Download pdf - New challenges and opportunities in nonclinical safety testing of biologics

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Regulatory Toxicology and Pharmacology xxx (2014) xxx–xxx

YRTPH 3029 No. of Pages 8, Model 5G

23 April 2014

Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier .com/locate /yr tph

Workshop Report

New challenges and opportunities in nonclinical safety testingof biologics q

http://dx.doi.org/10.1016/j.yrtph.2014.04.0050273-2300/� 2014 Published by Elsevier Inc.

q New challenges and opportunities in nonclinical safety testing of biologics werediscussed at the 3rd European BioSafe Annual General Membership meeting inNovember 2013, addressing scientific, strategic and experimental approaches inToxicology and Pharmacokinetics.⇑ Corresponding author.

E-mail address: [email protected] (A. Baumann).

Please cite this article in press as: Baumann, A., et al. New challenges and opportunities in nonclinical safety testing of biologics. Regul. Toxicol. Pha(2014), http://dx.doi.org/10.1016/j.yrtph.2014.04.005

Andreas Baumann a,⇑, Kelly Flagella b, Roy Forster c, Lolke de Haan d, Sven Kronenberg e, Mathias Locher f,Wolfgang F. Richter e, Frank-Peter Theil g, Marque Todd h

a Bayer Pharma AG, Berlin, Germanyb Genentech, South San Francisco, USAc CiToxLAB, Evreux, Franced MedImmune, Cambridge, UKe F. Hoffmann-La Roche Ltd, Basel, Switzerlandf Covagen AG, Zuerich, Switzerlandg UCB Pharma, Braine-l’Alleud, Belgiumh Pfizer, La Jolla, USA

a r t i c l e i n f o

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Article history:Received 28 March 2014Available online xxxx

Keywords:BiologicsPharmacokineticsNon-clinical safetyMinipigsPEGylated proteinsDistributionBispecifics

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a b s t r a c t

New challenges and opportunities in nonclinical safety testing of biologics were discussed at the 3rdEuropean BioSafe Annual General Membership meeting in November 2013 in Berlin:

(i) Approaches to refine use of non-human primates in non-clinical safety testing of biologics andcurrent experience on the use of minipigs as alternative non-rodent species.

(ii) Tissue distribution studies as a useful tool to support pharmacokinetic/pharmacodynamic (PKPD)assessment of biologics, in that they provide valuable mechanistic insights at drug levels at thesite of action.

(iii) Mechanisms of nonspecific toxicity of antibody drug conjugates (ADC) and ways to increase thesafety margins.

(iv) Although biologics toxicity typically manifests as exaggerated pharmacology there are somereported case studies on unexpected toxicity.

(v) Specifics of non-clinical development approaches of noncanonical monoclonal antibodies (mAbs),like bispecifics and nanobodies.

� 2014 Published by Elsevier Inc.

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1. Introduction

BioSafe is the Preclinical Safety expert group of the Biotechnol-ogy Industry Organization (BIO), which has been tasked with themission to serve as a resource for BIO members and BIO staff byidentifying and responding to key scientific and regulatory issuesrelated to the preclinical safety evaluation of biopharmaceuticalproducts. Beyond its general membership meetings in the U.S.,BioSafe has started to run in parallel yearly European Meetings

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to foster face to face discussions with European colleagues of BIOmember companies.

The 3rd Annual BioSafe European General Membership meet-ing was hosted by Bayer Pharma on November 18–19, 2013 inBerlin. The 80 scientists (65 from Europe, 15 from U.S. andJapan) – with toxicology, pathology or pharmacokinetic back-ground – represented global big pharmaceutical/biotechnologycompanies, small biotechnology companies and individualcontract research organizations. At this year’s meeting newchallenges in non-clinical development of biologics werediscussed, including animal use and species selection, unex-pected toxicities, distribution behavior and specifics of antibodydrug conjugate and non-traditional mAb development. At eachsession, case examples were presented followed by podiumdiscussions.

rmacol.

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2. Animal use in biologics development

With the increasing importance of biologics in drug develop-ment, non-human primates (NHP) have been identified as the mostsuitable and relevant toxicology species leading to a higherdemand of this species for non-clinical safety testing of biologics.Beyond the increasing ethical and public pressure to explore andadvance approaches to reduce the number of NHPs (Bluemel,2012), it was recently questioned why NHPs are used in biologicsdevelopment, when pharmacology-mediated adverse effects ofmonoclonal antibodies (mAbs) are highly predictive from in vitrostudies (Van Meer et al., 2013). In the introduction of this sessionchaired by Jenny Sims (Integrated Biologix) and Andreas Baumann(Bayer Pharma), it was questioned if this statement is only a futuredream or if it has some realistic components. If pharmacology-mediated adverse effects and PKPD relationships are understoodwith short-term animal studies, what is gained from furtherchronic toxicity studies. The following two lectures reviewedapproaches to refine NHP use in non-clinical safety testing inbiologics development without compromising the risk benefitassessments for human use.

Kathryn Chapman (U.K. National Centre for 3R’s, NC3Rs)presented approaches to minimize the use of NHPs in biologicsdevelopment. There has been particular interest in animal use inbiologic testing since it was recognized that the NHP may be theonly relevant non-clinical toxicology species for many of theseproducts. The NC3Rs, in collaboration with up to 30 organizationsfrom the pharmaceutical, biotechnology, contract research andregulatory environment, have facilitated cross-company data-sharing initiatives to minimize the increase in NHP use(Chapman et al., 0000). These evidence-based approaches havefed into regulatory addendums e.g., ICH S6 (R1) and ICH M3 (R2)and continue to support the field in using appropriate studydesigns to answer the scientific questions at hand. Two currenthot topics in this area with a focus on the 3Rs (replacement, refine-ment and reduction of animals in research) are (i) how oftenrodent models can support biologic development and (ii) howand when recovery animals should be included on studies. Unpub-lished data shows that company portfolios for mAbs range fromhaving no products with rodent potency to a third of their pipelinehaving the potential to use the rodents for some studies. This islinked to therapeutic area, for example less frequent rodentpotency for immunology products. Also the company strategy inscreening for rodent potency in candidate selection and develop-ment varies depending on therapeutic area. There are case studiesshowing that rodent models do support biologic programs andhave the potential to provide more relevant data and reduce theuse of NHP on some occasions. With the revision of ICH S6 (R1)Guideline (Preclinical Safety Evaluation of Biotechnology-DerivedPharmaceuticals), which describes the potential for only usingthe rodent in (sub) chronic studies if the toxicity profile of therodent and NHP is the same in short term studies, the predictionis that the rodent will be used more for development of theseproducts. In addition, technological advances such as microsam-pling mean that rodent data is likely to be used more frequentlyto support clinical trials.

The use of recovery animals has been identified as another areawhere animal use is increasing. Often recovery animals areincluded on all studies conducted and more than one dose group.The question is whether the reasons behind this are scientificallydriven or whether it is upward creep. A cross-company data shar-ing group has looked at 259 studies from 137 compounds and 22companies and these data show wide variation in the number ofrecovery animals used. Analysis shows that there are opportunitiesto reduce the use of recovery animals in certain circumstanceswhich would not impact drug development.

Please cite this article in press as: Baumann, A., et al. New challenges and oppor(2014), http://dx.doi.org/10.1016/j.yrtph.2014.04.005

In addition to the in vivo approaches there is also ongoing workto identify the benefits and limitations of in vitro technologies toassess the safety profile of biologics. A holistic, integrated approachto get the best data from the most appropriate technology orspecies is a ‘must have’ in the future of biologics products.

Lauren Black (Charles River Laboratories) gave some furtherinsights on the use of satellite groups and blood sample volumereduction. For many years different animals were used for toxicityevaluation (satellite animals) than those assayed for blood levels(toxicokinetic = TK groups). Assays of the TK were done in satellitegroups because the analytical methods were not sensitive andrequired up to 500 ll of blood to be drawn for each sample. Suchhigh blood volumes would deplete the rodent’s hematocrit ifdrawn multiple times from the same animal, and this would con-found interpretation of toxicity if performed in the ‘‘main study’’animals designated for pathology. So, until very recently, therodent numbers used for satellite TK and or pharmacodynamics(PD) could end up being half of the animals utilized on the study,and total number is often high (�300). The satellite animals werenot used for any other endpoints, other than TK or PD (no pathol-ogy and no intercurrent clinical pathology). In contrast to rodentstudies, large animal experiments are conducted much moretranslationally, where self baselines are routinely available. Inthese cases, far fewer large animals are used (�30).

It would be optimal to gain all data from each (rodent) animalutilized in toxicology studies, and correlate a given animal’stoxicity and PD measures, with its own TK. The only way to achievemore insight from each animal, depends on two advances; first –refined, methods for taking repeated blood draws from the rodent;and second – developing assay methods that utilize far smallersamples of blood (Powles-Glover et al., 2014).

This way, dynamic insights into animals TK, PD, and clinicalpathology effects might be gained, without undue stress to the ani-mal, or confounding toxic effects from repeated blood draws. Suchadvances have been developed in many labs using capillary basedmicrosampling of only 32 ll of blood, generally referred to asmicrosampling. Micro-ELISA methods have also been developed,and have not posed severe technical hurdles; typically, serum frombiologic drug-treated animals must be diluted anyway, to enableassays to fall within standard curves.

With improved insight, interpretation, and translation, moreinsightful toxicity studies may be designed in rodents, whichmay alleviate some need for NHP work.

3. Use of minipigs in non-clinical safety testing with biologics –Quo vadis ?

Minipigs are increasingly used as non-rodent species fortoxicity testing of pharmaceuticals, in particular in Europe(Svendsen, 2006; Ganderup et al., 2012). However, the focus islargely on small molecule-based therapeutics and dermal adminis-tration (Ganderup, 2011); only few data exist on repeat-dose IVadministration of biologics. The session chaired by Sven Kronen-berg (Hoffmann-La Roche) and Roy Forster (CiToxLAB) providedan overview about the use of minipigs in safety testing of biologics.A gap analysis on the use of mAbs in the minipig by Kronenbergemphasized that the minipig immune system has a largely analo-gous structure and function to the human immune system (Bodeet al., 2010), but a better understanding on how sensitive minipigsare towards infusion-related reactions and FccR-mediated effectorfunction is yet missing. This includes possible (side) effects of IVadministration of mAbs, such as cytokine release, complementactivation and ADCC. Some of the effects can be caused also bypolymer excipients used in biologics formulations: minipigs,similar to dogs, show acute cardio-pulmonary reactions to some

tunities in nonclinical safety testing of biologics. Regul. Toxicol. Pharmacol.


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Table 1Case examples of therapeutic proteins administered to minipigs (CiToxLAB data).

General toxicologySynthetic peptide (iv)Enzyme (oral)Peptide hormone (iv)Protein hormone (iv, sc)Immune modulator (iv)Peptide hormone (sc/im)Nanoparticulate (sc)Protease (local, ocular)Immunoglobulin (multiple)Blood substitute (infusion)

PK & ADMERecombinant proteinMonoclonal antibody (iv)Clotting factor (multiple)Monoclonal antibody (local, ocular)

Reprotox: seg IIPeptide hormone (iv)hormone (iv)

Local toleranceRecombinant protein (local)

A. Baumann et al. / Regulatory Toxicology and Pharmacology xxx (2014) xxx–xxx 3


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IV administered drug carriers and polymers (liposomes/lipid-basedexcipients) due to complement activation, which do not reproducein the ‘‘normal’’ human physiological response to such medication(Szebeni et al., 2007, 2012). However, different to dogs, no undueeffects were seen when typical biologics excipients such as polox-amer and polysorbate 20 and 80 were intravenously administeredto the minipig (Festag et al., 2013), supporting the use of minipigsfor typical mAb formulations. A major gap highlighted was theobvious lack of placental transfer of macromolecules (Szebeniet al., 2007) in the minipig that may limit their role in developmen-tal toxicity testing of mAbs. The lack of placental transfer ofmacromolecules in minipigs is due to their tight placental barrier;any potential contribution of porcine FcRn in the placental transferof human mAbs is unknown. Of note, affinities of various humanIgG’s to minipig FcRn are comparable to human FcRn (Zhenget al., 2012). The placental transfer of antibodies is key to the riskof developmental toxicity even though non-placental transfermechanisms are described in humans (DeSesso et al., 2012). Inminipigs, newborns receive maternal IgG via the colostrum. Itremains unclear whether a cross-reactive teratogenic monoclonalantibody would exert dysmorphogenic effects in the minipig.Other challenges presented, e.g. the rapid body weight gain ofminipigs, would require more flexible testing strategies (shorterstudies and use of younger animals until scale-up of manufacturingis in place). However, tissue cross reactivity testing (if done inminipig) as well as safety pharmacology and fertility endpointsin repeat-dose studies could be carried out in minipigs similar toNHPs.

Jessica Vamathevan (GSK) gave an introduction to the value ofgenomic data in the validation of pharmacological and toxicologicalmolecular targets. Whole genome DNA sequence data is increas-ingly available for animal model species. Sequence data for theGöttingen minipig (79� coverage) were recently generated byGSK (Vamathevan et al., 2013). The sequence data has been placedin the public domain through GenBank, DDBJ and EMBL. The gen-ome size and content of the minipig is comparable to humans asis the number of genes (minipig 18,150 genes; humans 21,656genes). Nevertheless, the evolutionary pressures influencing genesmay differ and the consequences may be varied. Genomic datacan be used to better understand pharmacological target genes.Sequence data can readily indicate conserved (high homology)genes (such as VEGFA or CTLA-4), poorly conserved low homologygenes (such as IL6-R), duplicated and/or truncated genes (such asthe non-functional porcine DHFR gene) or genes that have under-gone species-specific selection (e.g., TRPV-1 in minipig). It is there-fore possible with sequence data to ask a series of questions about atarget gene: Is the gene present in the selected species? Is thesequence well conserved? Has the gene undergone any positiveselection or functional divergence. Future approaches will involvethe integration of expression data (e.g., RNA Seq) into theseapproaches to help understanding the value of this animal model.

Subsequently, Michael Otteneder (Hoffmann-La Roche) gave anoverview on disposition and absorption kinetics of human mAbs inminipigs. In essence, several human mAbs tested showed IgG likePK properties (low clearance, long half-life and low volume ofdistribution) and thereby good translation to humans (Zhenget al., 2012). For both systemic (IV) and local (ocular or SC) admin-istration the minipig is a good translational model based on Rocheexperience. Otteneder also summarized data of an ADCC assayusing minipig PBMC that show a different response towardstherapeutic mAbs (IgG1, afucosylated IgG1 and IgG2) than humanblood cells. This may be due to different affinities of human IgG toporcine FccR and/or different expression levels of FccR immunecell populations. The results warrant further testing to understandif the minipig may respond differently than primates in terms ofeffector function.


In the last talk, Andrew Makin (CiToxLAB Scantox) focused onpractical considerations for the use of minipigs with biologics, aswell as some examples and case histories. On the practical side,concerns focus on the size of minipigs (test item requirements)and the issue of venous access for administration and or bloodsampling (now largely overcome with the availability of new cath-eter and access port materials). Case histories were given related tothe value of a minipig segment II (teratology) study for the evalu-ation of a peptide-based drug for metabolic disease, and the use ofminipigs in the efficacy and safety evaluation of a recombinantbone morphogenetic protein. CiToxLAB experience with biologicstesting in minipigs is summarized in Table 1, and includes severalproducts (such as peptides, protein hormones, monoclonal anti-bodies and clotting factors). In no case was the minipig subse-quently judged to be an inappropriate model. Most of thisinformation is not in the public domain, and it was concluded thatif just some of these studies/projects were published, we wouldprobably have a different impression of the value of minipigs inthe testing of biologics.

In the concluding panel discussion there was agreement thatany ethical concern on use of NHPs will not be solved by introduc-ing the minipig as an alternative non-rodent species despite beingmore perceived as a food and farm animal by the general public.The minipig’s capacity for pain and suffering is the same as thedog or the NHP (Webster et al., 2010). The minipig, however, haspractical advantages in housing and handling, and good animalwelfare is easier to achieve for minipigs as they do not need muchspace and are not athletic like dogs, nor arboreal like NHPs. Despitesome challenges (including lack of published experience, uncer-tainties regarding effector function, lack of placental transfer,quantities of test material, need for easy venous access), theminipig could serve in the future as a potential alternative speciesespecially where the NHP may be less appropriate from a biologyand non-clinical safety perspective. The published genomic datafrom the minipig may help as a first step for target validation.

4. Distribution of biologics

ADME studies are increasingly used to assess the in vivobehavior of biologics and to link those properties with therapeuticeffects and unwanted side effects. Since biologics are given mainlyparenterally, the focus of ADME is primarily on their distribution.The session, chaired by Frank-Peter Theil (UCB) and Wolfgang



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Richter (Hoffmann-La Roche), gave an overview on when distribu-tion studies are warranted, the available experimental approaches,the appropriate interpretation of experimental data, the use ofmodeling approaches and their application in PKPD assessments.

Wolfgang Richter gave an overview as to why distributionstudies can be of relevance for biologics. For small molecules,tissue distribution studies are usually done according to regulatoryrequirements, e.g., to allow dosimetry calculations for human massbalance studies. Due to a rapid equilibration of drug levels betweenplasma and tissues, their PKPD assessment can be done usuallywithout information on tissue levels. By contrast, for biologicsthere is usually no need to conduct distribution studies for regula-tory purposes. Due to the different tissue distribution behavior ofbiologics, however, serum levels are not necessarily predictive fortissue levels. Therefore, tissue distribution data can be very usefulto support PKPD assessment in that they provide valuable mecha-nistic insights of drug levels at the site of action, e.g., in tumors. Inaddition, they may be used for mechanistic investigations, e.g., toexplore sites of unexpected rapid drug clearance or for character-ization of target mediated drug disposition (TMDD). Overall, tissuedistribution studies are a powerful tool to learn about the disposi-tion and PKPD of biologics. The complexities of distributionstudies, however, need to be understood for an appropriate inter-pretation of experimental results.

Andy Boswell (Genentech) summarized experimental tech-niques as well as the selection of appropriate radioprobes andshowed applications of the various techniques in selected caseexamples. Tissue distribution studies can be conducted invasivelyor in a non-invasive manner e.g., by single photon emission com-puted tomography (SPECT) or positron emission tomography(PET). Tissue distribution studies may help to determine receptoroccupancy in tissues, which is usually determined by free druglevels in the tissue interstitial space. Such free drug levels can beestimated from total tissue drug levels obtained in distributionstudies by correcting for drug in residual blood in tissues anddividing the remaining amount of drug by the interstitial volume(Boswell et al., 2012). Residual blood in tissues and the fractionalvolume of interstitial space can be determined using suitableradioprobes (99Tc-labeled red blood cells and with the extra-cellular marker 111In-DTPA) (Boswell et al., 2010, 2011). 86RbCl isused for determination of blood flows. Biologics can be radio-labeled with either non-residualizing labels (e.g., 125I) or residual-izing labels (e.g., 111In). A novel 125I-labeling approach waspresented, which converts 125I to a residualizing label (Boswellet al., 2013a). Various case examples were presented. Use of 86RbClas a radioprobe showed that anti-VEGF treatment reduces tumorblood flow, while blood flow in muscle stayed unchanged(Pastuskovas et al., 2012). For an ADC, the small intestine couldbe identified as the site of TMDD by studying the distribution ofthe 111In-labeled ADC (Boswell et al., 2013b). Predosing with thenaked antibody could block intestinal and liver uptake, but nottumor uptake of the ADC. For another antibody, lack of tumordistribution was identified as the root cause for lack of anti-tumorefficacy.

Jay Tibbitts (UCB) discussed the importance of understandingbiotherapeutic distribution. The mechanism of distribution ofbiologics differs from that of small molecules. Distribution occursmainly via the paracellular route and is influenced by size andcharge of the biotherapeutic. An exception to this is the FcRn-mediated transcellular transport of antibodies postulated forcertain tissues (Garg et al., 2007). Convection and diffusion playkey roles in distribution, the balance of both processes dependson molecular weight and extracellular matrix composition intissues (Reddy et al., 2006). The nonspecific distribution of anti-bodies is generally well understood, is similar across species, butdiffers between tissues (Shah et al., 2013). Tissue distribution data


may help to manage target risk (Tijink et al., 2006) or to optimizemolecular formats (Dennis et al., 2007). Distribution in diseasedtissues may differ from that in healthy tissues (Palframan et al.,2009) suggesting the value of conducting distribution studies indiseased tissues to better understand relationships betweenplasma and the effect site. The understanding of such processesmay provide better insights for therapeutic opportunities. ForADCs, tissue distribution data can be used to optimize moleculesand to explore differences in tissue levels of ADC and the freecytotoxin (Alley et al., 2009). Overall, opportunities exist to usedistribution data to improve PKPD understanding and translationin both healthy and disease states.

Hans Peter Grimm (Hoffmann-La Roche) summarized howmodeling can complement experimental approaches to assesstissue distribution. In the interpretation of tissue distribution dataand their use in PKPD assessment, it is important to know (i) whatis measured, (ii) which tissue sub-compartment is assessed and(iii) in which tissue sub-compartment the drug needs to be to exertits effect. From a PKPD point of view, tissues may comprise numer-ous sub-compartments wherein the drug can reside: capillaryblood, extracellular space, bound to membrane target, intracellu-larly after non-specific uptake and intracellularly followingtarget-mediated uptake. A certain space in tissues is inaccessibleto drug (inaccessible space). Modeling can help to extract moredetailed information on drug levels in tissue sub-compartmentsfrom tissue distribution data [Grimm et al., European BioSafeMeeting 2012, in preparation] and to potentially link them to drugeffects. For a successful modeling of tissue penetration, furtherinformation is required or has to be derived on vascular and inter-stitial volume fractions, dynamic equilibria in the extravasationand interstitial transport as well as on the target-mediated drugdisposition in tissues. Understanding of these processes allowsthe modeling of distribution and finding of the appropriate balancebetween tissue penetration and elimination of a biologic. Thus itwas demonstrated that the highest tumor penetration is notobtained with a small, well tissue-penetrating Fab molecule, butrather with an IgG having a long residence time in the body(Schmidt et al., 2009).

Overall, the session demonstrated the utility of state of the arttissue distribution assessments to enhance the understanding ofPKPD and safety of biologics.

5. Challenges in non-clinical ADC development

This session covered challenges and future directions for thedevelopment of ADCs. ADCs typically comprise a monoclonalantibody directed at a cell surface target and a cytotoxic smallmolecule conjugated to the antibody via a chemical linker. Casestudies presented highlighted the diversity of challenges associ-ated with these molecules. A greater emphasis is being placed onbetter understanding the mechanisms of nonspecific toxicity inan attempt to increase the safety margins of these promisinganti-cancer molecules.

Marque Todd (Pfizer) introduced examples of toxicologyprograms used to support ADCs being developed for oncologyindications. The strategy was aligned with the ICH S9 (NonclinicalEvaluation for Anticancer Pharmaceuticals) and ICH S6(R1) guide-lines and a recent industry white paper (Roberts et al., 2013). Thesetoxicology programs are focused on characterizing the ADC andsecondarily, the cytotoxic drug. Studies typically include up to3-month repeat-dose toxicity studies in relevant species and safetypharmacology, genotoxicity, and tissue cross-reactivityassessments.

There are currently 3 marketed ADCs brentuximab vedotin,ado-trastuzumab emtansine, and gemtuzumab ozogamicine) and



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much was learned about the non-clinical and clinical toxicities ofADCs from these molecules (Ado-Trastuzumab Emtansine(Kadcyla™), 2012; Brentuximab Vedotin (Adcetris™), 2011;Gemtuzumab Ozogamicine (Mylotarg™), 1999). All of these agentshave shown some degree of hematological and hepatic toxicitiesand, with microtubule disrupting ADCs, peripheral neuropathy.There has been relatively good concordance between the non-clinical and clinical safety findings. Most of these toxicities arebelieved to be non-specific and not related to target antigenbinding. There is still much unknown about the biodistributionand uptake of ADCs by tissues not expressing target antigens.

Alternative strategies are being pursued to reduce non-specifictoxicity and increase the therapeutic margins of ADCs. Identifyingbetter targets has been difficult due to a disconnect between ADCactivity and target expression and pharmacology. Efforts are beingfocused on selecting antigen targets that rapidly and preferentiallyinternalize in tumor cells. Site-specific conjugation of the cytotoxicdrug to the antibody is another strategy that may improve the PK,safety and stability of ADCs (Junutula et al., 2008; Strop et al.,2013). Tolerability may also be improved by modifying the dosingregimen in the clinic, as shown with gemtuzumab ozogamicin(Sievers et al., 2001; Castaigne et al., 2012). Other approaches tomitigating toxicity include modifying the ADC structure itself, asin the case of a probody technology developed to mask on-targetantigen binding to reduce toxicity to normal tissues, possiblyallowing the development of targets with broader tissue distribu-tion (Desnoyers et al., 2013).

Ruprecht Zierz (Bayer) presented a case study of an anti-mesothelin–SPDB-DM4 ADC. Mesothelin is expressed on normalpericardium, peritoneum, and pleura and its physiological role isunknown. Since mesothelin is overexpressed on the cell surfaceof a number of human tumors, targeting mesothelin may offerthe potential for an effective tumor-specific therapy (Schatzet al., 2012; Kreft et al., 2013).

On-target toxicity cannot be investigated in non-clinical modelsbecause this ADC only binds human mesothelin. However, theantigen-independent, off target toxicity of the ADC was assessedin rats and cynomolgus monkeys. The first-in-man (FiM) studydesign included a dose regimen consisting of a single dose givenonce every 3 weeks. As such, the toxicology program consisted of(1) single dose general toxicity/toxicokinetic (including recovery),CNS, and respiratory toxicity studies of the ADC and unconjugatedDM4 in rats and (2) a general toxicity/toxicokinetic (includingcardiovascular safety pharmacology and recovery) study of theADC in monkeys. Tissue cross-reactivity studies of the ADC werealso performed with rat, monkey, and human tissues in order toidentify other potential target organs in addition to the knownmesothelial organs.

Toxicology results supported initiation of the FiM study with arecommended starting dose of 0.15 mg/kg/dose and dose escala-tion to a maximally tolerated dose (MTD) of 6.5 mg/kg/dose. Theanti-mesothelin ADC was well tolerated up to the MTD withpromising signs of clinical efficacy at 5.5 and 6.5 mg/kg (Bendellet al., 2013). Most of the toxicities occurring in patients wereidentified in non-clinical studies (e.g., liver toxicity and peripheralneuropathy) or were expected based on experience with otherDM4 containing ADCs (e.g., reversible corneal toxicity). All effectswere considered to be off-target, antigen-independent toxicities.Further clinical cohorts were initiated to evaluate efficacy of thisanti-mesothelin ADC.

Kelly Flagella (Genentech) highlighted key determinants oftoxicity of ADCs and presented non-clinical examples of the impactof linker chemistry, conjugation site, antigen selection and drugmechanism of action on the toxicities of ADCs. One of the keytenets of ADCs is the possibility of improving the therapeutic indexwith the targeted delivery of potent cytotoxic small molecule


drugs. This concept was shown with nonclinical examples, includ-ing a study showing that ado-trastuzumab emtansine, but notunconjugated DM1, reduced tumor growth in a mouse efficacymodel.

The cleavability of the linker affects deconjugation of the ADCand subsequently, impacts toxicity, as illustrated in studies ofanti-CD22 conjugates in rodents (Polson et al., 2009). Toxicitieswere observed with anti-CD22-SPP-DM1 (cleavable) and notanti-CD22-MCC-DM1 (non-cleavable) in rats. Slower drug decon-jugation was observed with anti-CD22-MCC-DM1 suggesting thatits tolerability was related to reduced circulating DM1.

Conventional conjugation technologies result in a heterogenousmixture of ADCs with different drug-to-antibody (DAR) speciesKaur et al., 2013. The extent of drug loading is associated with dif-ferent PK, efficacy and safety profiles of ADCs (Hamblett et al.,2004; Wang et al., 2005). This was illustrated by observations thatpurified anti-HER2-MMAF ADCs with 2, 4, or 6 MMAF moietiescaused increased toxicity with increasing drug substitution of theADC in rats. The development of site-directed conjugation methodsresulted in the generation of more homogenous mixtures of ADCscomprised mainly of DAR 2 species (Junutula et al., 2010) and arereferred to as THIOMAB drug conjugates (TDCs). ADCs and TDCshave been shown to have different PK and safety profiles in non-clinical species, which were dependent on the site of conjugation(Junutula et al., 2008, 2010; Shen et al., 2012). Both the catabolismand deconjugation of some TDCs were slower than ADCs in rats(Junutula et al., 2008). Improved tolerability has also beenreported, as was shown with the reduced neutropenia observedin NHPs with a TDC compared to that of an ADC (Junutula et al.,2008).

The presence of antigen-dependent toxicities may be depen-dent on the drug mechanism of action, as well as the antigen itself.In one example, the safety implications of targeting an intestinalstem cell antigen (Barker et al., 2007) with an ADC were evaluatedin rats using comparable doses of anti-LGR5 ADCs comprised ofeither microtubule disrupting (MMAE) or DNA damaging(NMS818) agents. Gastrointestinal toxicity was observed in ratsgiven anti-LGR5-NMS818, but not anti-LGR5-MMAE, suggestingthat this target-dependent toxicity was, at least in part, dependenton the drug mechanism of action. There are many factors, includ-ing drug potency, mechanism of action, and PK, that can affectthe susceptibility of normal tissue to a cytotoxic ADC. The entiretoxicology and efficacy profile must be considered when selectingthe optimal linker drug design.

6. BioSafe survey on PEGylated proteins (PEGproteins)

Jenny Sims (Integrated Biologix) and Andreas Baumann (Bayer)summarized the status of a survey on use of PEGproteins initiatedby the BioSafe Leadership committee after discussion of this topicat the 2nd European Biosafe General Membership Meeting in 2012(Kronenberg et al., 2013). This survey, was prompted by theincrease in internal as well inter-company discussions along withrecent Health Authority concerns expressed relating to the disposi-tion and safety of PEGproteins (Baumann et al. (submitted forpublication), EMA response to PDCO, http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/11/WC500135123.pdf). The aim of the survey was to collate case stud-ies of company experiences with PEG proteins in development byanonymized information and share with contributing companiesto enable a wider knowledge of the issues. The survey question-naire comprised 17 questions, related to the nature of the productsincluding of the chemical nature of the PEGs, disposition and tox-icology studies performed as well as any special investigationsconducted including PEG alone, (planned) clinical use including


http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/11/WC500135123.pdf




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dosing regimen and molar PEG intake over a specified time period,immunogenicity monitoring and finally, contacts and questionsobtained from Health Authorities during product development.Although information on product details was limited as well asinformation from regulatory discussions, the information on toxi-cology programs was extensive. The products were heterogeneousranging from replacement molecules to targeting products with anoverall molecular weight ranging from 25 to 230 kDa (includingPEG). The molecular weight of the PEG moiety conjugated to theproteins ranged from 20 to 60 kDa. Compounds are almost devel-oped for chronic indications, the PEG load (e.g., with single/weeklydose or life-long treatment) was only given for a few products. Thecomplete results of this survey are expected to be publishedshortly.

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7. Unexpected toxicity – beyond exaggerated pharmacology

In a session chaired by Frank Brennan (UCB) and Lolke deHaan (MedImmune) a number of case studies were presentedon unexpected toxicity of biologics. Simon Henderson (MedIm-mune) opened the session with 2 case studies. In the first casestudy, the toxicity profile of a monoclonal antibody (mAb) direc-ted against Plasminogen Activator Inhibitor 1 (PAI-1) wasdescribed. Non-GLP 1 month repeat dose toxicity studies wereconducted in rat and cynomolgus monkey. These studies showeddose dependent suppression of PAI-1, in the absence of significanttoxicity. Subsequently, to support clinical development, 3 monthrepeat dose toxicity studies in rat and cynomolgus monkey wereconducted. In the rat, treatment with the mAb was associatedwith an increase in the incidence and severity of cardiomyopathyat all dose levels. This was associated with hemorrhage, inflam-mation, accumulation of pigmented macrophages and fibrosis,of which cardiac fibrosis was not reversible and a No ObservedAdverse Effect Level (NOAEL) could not be determined. Further-more, during the dosing phase of the study, Xu et al. (2010)published that PAI-1 knock out (KO) mice develop very similarcardiac pathology, providing additional support for the findingsbeing pharmacologically mediated. In the cynomolgus monkey,treatment with the mAb was associated with dose-dependentsuperficial bruising which was apparent as early as week 2 ofthe study while in recovery animals, bruising completelyresolved. Given that this observation is consistent with thefibrinolytic pharmacology of the mAb, and the phenotype ofPAI-1 deficient human individuals, these findings were notconsidered adverse. However, in 2 low dose animals, cardiaclesions reminiscent of the lesions seen in the rat were apparent.These findings were not consistent with background cardiaclesions seen in the cynomolgus monkey (as reported by(Chamanza et al., 2010), and a NOAEL was not established. Inthe second case study, non-clinical safety studies in the rat andcynomolgus monkey with a genetically engineered metallopepti-dase, neprilysin, fused to albumin to extend plasma half-life(Henderson et al., 2013) were described. Given the concern overdegradation of ‘off target’ peptides, potential adverse effects onthe cardiovascular, endocrine and gastrointestinal systems wereassessed. In both studies, the fusion protein was well tolerated,and not associated with any adverse findings. However, in therat as well as the cynomolgus monkey, treatment was associatedwith marked prolongation of activated partial thromboplastintime (APTT). Subsequent in vitro analysis showed that this effectwas mediated by fibrin degradation. Thus, while degradation ofb-amyloid – the intended peptide target – and other knownpeptide substrates for neprilysin was not associated with adversefindings, these studies revealed a previously unknown substratefor the peptidase.


The second presentation was by Mary McFarlane (MedImmune)and described the unexpected toxicity observed with a highaffinity mAb directed against IgE. Repeat-dose toxicity studies incynomolgus monkeys were conducted to support clinical develop-ment. No adverse findings were observed in a 1 month repeat dosetoxicity study at dose levels up to 150 mg/kg/week. In contrast, in asubsequent 6 month study, hypertrophy of the pituitary gland wasseen in all females at 150 mg/kg/week (high dose) and in 2/3animals at 50 mg/kg/week (low dose). This finding was still presentat the end of a 13-week recovery period in the high dose grouponly. These changes were not associated with any down-streamfunctional effects in endocrine organs. Subsequent investigativestudies confirmed the absence of hyperplasia, found no evidenceof mAb accumulation in pituitary glands, and demonstrated lackof binding of the mAb to circulating female pituitary hormones.In conclusion, therefore, no mechanistic basis could be identifiedfor the observed pituitary hypertrophy in females.

In the final presentation, Annick Cauvin (UCB) presented on anenhanced pre- and post-natal development (ePPND) study incynomolgus monkeys conducted with an anti-cytokine mAb. Thisstudy was conducted against the backdrop of data showing that(i) KO mice for the same target were healthy and fertile andshowed no reproductive abnormalities, (ii) an antibody targetingthe same pathway was not associated with toxicity in an embryof-etal development (EFD) study in cynomolgus monkeys, and (iii)antibodies targeting the same pathway were not associated withadverse findings in reproductive and developmental studies inmice. General toxicity studies conducted with the anti-cytokinemAb in the cynomolgus monkey were uneventful. In the ePPNDstudy, mAb treatment was well tolerated during pregnancy. How-ever, mortality was observed at delivery in some treated maternalanimals, without premonitory signs. Furthermore, the duration ofgestation was significantly increased. Findings suggested anincreased incidence of difficult delivery (dystocia) associated withplacental retention and, in some cases, significant blood loss. Thesewere all considered target-related. Increased incidence of perinatalmortality was also observed in infants from treated mothers.Evaluation indicated a lack of care/feeding and/or head injuryconsecutive to dystocia and therefore indirect relation to treat-ment. Surviving infants developed normally with a functionalimmune system. None of the findings of this ePPND study wereobserved in mice, based on a different in physiology of parturition.In conclusion, species selection for reproductive and developmen-tal toxicity studies requires consideration of target involvementand modulation as a function of the physiological state. The cyno-molgus monkey, but not mice, predicted for a parturition risk forwomen.

Overall, this session demonstrated that biologics can be associ-ated with significant adverse effects. While effects were mostlypharmacologically mediated, they were not necessarily alwayspredicted, and identified previously unknown consequences ofthe respective pharmacologies.

8. Non-traditional mAbs – PK and safety implications

Non-traditional (non-canonical) mAbs like ADCs, but alsobispecifics, engineered antibodies and antibody fragments areincreasingly developed especially in indications like cancer. Thecanonical bivalent, monospecific, full-length IgG represents onlyabout half the anticancer mAbs in development (Reichert andDhimolea, 2012).

Mathias Locher (Covagen) presented the FynomAb technology.FynomAbs are bispecific or trispecific IgG-Fynomer-fusion proteinswhere a set of Fynomers adds a second or third binding modality tothe antibody. Fynomers are small (7 kD) fully human proteins,



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derived from the SH3 domain of the human Fyn kinase. The SH3domain is the subunit of the Fyn-kinase that initiates theprotein–protein interaction of the kinase. This protein–proteininteraction is meditated by 2 loops within the SH3 domain. Thefusion site of the Fynomers to a IgG molecule can be controlled(N terminal, C terminal, heavy chain, light chain) allowing the cre-ation of all possible bispecific architectures. Bispecific or trispecificFynomAbs usually exhibit a different mode of action compared tothe mono-specific IgG or even a combination of respective antibod-ies. Pharmacokinetics of FynomAbs where shown to be IgG-likeand the functionality of the Fc-part of the antibody is alwaysretained. COVA322, an anti-TNF/anti-IL17A bispecific FynomAb.was well tolerated in a 4 week cynomolgus monkey toxicologystudy and the clinical trial application to start a first into patientstudy early in 2014 has been filed.

Benno Rattel (Amgen) presented new insights into the therapyof ALL patients with blinatumomab, a bispecific T-cell engager,commonly referred to as a BiTE� antibody. The molecule is com-prises two different flexibly-linked single-chain antibodies, onedirected against CD19 and one targeting CD3. Blinatumomabtransiently links CD19-positive tumor cells with resting polyclonalT-cells for induction of a surface target antigen-dependent re-directed lysis of tumor cells, closely mimicking a natural cytotoxicT-cell response. The non-clinical pharmacological and pharmacoki-netic characterization was shown to be predictive for patients, andtreatment with blinatumomab as a monotherapy has shown an80% complete molecular response rate and prolonged leukemia-free survival in patients with minimal residual B-lineage acutelymphoblastic leukemia (MRD+B-ALL) Topp et al., 2011. After startof infusion, B-cell counts dropped to <1 B cell/L within an averageof 2 days and remained essentially undetectable for the entiretreatment period. By contrast, T-cell counts in all patients loweredrapidly within <1 day but recovered to baseline within a few days.T cells then expanded and on average more than doubled overbaseline within 2–3 weeks under continued infusion of blinatumo-mab (Klinger et al., 2012). A significant percentage of reappearingCD8 and CD4 T cells newly expressed activation marker CD69.

In a second phase II study in patients with (r/r) B-precursor ALL,88% of responders (n = 25/36) achieved molecular remission.Blinatumomab was shown to be active in patients, elderly andpediatric, who have exhausted all established therapies. Surpris-ingly, blinatumomab was also active in heavily immune-compro-mised patients with almost undetectable peripheral T cells.

Judith Baumeister (Ablynx) presented the so-called Nanobodies.Nanobodies are next generation biologics derived from variabledomains of naturally occurring mammalian heavy-chain only anti-bodies. Nanobodies as singular domains retain full functionalityand can be formatted flexibly to yield multi-specific, multi-valentor bi-paratopic molecules as is required to obtain optimal targetengagement. Pharmacokinetic properties can be modulated byincorporation of albumin-binding moieties conferring long half-lifeif desired for a given indication. PKPD properties of Nanobodiesdepend on their format as was detailed in two case studies: (i) Cap-lacizumab is a 28 kDa bivalent Nanobody targeting von WillebrandFactor (vWF) with both its moieties. Caplacizumab is in Phase IIclinical development for adjunct treatment of acquired thromboticthrombocytic purpura (TTP), a life threatening condition secondaryto hyperactive vWF. Caplacizumab avidly binds to its target and isretained in circulation in complexed form. Unbound drug is rapidlycleared via renal clearance thus minimizing overdosing potential.Non-clinically, reduction of vWF and consequently FVIII levelswas observed, together with mild signs of a bleeding tendency(epistaxis, menorrhagia, hematoma at injection sites), in line withexpected pharmacology. The PK behavior (short retention time andtimely wash out) benefits the mode of action and intended indica-tion. As a second example (ii) a bispecific trivalent molecule


(41 kDa) was described, which binds to its target with twomoieties and binds albumin via a third. Binding to albumin confersprolonged half-life and PK properties comparable to monoclonalantibodies in that the dominant linear clearance mechanismfollows that of albumin in the researched non-clinical species orman and a second, nonlinear clearance in the example contributedthrough TMDD.

9. Conclusions

Although approaches for non-clinical testing of biologics areoften planned case by case, trends with regard to more standard-ized programs are emerging, driven by lessons learned and onthe basis of evolving regulatory guidance (Baumann, 2009). Knowl-edge exchange between experts including different experiences iscritical in this evolving area. This meeting brought together expertsfrom different angle of non-clinical development of biologics,toxicologists as well as PK scientists. Beyond lectures, podium dis-cussions were held after most sessions to get more insights intoindividual case examples and our current understanding. Thisapproach gets more importance since there is still limited or noaccess to primary company data bases for retrospective analysisof (failed) development studies. Finally, further initiatives havebeen discussed, like support of symposia in the N3Rs area withcase examples or compiling of existing data for better support ofspecies selection, which will be discussed at the next meeting.

Conflict of interest

None declared.

Acknowledgments

The authors would like to thank all speakers (contact listonline) for their contributions and members of the BioSafe Leader-ship committee for valuable comments during review of thismanuscript.

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http://dx.doi.org/10.3109/10408444.2011.653487

http://dx.doi.org/10.1177/1091581812462039

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http://dx.doi.org/10.1016/j.addr.2012.07.005

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http://dx.doi.org/10.4161/mabs.25234

http://dx.doi.org/10.4161/mabs.25234


