand female fertility, embryofetal development, andpre- and postnatal development studies. In addition,special immunotoxicology evaluations were con-ducted in a 1-month subcutaneous immunotoxicitystudy and as part of the pre-and post-developmentaltoxicity study to assess the effect of muM17 onimmune function.

Prior to initiation of the preclinical studies, thepharmacology of muM17 was characterized and theactivity of muM17 was demonstrated to be compara-ble with efalizumab in binding and pharmacodynamicassays. Characterization experiments included themeasurement of binding affinity of muM17 to murineCD11a, in vitro activity of muM17 using the murinemixed lymphocyte reaction, and in vivo activity in amouse model of delayed type hypersensitivity. Inaddition, muM17 was shown to have a comparablepharmacodynamic effect as efalizumab in inducingdownmodulation of CD11a on peripheral bloodT cells following ligand binding.

Results from preclinical studies using muM17were consistent with distribution of the target antigenand pharmacology of blocking CD11a/ICAM-1 (intra-cellular adhesion molecule-1) interactions. In thetissue cross-reactivity study, the pattern of stainingusing a panel of murine tissues was similar to resultsobserved with efalizumab on a panel of humantissues. Results from themale and female fertility, andembryofetal development studies, demonstrated no

effects on either fertility or fetal development fromadministration of muM17 to CD-1 mice. Consistentwith these findings, no toxicities were observed ineither the 1- or 6-month toxicity studies.

The results from the 1-month subcutaneousimmunotoxicology study demonstrated administra-tion of muM17 to young adult mice as 4 weekly dosesresulted in decreased IgM and IgG responses tosheep red blood cells (SRBC), a T-cell-dependentantigen, immediately following the dosing phase.This result is consistent with the biology of blockingLFA-1/ICAM-1 interactions. After serum levels ofmuM17 decreased below the level of detection fol-lowing a 4-week recovery period, humoral immuneresponses to SRBC were comparable with controls.Similar decreases in humoral immune responses toSRBC were demonstrated in F1 generation miceexposed to muM17 via the dam during gestation andlactation in the pre- and postnatal developmentstudy. However, in contrast to the normalization ofhumoral immune responses observed in the youngadult mice following clearance of muM17, humoralimmune responses to SRBC in the F1 mice remaineddecreased relative to age-matched vehicle controlanimals following a 22-week recovery period.

Additional data regarding placental transfer andantigenicity of the surrogate antibody were obtainedfrom the toxicology studies. Placental transfer ofmuM17 was confirmed in the reproductive toxicitystudies. Fetal and maternal serum concentrationswere approximately proportional whereas muM17concentrations in amniotic fluid were considerablylower than maternal serum. Antigenicity of muM17was shown to be low in all studies with an incidenceof less than 1% for the entire surrogate moleculeprogram.21 The adverse safety events or toxicitiesobserved in the clinic with efalizumab are reportedto include infection, malignancy, immune thrombo-cytopenia, and hemolytic anemia.31 Approximately2 years after marketing approval a Dear Health CareProfessional letter was authored notifying prescrib-ing physicians of potential toxicities.32 Most of theadverse events observed in the clinic were consistentwith the drug’s mechanism of action but were notpredicted based on the safety profile of the murinesurrogate antibody.

The surrogate antibody program utilized the con-sistent biology of LFA-1/ICAM-1 between species tobuild a robust preclinical program that was success-fully accepted by regulatory agencies in the UnitedStates, European Union, and Japan. It is important

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to recognize that, because the use of chimpanzees isgreatly restricted and therefore of limited use forassessing safety, the mouse surrogate molecule effec-tively represented the primary safety evaluation; itwas not developed to allow evaluation of a second,rodent species. At first pass, use of the clinicalcandidate in chimpanzees may appear to be the morerelevant approach to safety assessment comparedwith the use of a surrogate in rodents. However, asaccess to chimpanzees becomes further restrictedin the future due to both supply (decreased breedingin United States33) and ethical considerations (sup-ported by proposal of the 2008 Great Ape ProtectionAct to the US Congress and adoption of similarlegislation in several EU countries), it will be vitalto provide sufficient scientific evidence regardingrelevance of the surrogate molecule in regulatorysubmissions, as well as complimentary data (ie, invitro assays) necessary to adequately bridge surrogatedata and relevance to humans.

Infliximab

Infliximab is an anti-human TNF-a (TNF) mAb thatwas first approved in the United States in 1998 forthe treatment of Crohn’s disease. Infliximab is a chi-meric IgG1 mAb that binds to human TNF and ishighly species specific (cross-reacts only with humanand chimpanzee TNF). Therefore, an analogousanti-TNF mAb (cV1q) that selectively inhibits thefunctional activity of mouse TNF was developed toassess chronic and reproductive toxicity.24 Similarto efalizumab, the murine surrogate antibody wasof the IgG2a isotype to match the potential effectorfunctions of an IgG1 isotype in humans. This surro-gate molecule inhibited disease activity in murinemodels of Crohn’s disease, and was thus demon-strated to be pharmacologically similar to infliximab.For registration, an embryofetal development toxicitystudy, a combined male and female fertility study,and a chronic, 6-month toxicity study in CD-1 micewas performed with the murine surrogate. Animmune response to the anti-murine mAb diddevelop in most animals, but this immune responsedid not affect exposure of the animals to the mAb(ie, no change in PK parameters). A nonterminalacute tolerability study was also conducted in chim-panzees with the anti-human TNF mAb.

Pre- and postnatal development studies in miceusing the murine surrogate antibody were also con-ducted postmarketing.34 In addition to all of the

standard evaluations of postnatal development, anevaluation of immune function was conducted in theF1 mice. When the results from the studies con-ducted with mice using the surrogate are comparedwith published information on TNF-deficientmice,35 the results are generally similar but not iden-tical. Although the genetically deficient mice andsurrogate-targeted mice show a general concordancewith regard to effects on fertility and embryofetaldevelopment, the genetically deficient mice show alack of splenic germinal centers and reduced func-tional immune responses that are not observed in thesurrogate molecule-treated mice. Therefore, thegenetically deficient mice are useful models forunderstanding the biology of TNF, but are imperfectmodels for evaluating the safety of mAb treatment.

This example illustrates an overall approach foran mAb that showed cross-reactivity only to humansand chimpanzees. This approach allowed for the safedosing of infliximab in clinical trials and was alsoacceptable to the regulatory agencies in the UnitedStates, European Union, and Japan. The adverseeffects of infliximab that have been observed in theclinic have been mostly related to selective downmo-dulation of immune responses leading to an increasein some opportunistic infections, which is directlyrelated to the pharmacology of the molecule. Asexpected, infections were not seen in the normalhealthy animals used in the toxicology studies, buthave been seen in TNF-a-deficient animals that arechallenged with specific pathogens. Therefore, tak-ing the entire weight of evidence into consideration,the human toxicities were predictable based uponthe nonclinical information and the pharmacologyof the molecule.

Although the toxicity studies conducted in micewith the surrogate molecule were acceptable to sup-port the clinical use of a human mAb with cross-reactivity only in humans and chimpanzees, themouse studies were not acceptable to the regulatoryagencies to support the clinical use of anotherhuman anti-TNF mAb that was pharmacologicallyactive in humans, chimpanzees, and macaques. Forthe latter mAb, regulatory agencies requiredembryofetal and postnatal development studies incynomolgus monkeys with the humanized mAb,36

despite DART studies previously conducted with asurrogate mAb, and significant chronic-use clinicalexperience with the original chimeric mAb, as wellas several other anti-TNF therapies. Therefore,although it has been proposed that surrogate

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molecules could be used to reduce the use ofnonhuman primates,37 to ensure the regulatoryacceptance of a surrogate molecule-only approachfor a clinical candidate that shows cross-reactivityto nonhuman primates, a dialogue must be con-ducted between the company and the agencies toensure that all parties are in agreement with thescientific justifications for the approach.

Interferon-g

IFN-g is an immunomodulatory cytokine that ishighly species specific, with pharmacologic activityonly in humans and nonhuman primates. In sub-chronic toxicity studies in cynomolgus monkeys, theclinical side-effects that were observed (fever,lethargy, anorexia, and changes in hematology andchemistry parameters) were comparable with clinicaleffects seen in humans.23 However, a neutralizingantibody response was seen that attenuated theresponse in a 13-week study compared with the4-week monkey study. A murine version of IFN-gwasutilized in a 4-week mouse study, where the nature ofthe treatment-related findings and organ systemsaffected were similar to the observations in cynomol-gus monkeys treated with the human protein, and yetno neutralizing antibodies developed.38 Thus, in thiscase the murine surrogate was not developed to allowsafety evaluation in a second, rodent species, butrather to better characterize the response without theconfounding factor of neutralizing immunogenicity.

In developmental toxicity studies, both thehuman IFN-g and the murine surrogate moleculewere abortifacients in cynomolgus monkeys andmice, respectively. The murine surrogate moleculewas also used to evaluate potential developmentaland reproductive capacity of juvenile animals associ-ated with chronic treatment, because juvenilepatients with chronic granulomatous disease arethe main patient population (Actimmune; http://www.fda.gov/cder/foi/label/2007/103836s5098LBL.pdf). In this study, mice were treated with daily doses(0, 0.02, 0.2, or 2 mg/kg/d) from postnatal day 8through day 60 to determine the effects on matura-tion, behavioral/functional development, and repro-ductive capacity.22 Male mice in the high-dosegroup had delayed sexual maturation, reduced epidi-dymal and testes weights, reduced sperm count andconcentration, and sperm abnormalities, and showedreduced mating performance and fertility despite theabsence of altered histopathology of the testes.

Motor activity was also decreased in all mice in thehigh-dose group. Although it is unknown whetherthese findings would be found in humans treatedchronically with IFN-g, this information does appearin the label for Actimmune (along with the caveat ofunknown significance). Prior to conducting thesestudies with the surrogate molecule, a careful reviewand comparison of data regarding biochemical prop-erties, biological activity, and disposition profiles ofboth proteins in similar test systems was performed.The characterization of the mouse surrogate mole-cule in this case allowed for further exploration ofthe reproductive and behavioral effects of IFN-g thatwould have been difficult to evaluate in the nonhu-man primate.

Products in Development

Keliximab, Clenoliximab

Keliximab is a primatized IgG1 mAb directed towarddomain 1 of human CD4.15,39 Clenoliximab is anIgG4 version, developed to reduce Fc interactionswith Fc receptors and thus mitigate T-cell depletionand cytokine release. Keliximab and clenoliximabshow cross-reactivity only to human and chimpanzeeCD4. To evaluate the preclinical safety of thesemolecules, the sponsors developed a transgenicmouse that expressed human CD4 in place of mouseCD4. Both monoclonal antibodies were shown to bepharmacologically active in these KO/KI mice.15

With this KI mouse, the human therapeutic antibo-dies could be tested for nonclinical safety. The KImouse was characterized by demonstration of appro-priate expression of human CD4 and by evaluation ofimmune system function following challenge withinfection or tumor cells. The preclinical safety stud-ies conducted with keliximab in the KI mice includedsingle and repeated dose toxicology studies, male andfemale fertility, embryofetal development, pre- andpostnatal development studies with functionalimmune response evaluation in the F1 generation,and host defense assays.15,39 A nonterminal acutetolerability study was also conducted in chimpan-zees. Toxicity studies conducted in the mice showedthe expected reduction in CD4 cells. The mice diddevelop an immune response toward the humanmAband in some instances anaphylactic reactionsoccurred. However, sufficient numbers of animalssurvived and exposure levels were sufficientlymaintained to adequately evaluate the toxicity.

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This example demonstrates a novel approach inwhich KI mice expressing the human target antigenwere developed so that the human protein could betested in preclinical studies. These methods have thedisadvantage that human proteins can be highlyimmunogenic in animals and thereby limit the dura-tion of the studies, impact the exposure of the animalto the therapeutic, or lead to adverse effects. Alsothis approach requires development and extensivecharacterization of the animal model, including gen-otyping of all animals used for the studies, to ensureappropriate expression of human CD4. In addition,the model tests only specific inhibition of theintended target and may not expose any secondarypharmacology attributable to closely related ordownstream targets associated with administrationof the human protein. However, when the clinicalcandidate is active only in humans and chimpanzees,data in KI mice treated with the clinical candidatecan be useful in assessing safety.

Fully Human mAb (Anti-Cytokine Receptor)

A fully human mAb targeting a cytokine receptor wasdeveloped for treatment of allergic inflammatoryresponses (data on file, Amgen Inc). Because theactivity of the clinical molecule was limited tohumans and chimpanzees, a surrogate moleculeapproach was taken for the safety program to supportclinical development, utilizing 2 well-characterizedchimeric anti-mouse and anti-monkey surrogateantibodies. These molecules have similar respectiveactivity for murine and cynomolgus monkey cytokineinhibition as does the clinical molecule for humancytokine inhibition. The characterization of thesemolecules included binding and functional activityin cell-based assays in vitro (murine and monkey)and in vivo pharmacologic activity (murine only).The safety program along with various noteworthyrationales behind the necessity for many of thestudies is described below.

Prior to the first in human (FIH) clinical trial, themolecules that were available to conduct preclinicalstudies included the clinical molecule, which onlycross-reacted with the target in humans and chimpan-zees, and the anti-murine surrogate antibody. Asingle-dose chimpanzee study was conducted primar-ily to model the pharmacokinetics of the clinical mole-cule in a relevant species, and a 4-week mousetoxicology study with the anti-murine surrogate wasconducted as the definitive safety study to support the

FIH trial. Because the clinical candidate moleculewas not evaluated in the toxicology studies wherehistopathology evaluation was available, a rabbit localtolerance study was conducted with the clinicalmolecule to assess the irritation potential of theformulation to support the FIH trial.

Information from the literature indicated thatthe cytokine of interest is important in the mainte-nance of pregnancy. In a previous embryo-fetaldevelopment study conducted with a human solublecytokine receptor that had the same pharmacologicactivity (antagonism of the cytokine) in cynomolgusmonkeys, an increased frequency of spontaneousabortions and stillbirths was observed.40 To deter-mine if a better model could be established to under-stand the mechanism of this toxicity, a mouse studywas conducted with a murine surrogate molecule.The cynomolgus monkey reproductive findings werenot reproducible in mice with a murine surrogate ofthe soluble cytokine receptor or with an anti-murinecytokine receptor antibody, suggesting that themouse was not an appropriate species for evaluationof the reproductive effects following inhibition ofthis pathway. Thus, the cynomolgus monkey surro-gate molecule was developed primarily to evaluatereproductive toxicity. The murine surrogate antibodywas utilized for fertility evaluation, because an effecton fertility was not expected based on information inthe literature. The complete package for reproduc-tive toxicology evaluation thus consisted of a fertilitystudy with the murine surrogate molecule in miceand an embryofetal and prenatal development studywith the cynomolgus monkey surrogate molecule inthe cynomolgus monkey.

Results from the anti-monkey surrogate moleculefor the reproductive toxicology evaluations as well asavailability of results from a 1-month repeated-dosecynomolgusmonkey toxicology study with themonkeysurrogate molecule were available at the time point forinitiation of the subchronic study. Although bothsurrogate molecules were similar to the clinicalcandidate, to limit resources originally dedicated to2 surrogate molecules, the cynomolgus monkeysurrogate molecule was the only one utilized toconduct the subchronic (3–month) and chronic(6-month) repeated-dose toxicology studies.

Fully Human mAb (Anti-Cytokine)

A fully human mAb targeting a cytokine was devel-oped for the treatment of inflammatory disease (data

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on file, Amgen Inc). The clinical molecule bound torecombinant human cytokine with high affinity, buthad lower affinity (approximately 30-fold) for thecynomolgus monkey cytokine. In addition, the clini-cal molecule could neutralize the ability of thehuman cytokine to stimulate human cells in a cell-based assay, but was not able to efficiently neutralizethe cynomolgus monkey cytokine. This demonstratesthat binding alone may not be sufficient todemonstrate species relevance. A murine surrogatemolecule was not available at the time of initiationof the preclinical safety program. Thus, to enablepreclinical studies in cynomolgus monkeys, a surro-gate antibody was developed by fusing the F(ab) por-tion of a mouse anti-human cytokine, known toneutralize the cynomolgus monkey cytokine, withhuman Fc. The chimeric surrogate molecule hadnearly as high an affinity for the human cytokine asdid the clinical molecule, and higher binding affinityfor the cynomolgus monkey cytokine compared withthe clinical molecule. The surrogate molecule alsoneutralized the ability of cynomolgus monkey cyto-kine to stimulate cynomolgus monkey cells in a man-ner similar to the neutralization of human cytokineactivity on human cells by the clinical molecule.Thus, the surrogate molecule was used in the mon-key for the nonclinical safety program.

Toxicology studies were conducted in cynomol-gus monkeys with the surrogate molecule to supportthe early stage clinical development plan. Prior to theFIH clinical trial, studies conducted included arepeated-dose study of 1-month duration and a safetypharmacology study (cardiovascular, respiratory, andcentral nervous system). Results from the 1-monthstudy indicated a pharmacodynamic effect with thesurrogate molecule in the monkey that would be pre-dicted based on one of the known activities for thecytokine of interest, as noted in the literature. Inter-estingly, this pharmacodynamic effect was not evi-dent in the FIH trial, indicating that there may bea species difference in response to inhibition of thecytokine of interest or that there is a differencebetween the monkey surrogate molecule and theclinical candidate. This example highlights thechallenges when using a surrogate molecule and con-flicting data are generated. Further work is thenneeded to understand whether the difference is dueto the use of a different molecule, or whether theactivity of that target is different in the animalspecies compared with humans, or between differentanimal species (as seen in the previous example).

Fusion Protein

A soluble lymphotoxin b receptor consisting of theextracellular domain of human lymphotoxin b fusedto theFc region of human IgG1 (LTbR-Fc) is currentlyin development for the treatment of rheumatoid arthri-tis. The pharmacologic activity is limited to humansand nonhuman primates. During development, ananti-murine lymphotoxin b receptor Fc IgG fusion sur-rogatemoleculewas generated to evaluate thepharma-cology of the soluble receptor in rodents.TheFc regionin themurine surrogatemoleculewas of the IgG2a iso-type to match the effector function of an IgG1 isotypein humans. Adult mice treated with the surrogateLTbR-Fc showed reduced immune responses andreduced disease activity in a number ofmurine diseasemodels.41 Mice that are genetically deficient in LTbhave also been generated and shown reduced immuneresponses and an absence of lymph nodes.42 Pregnantmice treatedwith the surrogateLTbR-Fcmoleculehadoffspring that lacked lymph nodes.43 However, admin-istration of the human LTbR-Fc to pregnant monkeys(a pharmacologically relevant species) was not associ-ated with an absence of lymph nodes.44 The differ-ences observed between the surrogate in the mouseand thehuman therapeutic in themonkeysmay be dueto differences in the timing of lymph node develop-ment relative to placental transport of antibodies inmice versusprimates, althoughmonkey fetal exposureswere not reported in this study.

This example illustrates how results obtained with asurrogatemolecule inmiceorextrapolatedfromKOmicemay not necessarily be representative of the results thatare likely to occur in nonhuman primates or humans,particularly in developmental toxicity studies in whichspecies differences are known to exist in the stages ofembryofetal development. In this case, the surrogate andthe genetically deficient animals identified the potentialhazard, but may have overestimated the human risk.

Questions Regarding the Use of SurrogateMolecules

If a Surrogate Molecule Is Used to AssessAny Aspect of Safety, Is It a Requirementto Conduct All Toxicology Studies in ThatSpecies?

The nonclinical safety programs for small moleculedrugs routinely require assessment of general toxicity

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in 2 species, including a rodent (usually the rat) anda nonrodent (usually the dog) (ICH M3 (R1)45). TheICH S6 guidance document for biopharmaceuticalsstates that ‘‘Safety evaluation programs shouldnormally include 2 relevant species. However, incertain justified cases, one relevant species maysuffice (e.g., when only one relevant species can beidentified or where the biological activity of thebiopharmaceutical is well understood).’’ Further-more, the guidance document goes on to say that‘‘even where two species may be necessary to charac-terize toxicity in short term studies, it may be possibleto justify the use of only one species for subsequentlong-term studies (e.g., if the toxicity profile in the2 species is comparable in the short term).’’ There-fore, for biopharmaceuticals that have a very specificmechanism of action that is well understood, such asmonoclonal antibodies and receptor fusion proteins,a 2-species assessment may not always be necessary.

For species-restricted biopharmaceuticals, themost common species used for nonclinical safetytesting is a nonhuman primate, usually a macaque.The toxicities that have been observed in macaquesfor biopharmaceuticals have generally been directlyrelated to the pharmacology of the molecule andoff-target toxicities are rarely observed. Because thesurrogate molecule has many limitations that havebeen mentioned previously, the surrogate moleculeprovides supportive information only in those situa-tions where there is no other option for nonclinicalsafety testing or where information can be obtainedin the rodent that cannot be adequately evaluatedin the nonhuman primate (eg, fertility). In cases inwhich a surrogate molecule is available and is consid-ered optimal for use in developmental and reproduc-tive toxicity evaluations, inclusion of standardtoxicity endpoints (ie, clinical pathology endpoints)in preliminary dose-ranging studies may complimentthe overall weight of evidence regarding drug safety.These may provide a useful comparison to thefindings seen in the general toxicology studies withthe clinical candidate.

Should a Surrogate Molecule BeDeveloped to Allow for Assessment ofCarcinogenicity?

The question of carcinogenicity is not so much oneof whether a surrogate molecule should be used forcarcinogenity testing, but whether 2-year bioassays

in rodents are relevant models for evaluatingcarcinogenic potential for biopharmaceuticals.46

Carcinogens generally fall into 3 major categories:genotoxic carcinogens, cellular proliferators, andimmune suppressants.47 Biopharmaceuticals have alarge molecular weight that precludes them from dif-fusing into cells and interacting with DNA. Therefore,biopharmaceuticals are unlikely to be genotoxiccarcinogens.

Biopharmaceuticals that induce cellular prolif-eration such as insulin-like growth factor and growthhormone are assumed to be associated with a greaterrisk of tumor development. Increased cellular prolif-eration can be detected with in vitro studies and maybe evident in repeated-dose toxicology studies ofsufficient duration. The absence of hyperplasia in arepeated-dose toxicity study may be indicative thatthe biopharmaceutical is unlikely to be carcinogenicbecause of increased proliferation, when consideredin context with the duration of dosing, level of anal-ysis, and known target biology. A recently publishedstudy has described the development of mouse- andrat-specific growth hormones for the evaluation ofcarcinogenic potential in 2-year bioassays assurrogate molecules for human growth hormone.48

The studies showed no increase in tumors in thetreated animals. However, it is not clear that thesenegative results in rodents will change either theperception or labeling of the product, even inthe context of 40 years of clinical experience usinghuman growth hormones.

Finally, immune suppressive agents areassumed to increase a patient’s susceptibility to cer-tain tumor types (especially lymphomas and skincancer) because of decreased host defense.49 Thishypothesis has been based upon clinical experience,not upon 2-year bioassays, which are frequentlynegative for small molecule nongenotoxic immuno-suppressive agents. Immunosuppressive drugs andimmunomodulating biopharmaceuticals carrywarnings on their product labels for a potentialincreased susceptibility to tumors. In a 2-year bioas-say, mice infected with both mouse leukemia virusand mammary tumor virus and then treated withabatacept, which inhibits T-cell activation, werefound to develop lymphomas and mammary tumors,respectively.50 Therefore, this study reinforces thehypothesis that immunosuppression can increasesusceptibility to oncogenic viruses but did notprovide new information and did not change thewarnings in the product label.

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The carcinogenic risk assessment for biopharma-ceuticals can best be made based upon an under-standing of the biology of the molecule. Anexample of a rational, scientific-based assessmentof carcinogenicity potential in the absence of animalcarcinogenicity testing has been published forinterleukin-10.51 Overall, for the majority of bio-pharmaceuticals, there is little rationale for conduct-ing 2-year bioassays for biopharmaceuticals witheither the human therapeutic (if it shows rodentcross- reactivity) or with a surrogate molecule,because the assessment of carcinogenic potentialand communication of risk could be achieved byother means (ie, in vitro data, pharmacology data,appropriate wording in the product label, etc). Inthe end, assessment of carcinogenic potential shouldbe made on a case-by-case basis considering therelevance of carcinogenicity testing in the contextof target biology, to adequately communicate carci-nogenic risk to patients.

How Should the Relevance of theSurrogate Molecule to the ClinicalCandidate Be Determined? What Is theImportance of Understanding Target-Mediated Pharmacologic Effects VersusCompound-Related Off-Target Effects?

It is important that any compound selected for use asa surrogate molecule be shown to be pharmacologi-cally similar to the development compound, because,if not, it is possible that erroneous conclusionsabout safety could be reached. A few examples aredescribed below.

In the first example, a sponsor was developing anIgG1 Fc-mutated humanized mAb against a chemo-kine receptor present on T cells and monocytes thatwas believed to be important in leukocyte traffickingand activation in inflammatory and autoimmune dis-eases (C. Horvath, personal communication, 2009).The antibody had been tested extensively in vitrowith human cell systems and demonstrated to be aligand-binding antagonist with no signaling throughthe receptor and no target cell-depleting effects.These properties were confirmed in early clinicaltrials with the humanized mAb. To potentiallyaddress reproductive toxicology and host resistanceconcerns related to target blockade, an anti-rodentmAb was desired for use as a surrogate molecule fortoxicity testing. One such anti-rodent IgG2 mAb had

been published by an academic laboratory to beeffective in several animal models of autoimmunedisease and to induce no target cell depletion. ThismAb was subsequently in-licensed by the sponsor foruse as a development surrogate molecule and smallquantities were synthesized for evaluation. Theanti-rodent mAb was administered to normal miceand target cells were immunophenotyped and evalu-ated for receptor expression and blockade by flowcytometry. Unlike the development mAb, the surro-gate mAb resulted in depletion of all target cellswithin the circulating blood, as well as spleen tissue,within 15 min of dosing. T cells and monocytes didnot begin to return to the circulation until approxi-mately 3 days later. Upon reviewing the publicationsthat demonstrated efficacy with this rodent mAb, itwas found that the cell-depleting properties were notknown because early time points after dosing had notbeen evaluated. In light of this information (celldepletion with the surrogate molecule vs no targetcell depletion with the clinical molecule), the spon-sor questioned whether the results with the surrogatemAb in models of autoimmune disease would be reli-able indicators of the results that might be expectedfor the development mAb in human autoimmunediseases. An attempt to alter the cell-depletingproperties of the surrogate molecule by antibodyengineering was then undertaken. Despite almost2 years of dedicated efforts, the cell-depleting prop-erties of this mAb could not be eliminated by isotypeswitching or Fc mutations. The sponsor concludedthat the anti-rodent mAb could not be used as asurrogate molecule because either the surrogatemAb was not pharmacologically similar to the anti-human mAb (eg, different Fc-Fc receptor inter-actions) or the rodent species was not biologicallysimilar to humans (eg, different target expressionand/or function). In this example, a safety profilegenerated with the surrogate mAb may have over-estimated potential concerns (eg, extent of immuno-modulation) because the surrogate molecule resultedin destruction of the target cells, rather than block-ade of a specific cellular function.

The second example is a recent one in whichreliance on results of a surrogate mAb may have con-tributed to an underestimation of the potential safetyconcerns for the development mAb. TGN1412 was ahumanized IgG4 mAb directed against CD28 onT cells being developed for oncology and autoimmunediseases. In vitro, the TGN1412 ‘‘superagonist’’ mAbdirectly activated human T cells in the absence of a

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second costimulatory signal and resulted in cytokinerelease and T-cell proliferation, with a bias toward aregulatory immunophenotype (Treg cells). However,when dosed to normal volunteers, TGN1412 resultedin massive cytokine release (a ‘‘cytokine storm’’),accompanied by rapid, severe T-cell depletion (within1 hour, the earliest time point at which T-cell countswere measured) associated with headache, rigors,myalgia, hypotension, tachycardia, fever, and multior-gan failure.52 This response of T-cell depletion andcytokine release had not been described in preclinicalstudies. The pharmacologic effect of CD28 stimula-tion had been evaluated extensively in rats with a sur-rogate anti-rat CD28 mAb, JJ316.53 This mAb wasshown to be effective in several animal models ofautoimmune disease, such as adjuvant-inducedarthritis (AA) and experimental allergic encephalo-myelitis (EAE), but not collagen-induced arthritis(CIA). In normal rats, the surrogate mAb was associ-ated with marked increases in blood and spleen T-cellcounts (up to*20-fold) and marked expansion (up to*6-fold) of lymphoid tissues, such as spleen andlymph nodes, within 3 days.54,55 No adverse effectsrelated to cytokine release were reported in this study.The results generated with the surrogate mAb there-fore suggested that administration of an anti-CD28mAb could be well tolerated.

Because TGN1412 (or an IgG1 version,TGN1112) recognized CD28 in rhesus and cynomol-gus monkeys, it was evaluated in these species andwas reported to be efficacious in a monkey modelof rheumatoid arthritis (CIA). In toxicology studiesin monkeys, TGN1412 was associated with minimalcytokine release and, at approximately 2 weeks afterdosing, with only mild increases in blood T-cellcounts (*2-fold) and minimal evidence of expansionof lymphoid tissues.56-58 These results for TGN1412in monkeys were in contrast to those obtained withJJ316 in rats, where profound lymphocytosisoccurred. Although a cytokine storm did not occurin monkeys, close examination of the reported lym-phocyte counts suggests that T cells may have beendepleted in monkeys after dosing (as they were inhumans). It is not possible to confirm this becauseearly postdose time points were not evaluated.T-cell depletion was, however, a prominent findingwhen TGN1412 was administered to another speciesexpressing human CD28! T cells. When H2d Rag2–/–gc"/" KO mice are irradiated and their bonemarrow reconstituted with human CD34! fetal liver(stem) cells, they develop human immune systems

(HIS) with all major human myeloid and lymphoidcellular compartments, including CD28! human Tcells. When the original mouse precursor ofTGN1412 (mAb 5.11A1) was administered to thesemice, they developed rapid, profound T-cell deple-tion that persisted through 60 days.59 These resultsare in contrast to those obtained with the anti-rodent surrogate mAb. Thus, perhaps testing of thedevelopment compound in this ‘‘surrogate species’’expressing human T cells was more representativeof the potential safety concerns for the developmentmAb. These examples highlight some of the caveatsthat must be considered when electing to supportdevelopment products with surrogate compounds.Further, because the generation of surrogate mole-cules for toxicology studies is a time- and resource-consuming effort, use of a surrogate molecule shouldbe warranted only in special cases. Surrogates stud-ies should not be conducted based on ‘‘no effect’’ innonhuman primate studies if no toxicity was pre-dicted based on super pharmacology or the absenceof the target in a normal animal.

Discussion/Conclusions

This review illustrates that alternative approaches,including animal models of disease, KO/Tg or huma-nized mice, or surrogate molecules, can be appropri-ate to improve the predictive value of preclinicalsafety assessments for species-specific biopharma-ceuticals, although many caveats must be considered(Tables 3 and 4). Surrogate molecules may be partic-ularly useful for repeated-dose toxicity studies (incases where the molecule cross-reacts only with thehuman and chimpanzee target), or for specialty stud-ies such as fertility toxicity testing, immunotoxicitytesting, host resistance models, and so forth (in caseswhere the molecule cross-reacts with primates, butnot rodents). However, having a surrogate moleculeavailable does not obligate evaluation of a ‘‘secondspecies’’ or the conduct of carcinogenicity studies.Rather, alternative approaches should only be usedto evaluate safety when the clinical candidate cannotbe readily used to identify the hazard in an appropri-ate nonclinical species.

There are many issues to be considered whenpursuing alternative approaches with a surrogatemolecule. Indeed, it is critical to sufficiently under-stand the similarity and relevance of the surrogatemolecule to the clinical candidate (eg, based on

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Tab

le3.

Specific

Issues

WithNon

clinical

Evaluationof

PK/PD/Toxicityof

Biopharmaceu

ticalsLackingCross-R

eactivityin

Traditional

Species

(eg,

Ratsan

dDogs)

Develop

men

tStrategy

TestHuman

TestArticle(C

linicalCan

didate)

inNHPSpecies

TestSurrogateRod

entTestArticle

(Hom

ologue)

inRod

entSpecies

TestKnocko

utMiceWithGen

etic

Deletion

ofTarget

Pro

Con

Pro

Con

Pro

Con

Speciesch

aracteristics

Relativeexpen

seExpen

sive

Inexpen

sive

Can

bevery

expen

sive

Numbersof

anim

alsan

dsampling

Allow

srepeated

sampling

Gen

erally

requires

low

n/group;generally

not

pow

ered

forstatistical

sign

ifican

ce;

individualsmay

providetoxicitysign

als

Allow

shighn/group;may

bepow

ered

forstatisti-

calsign

ifican

ce

Usually

doesnot

allow

repeated

sampling

Allow

shighn/group;

may

bepow

ered

forstatistical

sign

ifican

ce

Usuallydoesnot

allow

repeatedsampling

Pop

ulation

andindividual

anim

alch

aracterization

Heterogen

eouspop

ula-

tion

(likehuman

s?);

may

needto

screen

for

selected

attributes

Hom

ogen

eouspop

ulation

Hom

ogen

eous

pop

ulation

May

haveto

phen

o-typeor

genotype

individuals

Anim

alavailability

Gen

erally

good

Gen

erally

good

Gen

erally

poo

r;lim-

ited

number

ofvendors;may

requirespecial

considerations,

includingscale-up

ofproduction

Opportunisticor

back-

grou

ndinfections

Occasionally

present;

serology

testingmay

not

bereliab

le

Gen

erally

not

anissue;

barrier

derived

orVAF

strainsavailable

Occasionally

present;

especiallyiffrom

acad

emia;may

needto

be

rederived

Other

usesforspecies+

treatm

entwithtest

article

Allow

stestingof

other

clinical

indications(lim

-ited

number

ofdisease

mod

els),

immunotoxicity,

chronic

toxicity

andreproductive

anddevelop

men

-taltoxicity

testing

Gen

erally

not

usedfor

hostresistan

ceassays,

tumor

surveillan

ceassays

orcarcinogen

i-city

testing;

noindus-

trystan

dardforNHP

immunotoxicityor

immunom

odulation

assays

Allow

stestingof

other

clinical

indications

(disease

mod

els),

immunotoxicity,

host

resistan

ceassays,

tumor

surveillan

ce,

chronic

toxicity,repro-

ductivean

ddevelop

-men

taltoxicity

and

carcinogen

icity

Allow

stestingof

other

clinical

indications(dis-

ease

mod

els),

immunotoxicity,

hostresistan

ceassays,tumor

surveillan

ce,

chronic

toxicity,

reproductivean

ddevelop

men

tal

toxicity

andcarci-

nogen

icity.

May

allow

testingof

replacemen

ttherap

ies.

(con

tinued)

246




Tab

le3.(con

tinued

)

Develop

men

tStrategy

TestHuman

TestArticle(C

linicalCan

didate)

inNHPSpecies

TestSurrogateRod

entTestArticle

(Hom

ologue)

inRod

entSpecies

TestKnocko

utMiceWithGen

etic

Deletion

ofTarget

Pro

Con

Pro

Con

Pro

Con

Reagents,assays,meth-

ods,controls(eg,

clini-

calor

anatom

icpathology)

Gen

erally

available,

oftenad

apted

from

human

Gen

erally

available,often

differentfrom

human

Gen

erally

available,

may

bedifferent

from

wild-type

mice

CRO

capacityan

dcapab

ilities

Gen

erally

good

Gen

erally

good

May

requirespecial

considerations;

CROsreluctan

tto

acceptan

imals

from

non

-approved

vendors

Historicalcontrols

Gen

erally

available

Gen

erally

available

May

not

beavailable;

may

needto

gener-

ate;

oftencreated

innon

trad

itional

strains;noindustry

consensusfor

‘‘phen

otyping’’k

/oTargetbiology

inselected

species

Tissueexpressionan

dfunction

Targetexpression

andfunctionin

NHPsmay

be

comparab

leto

human

s(shou

lddem

onstrate

bio-

logicalactivity

ofclinical

candidate)

Must

dem

onstrate

that

roden

tspe-

cies

target

biology

isrelevantfor

human

s

Rod

entspeciesk/o

target

biology

oftendescribed

(validated

?)in

literature

Gen

etic

deletionor

insertionof

atarget

may

not

represent

chronic

blockad

eor

replacemen

tby

test

article;

redun-

dan

tpathways,

falsebiology

etc

Roleof

target

indisease

mod

els

NHPmod

elsof

disease

aregenerally

less

common

than

roden

tmod

els

Roleof

roden

ttarget

indisease

mod

elsoften

described

inliterature

Gen

erallynot

know

nifpredictive

ofrole

inhuman

diseases

Roleof

roden

ttarget

indisease

mod

els

oftendescribed

inliterature

Gen

erally

not

know

nifpredictive

ofrole

inhuman

diseases

Testarticle

man

ufacture

Supply

Allow

suse

ofclinical

candidate

Req

uires

largeam

ountof

clinical

candidate

Req

uires

smallam

ountof

hom

ologue

Req

uires

develop

-men

tof

hom

olo-

guewithGMP-

like

man

ufacture

Characterization

Relieson

existing

bioch

emical

char-

acterization

and

lotreleasespecs

Req

uires

develop

-men

tof

new

bio-

chem

ical

charac-

terization

andlot

releasespecs;may

not

represent

clinical

candidate

(con

tinued)

247




Tab

le3.(con

tinued

)

Develop

men

tStrategy

TestHuman

TestArticle(C

linicalCan

didate)

inNHPSpecies

TestSurrogateRod

entTestArticle

(Hom

ologue)

inRod

entSpecies

TestKnocko

utMiceWithGen

etic

Deletion

ofTarget

Pro

Con

Pro

Con

Pro

Con

Impurities

orcontaminan

tsAllow

stestingof

product-related

impurities,con-

taminan

ts,or

other

characteris-

tics

ofclinical

candidate(eg,

glycosylation,

hostcellproteins)

Hom

ologuemay

haveprod

uct-

relatedim

purities,

contaminan

ts,or

other

differen

ces

from

hTA(eg,

gly-

cosylation

,host

cellproteins);m

aynot

represen

thTA

Testarticle

pharmacology

Invitroor

invivo

pharmacology

Affinity,

specificity,

activity

ofclinical

candidatein

NHPsmay

be

comparab

leto

human

s(m

ust

dem

onstrate)

Must

dem

onstrate

that

affinity,

spe-

cificity,activity

ofhom

ologuein

roden

tspeciesis

relevantfor

human

s

Poten

tial

secondary

andparalleleffects

from

genetic

man

ipulation

may

confoundresults

Reagents,assays,meth-

ods,controls(eg,

todetectPK,PD,im

mu-

nogen

icityrespon

ses)

Gen

erally

available,

oftenad

apted

from

clinical

can-

didatemethod

sforhuman

s

Req

uires

develop

-men

tof

new

reagen

ts,assays,

method

san

dcon-

trolsspecific

for

hom

ologuein

roden

tspecies

PK/PD

PK/PD

ofclinical

candidatein

NHPsmay

be

comparab

leto

human

s

Must

dem

onstrate

that

hom

ologue

PK/PD

inroden

tspeciesisrelevant

forhuman

s(com

-parab

leto

clinical

candidatein

NHPs?)

Immunogen

icity

Clinical

candidateoften

immunogen

icin

NHPs

Hom

ologuemay

be

immunogen

icin

roden

tspecies

Immunotoxicity

Gen

eral

issues

Noindustry

stan

dardfor

NHPim

munotox

orim

munom

odulation

assays;lack

ofhistori-

calcontrols,validated

assays,positivecon-

trols,etc;

difficu

lties

ininterpretingresults

since

smalln

May

beab

leto

apply

method

sad

aptedfrom

adultstudies(thisis

thetren

d)

Lackof

historical

controls,vali-

dated

assays,pos-

itivecontrols,etc;

difficu

ltiesin

interpreting

results

Gen

etic

deletionor

insertionof

atarget

may

not

represent

chronic

blockad

eor

replacemen

tby

clinical

candidate

CRO,Con

tractResearchOrgan

ization;GMP,Goo

dMan

ufacturingPractice;

hTA,human

test

article;

NHP,non

human

primate;

PD,pharmacod

ynam

ics;PK,pharmacok

inetics;

VAF,VirusAntibod

yFree.

248




Tab

le4.

Specific

Issues

WithNon

clinical

Evaluationof

Rep

roductivean

dDevelop

men

talToxicityof

Biopharmaceu

ticalsLackingCross-R

eactivityin

Traditional

Species(eg,

Rats,Mice,

andRab

bits)

Develop

men

tStrategy

TestHuman

TestArticle

(Clinical

Can

didate)

inNHPSpecies

TestSurrogateRod

entTestArticle

(Hom

ologue)

inRod

entSpecies

TestKnocko

utMiceWithGen

eticDeletionof

Target

Pro

Con

Pro

Con

Pro

Con

Rep

roductivean

ddevelop

men

tal

toxicity

Com

parativeplacentation

,physiology,

endocrinology

Bidiscoid

placenta;

moresimilar

tohuman

sthan

roden

tsare;

phy-

siologyan

den

do-

crinologysimilar

tohuman

sa

Minim

alexperience

with

smallmolecules

Differencesin

maternal-

fetaltran

sfer

oflarge

moleculesduring

gestation

CRO

capacityan

dcapab

ilities

Few

CROsofferNHP

DART;cu

rren

tly

limited

to*

2-3

Several

CROsoffer

roden

trepro/devel

toxicity;cu

rren

tly

Few

ornoCROshave

experience

Historicalcontrols

Lim

ited

historicalc

ontrol

data

MostCROsthat

offer

roden

trepro/devel

havehistorical

datab

ases

Lessdataformicethan

rats,little

datawith

proteinsin

mice

Unlikely

tobeavailable

Accep

tedgu

idelines

Noindustry

stan

dardsfor

NHPrepro/devel

toxi-

city

studydesigns

ICH

guidelines

for

roden

trepro/devel

toxicity

study

designs

ICH

guidelines

for

roden

trepro/

devel

toxicity

studydesigns

may

be

adap

table

Relevan

tcontrolsmight

includewildtypeor

heterozygou

smice;

nohistoricalrefer-

ence

forsomestrains

Studyduration

Mon

thsto

years

Weeks

tomon

ths

Weeks

tomon

ths

Rep

roductivean

ddevelop

men

tal

toxicity

(con

t.)

Utility

andinterpretation

May

beusefulto

screen

forunex-

pectedor

high

inciden

ceeven

ts(teratogen

icity)

Highspon

taneousab

or-

tion

rates;interpreta-

tion

difficu

ltifhigh

inciden

ceof

(neu

tra-

lizing)

immunogen

i-city

and/or

spon

taneousab

ortion

May

beusefulif

hom

ologueisphar-

macologicallyactive

inthissystem

and

relevantforclinical

candidatein

human

s

Industry

guidelines

for

comparab

ility(rele-

vance)of

hom

ologue

inroden

tspeciesnot

defined

,may

getdif-

ferentor

irrelevant

answ

erifhom

ologue

not

pharmacologically

comparab

le

May

allow

under-

stan

dingof

tar-

getrole

inrepro/

devel

Gen

etic

deletionor

insertionof

atarget

may

not

represent

chronic

blockad

eor

replacemen

tbydrug

(eg,

somekn

ocko

uts

areem

bryolethal,yet

may

not

beindicative

ofeffectsof

chronic

blockad

e) (con

tinued)

249




Tab

le4.(con

tinued

)

Develop

men

tStrategy

TestHuman

TestArticle

(Clinical

Can

didate)

inNHPSpecies

TestSurrogateRod

entTestArticle

(Hom

ologue)

inRod

entSpecies

TestKnocko

utMiceWithGen

eticDeletionof

Target

Pro

Con

Pro

Con

Pro

Con

Other

uses

Efficacymod

elsfor

testingclinical

candidateeffects

onreproductive

biology

Develop

men

tal

(ped

iatric)

immunotoxicity

Gen

eral

issues

Noindustry

stan

dardfor

NHPim

munotox

orim

munom

odulation

assays;lack

ofhistori-

calcontrols,validated

assays,positivecon-

trols,etc;

difficu

lties

ininterpretingresults

May

beab

leto

apply

method

sad

apted

from

adultstudies

(thisisthetren

d)

Lackof

historicalcon-

trols,validated

assays,

positivecontrols,etc;

difficu

ltiesin

inter-

pretingresults

Gen

etic

deletionor

insertionof

atarget

may

not

represent

chronic

blockad

eor

replacemen

tbydrug

a.Human

shavediscoid

placenta.

CRO,Con

tractResearchOrgan

ization;DART,develop

men

talan

dreproductivetoxicology;IC

H,International

Con

ference

onHarmon

ization;NHP,non

human

primate.

250




characteristics of the molecule, formulation, andpharmacologic activity). Examples have been givenin which surrogates have both under- or overpre-dicted the effects seen in humans, and produced dif-ferent results compared with the clinical candidatein nonhuman primates. However, the potential forover-and underprediction of human effects is a chal-lenge common to both traditional and alternativetoxicity assessments in any nonhuman species.When a difference between results or lack of predic-tivity occurs with a surrogate molecule, additionalquestions arise regarding whether the differencesbetween nonclinical and human studies are due topharmacological or molecular differences betweenthe surrogate and the biopharmaceutical, or simplydue to differences in species biology. Thus, it is crit-ical to fully understand and extensively characterizethe pharmacology of the surrogate molecule in theappropriate species both in vitro and in vivo, andensure that its pharmacology is as similar to that ofthe clinical candidate in humans as possible.Because the pharmacology is often poorly under-stood, the use of alternative approaches in the safetyassessment of biopharmaceuticals should be supple-mental and evaluated as part of a case-by-case,weight-of-evidence approach. However, any of thesealternatives can be valuable support for a biopharma-ceutical safety assessment program in which uniquechallenges of species specificity often make a stan-dard toxicology program inappropriate to conduct.

Acknowledgments

The authors appreciate the BioSafe leadership com-mittee, the PhRMA Biologics Technical Group, andthe FDA’s review of this manuscript and their criticalinput on these complex issues.

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