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Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph


Workshop Report

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New challenges and opportunities in nonclinical safety testing of biologics q

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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, Germany Genentech, South San Francisco, USA CiToxLAB, Evreux, France d MedImmune, Cambridge, UK e F. Hoffmann-La Roche Ltd, Basel, Switzerland f Covagen AG, Zuerich, Switzerland g UCB Pharma, Braine-l’Alleud, Belgium h Pfizer, La Jolla, USA b c

a r t i c l e

i n f o

Article history: Received 28 March 2014 Available online xxxx Keywords: Biologics Pharmacokinetics Non-clinical safety Minipigs PEGylated proteins Distribution Bispecifics

a b s t r a c t New challenges and opportunities in nonclinical safety testing of biologics were discussed at the 3rd European BioSafe Annual General Membership meeting in November 2013 in Berlin:

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(i) Approaches to refine use of non-human primates in non-clinical safety testing of biologics and current 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 the site of action. (iii) Mechanisms of nonspecific toxicity of antibody drug conjugates (ADC) and ways to increase the safety margins. (iv) Although biologics toxicity typically manifests as exaggerated pharmacology there are some reported case studies on unexpected toxicity. (v) Specifics of non-clinical development approaches of noncanonical monoclonal antibodies (mAbs), like bispecifics and nanobodies.


Ó 2014 Published by Elsevier Inc.

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1. Introduction


BioSafe is the Preclinical Safety expert group of the Biotechnology Industry Organization (BIO), which has been tasked with the mission to serve as a resource for BIO members and BIO staff by identifying and responding to key scientific and regulatory issues related to the preclinical safety evaluation of biopharmaceutical products. Beyond its general membership meetings in the U.S., BioSafe has started to run in parallel yearly European Meetings

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New challenges and opportunities in nonclinical safety testing of biologics were discussed at the 3rd European BioSafe Annual General Membership meeting in November 2013, addressing scientific, strategic and experimental approaches in Toxicology and Pharmacokinetics. ⇑ Corresponding author. E-mail address: [email protected] (A. Baumann).

to foster face to face discussions with European colleagues of BIO member companies. The 3rd Annual BioSafe European General Membership meeting was hosted by Bayer Pharma on November 18–19, 2013 in Berlin. The 80 scientists (65 from Europe, 15 from U.S. and Japan) – with toxicology, pathology or pharmacokinetic background – represented global big pharmaceutical/biotechnology companies, small biotechnology companies and individual contract research organizations. At this year’s meeting new challenges in non-clinical development of biologics were discussed, including animal use and species selection, unexpected toxicities, distribution behavior and specifics of antibody drug conjugate and non-traditional mAb development. At each session, case examples were presented followed by podium discussions.

http://dx.doi.org/10.1016/j.yrtph.2014.04.005 0273-2300/Ó 2014 Published by Elsevier Inc.

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

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2. Animal use in biologics development With the increasing importance of biologics in drug development, non-human primates (NHP) have been identified as the most suitable and relevant toxicology species leading to a higher demand of this species for non-clinical safety testing of biologics. Beyond the increasing ethical and public pressure to explore and advance approaches to reduce the number of NHPs (Bluemel, Q3 2012), it was recently questioned why NHPs are used in biologics development, when pharmacology-mediated adverse effects of monoclonal antibodies (mAbs) are highly predictive from in vitro studies (Van Meer et al., 2013). In the introduction of this session chaired by Jenny Sims (Integrated Biologix) and Andreas Baumann (Bayer Pharma), it was questioned if this statement is only a future dream or if it has some realistic components. If pharmacologymediated adverse effects and PKPD relationships are understood with short-term animal studies, what is gained from further chronic toxicity studies. The following two lectures reviewed approaches to refine NHP use in non-clinical safety testing in biologics development without compromising the risk benefit assessments for human use. Kathryn Chapman (U.K. National Centre for 3R’s, NC3Rs) presented approaches to minimize the use of NHPs in biologics development. There has been particular interest in animal use in biologic testing since it was recognized that the NHP may be the only relevant non-clinical toxicology species for many of these products. The NC3Rs, in collaboration with up to 30 organizations from the pharmaceutical, biotechnology, contract research and regulatory environment, have facilitated cross-company datasharing initiatives to minimize the increase in NHP use (Chapman et al., 0000). These evidence-based approaches have fed into regulatory addendums e.g., ICH S6 (R1) and ICH M3 (R2) and continue to support the field in using appropriate study designs to answer the scientific questions at hand. Two current hot topics in this area with a focus on the 3Rs (replacement, refinement and reduction of animals in research) are (i) how often rodent models can support biologic development and (ii) how and when recovery animals should be included on studies. Unpublished data shows that company portfolios for mAbs range from having no products with rodent potency to a third of their pipeline having the potential to use the rodents for some studies. This is linked to therapeutic area, for example less frequent rodent potency for immunology products. Also the company strategy in screening for rodent potency in candidate selection and development varies depending on therapeutic area. There are case studies showing that rodent models do support biologic programs and have the potential to provide more relevant data and reduce the use of NHP on some occasions. With the revision of ICH S6 (R1) Guideline (Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals), which describes the potential for only using the rodent in (sub) chronic studies if the toxicity profile of the rodent and NHP is the same in short term studies, the prediction is that the rodent will be used more for development of these products. In addition, technological advances such as microsampling mean that rodent data is likely to be used more frequently to support clinical trials. The use of recovery animals has been identified as another area where animal use is increasing. Often recovery animals are included on all studies conducted and more than one dose group. The question is whether the reasons behind this are scientifically driven or whether it is upward creep. A cross-company data sharing group has looked at 259 studies from 137 compounds and 22 companies and these data show wide variation in the number of recovery animals used. Analysis shows that there are opportunities to reduce the use of recovery animals in certain circumstances which would not impact drug development.

In addition to the in vivo approaches there is also ongoing work to identify the benefits and limitations of in vitro technologies to assess the safety profile of biologics. A holistic, integrated approach to get the best data from the most appropriate technology or species is a ‘must have’ in the future of biologics products. Lauren Black (Charles River Laboratories) gave some further insights on the use of satellite groups and blood sample volume reduction. For many years different animals were used for toxicity evaluation (satellite animals) than those assayed for blood levels (toxicokinetic = TK groups). Assays of the TK were done in satellite groups because the analytical methods were not sensitive and required up to 500 ll of blood to be drawn for each sample. Such high blood volumes would deplete the rodent’s hematocrit if drawn multiple times from the same animal, and this would confound interpretation of toxicity if performed in the ‘‘main study’’ animals designated for pathology. So, until very recently, the rodent 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 were not used for any other endpoints, other than TK or PD (no pathology and no intercurrent clinical pathology). In contrast to rodent studies, large animal experiments are conducted much more translationally, where self baselines are routinely available. In these cases, far fewer large animals are used (30). It would be optimal to gain all data from each (rodent) animal utilized in toxicology studies, and correlate a given animal’s toxicity and PD measures, with its own TK. The only way to achieve more 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 smaller samples of blood (Powles-Glover et al., 2014). This way, dynamic insights into animals TK, PD, and clinical pathology effects might be gained, without undue stress to the animal, or confounding toxic effects from repeated blood draws. Such advances have been developed in many labs using capillary based microsampling of only 32 ll of blood, generally referred to as microsampling. Micro-ELISA methods have also been developed, and have not posed severe technical hurdles; typically, serum from biologic drug-treated animals must be diluted anyway, to enable assays to fall within standard curves. With improved insight, interpretation, and translation, more insightful toxicity studies may be designed in rodents, which may 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 for toxicity testing of pharmaceuticals, in particular in Europe (Svendsen, 2006; Ganderup et al., 2012). However, the focus is largely on small molecule-based therapeutics and dermal administration (Ganderup, 2011); only few data exist on repeat-dose IV administration of biologics. The session chaired by Sven Kronenberg (Hoffmann-La Roche) and Roy Forster (CiToxLAB) provided an overview about the use of minipigs in safety testing of biologics. A gap analysis on the use of mAbs in the minipig by Kronenberg emphasized that the minipig immune system has a largely analogous structure and function to the human immune system (Bode et al., 2010), but a better understanding on how sensitive minipigs are towards infusion-related reactions and FccR-mediated effector function is yet missing. This includes possible (side) effects of IV administration of mAbs, such as cytokine release, complement activation and ADCC. Some of the effects can be caused also by polymer excipients used in biologics formulations: minipigs, similar to dogs, show acute cardio-pulmonary reactions to some


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

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IV administered drug carriers and polymers (liposomes/lipid-based excipients) due to complement activation, which do not reproduce in the ‘‘normal’’ human physiological response to such medication (Szebeni et al., 2007, 2012). However, different to dogs, no undue effects were seen when typical biologics excipients such as poloxamer and polysorbate 20 and 80 were intravenously administered to the minipig (Festag et al., 2013), supporting the use of minipigs for typical mAb formulations. A major gap highlighted was the obvious lack of placental transfer of macromolecules (Szebeni et al., 2007) in the minipig that may limit their role in developmental toxicity testing of mAbs. The lack of placental transfer of macromolecules in minipigs is due to their tight placental barrier; any potential contribution of porcine FcRn in the placental transfer of human mAbs is unknown. Of note, affinities of various human IgG’s to minipig FcRn are comparable to human FcRn (Zheng et al., 2012). The placental transfer of antibodies is key to the risk of developmental toxicity even though non-placental transfer mechanisms are described in humans (DeSesso et al., 2012). In minipigs, newborns receive maternal IgG via the colostrum. It remains unclear whether a cross-reactive teratogenic monoclonal antibody would exert dysmorphogenic effects in the minipig. Other challenges presented, e.g. the rapid body weight gain of minipigs, would require more flexible testing strategies (shorter studies and use of younger animals until scale-up of manufacturing is in place). However, tissue cross reactivity testing (if done in minipig) as well as safety pharmacology and fertility endpoints in repeat-dose studies could be carried out in minipigs similar to NHPs. Jessica Vamathevan (GSK) gave an introduction to the value of genomic data in the validation of pharmacological and toxicological molecular targets. Whole genome DNA sequence data is increasingly available for animal model species. Sequence data for the Göttingen minipig (79 coverage) were recently generated by GSK (Vamathevan et al., 2013). The sequence data has been placed in the public domain through GenBank, DDBJ and EMBL. The genome size and content of the minipig is comparable to humans as is the number of genes (minipig 18,150 genes; humans 21,656 genes). Nevertheless, the evolutionary pressures influencing genes may differ and the consequences may be varied. Genomic data can 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 homology genes (such as IL6-R), duplicated and/or truncated genes (such as the non-functional porcine DHFR gene) or genes that have undergone species-specific selection (e.g., TRPV-1 in minipig). It is therefore possible with sequence data to ask a series of questions about a target gene: Is the gene present in the selected species? Is the sequence well conserved? Has the gene undergone any positive selection or functional divergence. Future approaches will involve the integration of expression data (e.g., RNA Seq) into these approaches to help understanding the value of this animal model. Subsequently, Michael Otteneder (Hoffmann-La Roche) gave an overview on disposition and absorption kinetics of human mAbs in minipigs. In essence, several human mAbs tested showed IgG like PK properties (low clearance, long half-life and low volume of distribution) and thereby good translation to humans (Zheng et al., 2012). For both systemic (IV) and local (ocular or SC) administration the minipig is a good translational model based on Roche experience. Otteneder also summarized data of an ADCC assay using minipig PBMC that show a different response towards therapeutic mAbs (IgG1, afucosylated IgG1 and IgG2) than human blood cells. This may be due to different affinities of human IgG to porcine FccR and/or different expression levels of FccR immune cell populations. The results warrant further testing to understand if the minipig may respond differently than primates in terms of effector function.


In the last talk, Andrew Makin (CiToxLAB Scantox) focused on practical considerations for the use of minipigs with biologics, as well 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 blood sampling (now largely overcome with the availability of new catheter and access port materials). Case histories were given related to the value of a minipig segment II (teratology) study for the evaluation of a peptide-based drug for metabolic disease, and the use of minipigs in the efficacy and safety evaluation of a recombinant bone morphogenetic protein. CiToxLAB experience with biologics testing in minipigs is summarized in Table 1, and includes several products (such as peptides, protein hormones, monoclonal antibodies and clotting factors). In no case was the minipig subsequently judged to be an inappropriate model. Most of this information is not in the public domain, and it was concluded that if just some of these studies/projects were published, we would probably have a different impression of the value of minipigs in the testing of biologics. In the concluding panel discussion there was agreement that any ethical concern on use of NHPs will not be solved by introducing the minipig as an alternative non-rodent species despite being more perceived as a food and farm animal by the general public. The minipig’s capacity for pain and suffering is the same as the dog or the NHP (Webster et al., 2010). The minipig, however, has practical advantages in housing and handling, and good animal welfare is easier to achieve for minipigs as they do not need much space and are not athletic like dogs, nor arboreal like NHPs. Despite some challenges (including lack of published experience, uncertainties regarding effector function, lack of placental transfer, quantities of test material, need for easy venous access), the minipig could serve in the future as a potential alternative species especially where the NHP may be less appropriate from a biology and non-clinical safety perspective. The published genomic data from the minipig may help as a first step for target validation.


4. Distribution of biologics


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


Table 1 Case examples of therapeutic proteins administered to minipigs (CiToxLAB data). General toxicology Synthetic 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 & ADME Recombinant protein Monoclonal antibody (iv) Clotting factor (multiple) Monoclonal antibody (local, ocular) Reprotox: seg II Peptide hormone (iv) hormone (iv) Local tolerance Recombinant protein (local)

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Richter (Hoffmann-La Roche), gave an overview on when distribution studies are warranted, the available experimental approaches, the appropriate interpretation of experimental data, the use of modeling approaches and their application in PKPD assessments. Wolfgang Richter gave an overview as to why distribution studies can be of relevance for biologics. For small molecules, tissue distribution studies are usually done according to regulatory requirements, e.g., to allow dosimetry calculations for human mass balance studies. Due to a rapid equilibration of drug levels between plasma and tissues, their PKPD assessment can be done usually without information on tissue levels. By contrast, for biologics there is usually no need to conduct distribution studies for regulatory purposes. Due to the different tissue distribution behavior of biologics, however, serum levels are not necessarily predictive for tissue levels. Therefore, tissue distribution data can be very useful to support PKPD assessment in that they provide valuable mechanistic insights of drug levels at the site of action, e.g., in tumors. In addition, they may be used for mechanistic investigations, e.g., to explore sites of unexpected rapid drug clearance or for characterization of target mediated drug disposition (TMDD). Overall, tissue distribution studies are a powerful tool to learn about the disposition and PKPD of biologics. The complexities of distribution studies, however, need to be understood for an appropriate interpretation of experimental results. Andy Boswell (Genentech) summarized experimental techniques as well as the selection of appropriate radioprobes and showed applications of the various techniques in selected case examples. Tissue distribution studies can be conducted invasively or in a non-invasive manner e.g., by single photon emission computed tomography (SPECT) or positron emission tomography (PET). Tissue distribution studies may help to determine receptor occupancy in tissues, which is usually determined by free drug levels in the tissue interstitial space. Such free drug levels can be estimated from total tissue drug levels obtained in distribution studies by correcting for drug in residual blood in tissues and dividing the remaining amount of drug by the interstitial volume (Boswell et al., 2012). Residual blood in tissues and the fractional volume of interstitial space can be determined using suitable radioprobes (99Tc-labeled red blood cells and with the extracellular marker 111In-DTPA) (Boswell et al., 2010, 2011). 86RbCl is used for determination of blood flows. Biologics can be radiolabeled with either non-residualizing labels (e.g., 125I) or residualizing labels (e.g., 111In). A novel 125I-labeling approach was presented, which converts 125I to a residualizing label (Boswell et al., 2013a). Various case examples were presented. Use of 86RbCl as a radioprobe showed that anti-VEGF treatment reduces tumor blood flow, while blood flow in muscle stayed unchanged (Pastuskovas et al., 2012). For an ADC, the small intestine could be identified as the site of TMDD by studying the distribution of the 111In-labeled ADC (Boswell et al., 2013b). Predosing with the naked antibody could block intestinal and liver uptake, but not tumor uptake of the ADC. For another antibody, lack of tumor distribution was identified as the root cause for lack of anti-tumor efficacy. Jay Tibbitts (UCB) discussed the importance of understanding biotherapeutic distribution. The mechanism of distribution of biologics differs from that of small molecules. Distribution occurs mainly via the paracellular route and is influenced by size and charge of the biotherapeutic. An exception to this is the FcRnmediated transcellular transport of antibodies postulated for certain tissues (Garg et al., 2007). Convection and diffusion play key roles in distribution, the balance of both processes depends on molecular weight and extracellular matrix composition in tissues (Reddy et al., 2006). The nonspecific distribution of antibodies is generally well understood, is similar across species, but differs between tissues (Shah et al., 2013). Tissue distribution data

may help to manage target risk (Tijink et al., 2006) or to optimize molecular formats (Dennis et al., 2007). Distribution in diseased tissues may differ from that in healthy tissues (Palframan et al., 2009) suggesting the value of conducting distribution studies in diseased tissues to better understand relationships between plasma and the effect site. The understanding of such processes may provide better insights for therapeutic opportunities. For ADCs, tissue distribution data can be used to optimize molecules and to explore differences in tissue levels of ADC and the free cytotoxin (Alley et al., 2009). Overall, opportunities exist to use distribution data to improve PKPD understanding and translation in both healthy and disease states. Hans Peter Grimm (Hoffmann-La Roche) summarized how modeling can complement experimental approaches to assess tissue distribution. In the interpretation of tissue distribution data and their use in PKPD assessment, it is important to know (i) what is measured, (ii) which tissue sub-compartment is assessed and (iii) in which tissue sub-compartment the drug needs to be to exert its effect. From a PKPD point of view, tissues may comprise numerous sub-compartments wherein the drug can reside: capillary blood, extracellular space, bound to membrane target, intracellularly after non-specific uptake and intracellularly following target-mediated uptake. A certain space in tissues is inaccessible to drug (inaccessible space). Modeling can help to extract more detailed information on drug levels in tissue sub-compartments from tissue distribution data [Grimm et al., European BioSafe Q4 Meeting 2012, in preparation] and to potentially link them to drug effects. For a successful modeling of tissue penetration, further information is required or has to be derived on vascular and interstitial volume fractions, dynamic equilibria in the extravasation and interstitial transport as well as on the target-mediated drug disposition in tissues. Understanding of these processes allows the modeling of distribution and finding of the appropriate balance between tissue penetration and elimination of a biologic. Thus it was demonstrated that the highest tumor penetration is not obtained with a small, well tissue-penetrating Fab molecule, but rather 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 art tissue distribution assessments to enhance the understanding of PKPD and safety of biologics.


5. Challenges in non-clinical ADC development


This session covered challenges and future directions for the development of ADCs. ADCs typically comprise a monoclonal antibody directed at a cell surface target and a cytotoxic small molecule conjugated to the antibody via a chemical linker. Case studies presented highlighted the diversity of challenges associated with these molecules. A greater emphasis is being placed on better understanding the mechanisms of nonspecific toxicity in an attempt to increase the safety margins of these promising anti-cancer molecules. Marque Todd (Pfizer) introduced examples of toxicology programs used to support ADCs being developed for oncology indications. The strategy was aligned with the ICH S9 (Nonclinical Evaluation for Anticancer Pharmaceuticals) and ICH S6(R1) guidelines and a recent industry white paper (Roberts et al., 2013). These toxicology programs are focused on characterizing the ADC and secondarily, the cytotoxic drug. Studies typically include up to 3-month repeat-dose toxicity studies in relevant species and safety pharmacology, genotoxicity, and tissue cross-reactivity assessments. 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 of ADCs from these molecules (Ado-Trastuzumab Emtansine (Kadcyla™), 2012; Brentuximab Vedotin (Adcetris™), 2011; Gemtuzumab Ozogamicine (Mylotarg™), 1999). All of these agents have shown some degree of hematological and hepatic toxicities and, with microtubule disrupting ADCs, peripheral neuropathy. There has been relatively good concordance between the nonclinical and clinical safety findings. Most of these toxicities are believed to be non-specific and not related to target antigen binding. There is still much unknown about the biodistribution and uptake of ADCs by tissues not expressing target antigens. Alternative strategies are being pursued to reduce non-specific toxicity and increase the therapeutic margins of ADCs. Identifying better targets has been difficult due to a disconnect between ADC activity and target expression and pharmacology. Efforts are being focused on selecting antigen targets that rapidly and preferentially internalize in tumor cells. Site-specific conjugation of the cytotoxic drug 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 dosing regimen in the clinic, as shown with gemtuzumab ozogamicin (Sievers et al., 2001; Castaigne et al., 2012). Other approaches to mitigating toxicity include modifying the ADC structure itself, as in the case of a probody technology developed to mask on-target antigen binding to reduce toxicity to normal tissues, possibly allowing the development of targets with broader tissue distribution (Desnoyers et al., 2013). Ruprecht Zierz (Bayer) presented a case study of an antimesothelin–SPDB-DM4 ADC. Mesothelin is expressed on normal pericardium, peritoneum, and pleura and its physiological role is unknown. Since mesothelin is overexpressed on the cell surface of a number of human tumors, targeting mesothelin may offer the potential for an effective tumor-specific therapy (Schatz et al., 2012; Kreft et al., 2013). On-target toxicity cannot be investigated in non-clinical models because this ADC only binds human mesothelin. However, the antigen-independent, off target toxicity of the ADC was assessed in rats and cynomolgus monkeys. The first-in-man (FiM) study design included a dose regimen consisting of a single dose given once 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 unconjugated DM4 in rats and (2) a general toxicity/toxicokinetic (including cardiovascular safety pharmacology and recovery) study of the ADC in monkeys. Tissue cross-reactivity studies of the ADC were also performed with rat, monkey, and human tissues in order to identify other potential target organs in addition to the known mesothelial organs. Toxicology results supported initiation of the FiM study with a recommended starting dose of 0.15 mg/kg/dose and dose escalation to a maximally tolerated dose (MTD) of 6.5 mg/kg/dose. The anti-mesothelin ADC was well tolerated up to the MTD with promising signs of clinical efficacy at 5.5 and 6.5 mg/kg (Bendell et al., 2013). Most of the toxicities occurring in patients were identified in non-clinical studies (e.g., liver toxicity and peripheral neuropathy) or were expected based on experience with other DM4 containing ADCs (e.g., reversible corneal toxicity). All effects were considered to be off-target, antigen-independent toxicities. Further clinical cohorts were initiated to evaluate efficacy of this anti-mesothelin ADC. Kelly Flagella (Genentech) highlighted key determinants of toxicity of ADCs and presented non-clinical examples of the impact of linker chemistry, conjugation site, antigen selection and drug mechanism of action on the toxicities of ADCs. One of the key tenets of ADCs is the possibility of improving the therapeutic index with the targeted delivery of potent cytotoxic small molecule


drugs. This concept was shown with nonclinical examples, including a study showing that ado-trastuzumab emtansine, but not unconjugated DM1, reduced tumor growth in a mouse efficacy model. The cleavability of the linker affects deconjugation of the ADC and subsequently, impacts toxicity, as illustrated in studies of anti-CD22 conjugates in rodents (Polson et al., 2009). Toxicities were observed with anti-CD22-SPP-DM1 (cleavable) and not anti-CD22-MCC-DM1 (non-cleavable) in rats. Slower drug deconjugation was observed with anti-CD22-MCC-DM1 suggesting that its tolerability was related to reduced circulating DM1. Conventional conjugation technologies result in a heterogenous mixture of ADCs with different drug-to-antibody (DAR) species Kaur et al., 2013. The extent of drug loading is associated with different PK, efficacy and safety profiles of ADCs (Hamblett et al., 2004; Wang et al., 2005). This was illustrated by observations that purified anti-HER2-MMAF ADCs with 2, 4, or 6 MMAF moieties caused increased toxicity with increasing drug substitution of the ADC in rats. The development of site-directed conjugation methods resulted in the generation of more homogenous mixtures of ADCs comprised mainly of DAR 2 species (Junutula et al., 2010) and are referred to as THIOMAB drug conjugates (TDCs). ADCs and TDCs have been shown to have different PK and safety profiles in nonclinical species, which were dependent on the site of conjugation (Junutula et al., 2008, 2010; Shen et al., 2012). Both the catabolism and deconjugation of some TDCs were slower than ADCs in rats (Junutula et al., 2008). Improved tolerability has also been reported, as was shown with the reduced neutropenia observed in NHPs with a TDC compared to that of an ADC (Junutula et al., 2008). The presence of antigen-dependent toxicities may be dependent on the drug mechanism of action, as well as the antigen itself. In one example, the safety implications of targeting an intestinal stem cell antigen (Barker et al., 2007) with an ADC were evaluated in rats using comparable doses of anti-LGR5 ADCs comprised of either microtubule disrupting (MMAE) or DNA damaging (NMS818) agents. Gastrointestinal toxicity was observed in rats given anti-LGR5-NMS818, but not anti-LGR5-MMAE, suggesting that this target-dependent toxicity was, at least in part, dependent on the drug mechanism of action. There are many factors, including drug potency, mechanism of action, and PK, that can affect the susceptibility of normal tissue to a cytotoxic ADC. The entire toxicology and efficacy profile must be considered when selecting the 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 initiated by the BioSafe Leadership committee after discussion of this topic at the 2nd European Biosafe General Membership Meeting in 2012 (Kronenberg et al., 2013). This survey, was prompted by the increase in internal as well inter-company discussions along with recent Health Authority concerns expressed relating to the disposition and safety of PEGproteins (Baumann et al. (submitted for Q5 publication), 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 studies of company experiences with PEG proteins in development by anonymized information and share with contributing companies to enable a wider knowledge of the issues. The survey questionnaire comprised 17 questions, related to the nature of the products including of the chemical nature of the PEGs, disposition and toxicology studies performed as well as any special investigations conducted including PEG alone, (planned) clinical use including


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

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dosing regimen and molar PEG intake over a specified time period, immunogenicity monitoring and finally, contacts and questions obtained from Health Authorities during product development. Although information on product details was limited as well as information from regulatory discussions, the information on toxicology programs was extensive. The products were heterogeneous ranging from replacement molecules to targeting products with an overall molecular weight ranging from 25 to 230 kDa (including PEG). The molecular weight of the PEG moiety conjugated to the proteins ranged from 20 to 60 kDa. Compounds are almost developed for chronic indications, the PEG load (e.g., with single/weekly dose or life-long treatment) was only given for a few products. The complete results of this survey are expected to be published shortly.


7. Unexpected toxicity – beyond exaggerated pharmacology


In a session chaired by Frank Brennan (UCB) and Lolke de Haan (MedImmune) a number of case studies were presented on unexpected toxicity of biologics. Simon Henderson (MedImmune) opened the session with 2 case studies. In the first case study, the toxicity profile of a monoclonal antibody (mAb) directed against Plasminogen Activator Inhibitor 1 (PAI-1) was described. Non-GLP 1 month repeat dose toxicity studies were conducted in rat and cynomolgus monkey. These studies showed dose dependent suppression of PAI-1, in the absence of significant toxicity. Subsequently, to support clinical development, 3 month repeat dose toxicity studies in rat and cynomolgus monkey were conducted. In the rat, treatment with the mAb was associated with an increase in the incidence and severity of cardiomyopathy at all dose levels. This was associated with hemorrhage, inflammation, accumulation of pigmented macrophages and fibrosis, of which cardiac fibrosis was not reversible and a No Observed Adverse Effect Level (NOAEL) could not be determined. Furthermore, during the dosing phase of the study, Xu et al. (2010) published that PAI-1 knock out (KO) mice develop very similar cardiac pathology, providing additional support for the findings being pharmacologically mediated. In the cynomolgus monkey, treatment with the mAb was associated with dose-dependent superficial bruising which was apparent as early as week 2 of the study while in recovery animals, bruising completely resolved. Given that this observation is consistent with the fibrinolytic pharmacology of the mAb, and the phenotype of PAI-1 deficient human individuals, these findings were not considered adverse. However, in 2 low dose animals, cardiac lesions reminiscent of the lesions seen in the rat were apparent. These findings were not consistent with background cardiac lesions seen in the cynomolgus monkey (as reported by (Chamanza et al., 2010), and a NOAEL was not established. In the second case study, non-clinical safety studies in the rat and cynomolgus monkey with a genetically engineered metallopeptidase, neprilysin, fused to albumin to extend plasma half-life (Henderson et al., 2013) were described. Given the concern over degradation of ‘off target’ peptides, potential adverse effects on the cardiovascular, endocrine and gastrointestinal systems were assessed. In both studies, the fusion protein was well tolerated, and not associated with any adverse findings. However, in the rat as well as the cynomolgus monkey, treatment was associated with marked prolongation of activated partial thromboplastin time (APTT). Subsequent in vitro analysis showed that this effect was mediated by fibrin degradation. Thus, while degradation of b-amyloid – the intended peptide target – and other known peptide substrates for neprilysin was not associated with adverse findings, these studies revealed a previously unknown substrate for the peptidase.

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The second presentation was by Mary McFarlane (MedImmune) and described the unexpected toxicity observed with a high affinity mAb directed against IgE. Repeat-dose toxicity studies in cynomolgus monkeys were conducted to support clinical development. No adverse findings were observed in a 1 month repeat dose toxicity study at dose levels up to 150 mg/kg/week. In contrast, in a subsequent 6 month study, hypertrophy of the pituitary gland was seen in all females at 150 mg/kg/week (high dose) and in 2/3 animals at 50 mg/kg/week (low dose). This finding was still present at the end of a 13-week recovery period in the high dose group only. These changes were not associated with any down-stream functional effects in endocrine organs. Subsequent investigative studies confirmed the absence of hyperplasia, found no evidence of mAb accumulation in pituitary glands, and demonstrated lack of binding of the mAb to circulating female pituitary hormones. In conclusion, therefore, no mechanistic basis could be identified for the observed pituitary hypertrophy in females. In the final presentation, Annick Cauvin (UCB) presented on an enhanced pre- and post-natal development (ePPND) study in cynomolgus monkeys conducted with an anti-cytokine mAb. This study was conducted against the backdrop of data showing that (i) KO mice for the same target were healthy and fertile and showed no reproductive abnormalities, (ii) an antibody targeting the same pathway was not associated with toxicity in an embryofetal development (EFD) study in cynomolgus monkeys, and (iii) antibodies targeting the same pathway were not associated with adverse findings in reproductive and developmental studies in mice. General toxicity studies conducted with the anti-cytokine mAb in the cynomolgus monkey were uneventful. In the ePPND study, mAb treatment was well tolerated during pregnancy. However, mortality was observed at delivery in some treated maternal animals, without premonitory signs. Furthermore, the duration of gestation was significantly increased. Findings suggested an increased incidence of difficult delivery (dystocia) associated with placental retention and, in some cases, significant blood loss. These were all considered target-related. Increased incidence of perinatal mortality was also observed in infants from treated mothers. Evaluation indicated a lack of care/feeding and/or head injury consecutive to dystocia and therefore indirect relation to treatment. Surviving infants developed normally with a functional immune system. None of the findings of this ePPND study were observed in mice, based on a different in physiology of parturition. In conclusion, species selection for reproductive and developmental toxicity studies requires consideration of target involvement and modulation as a function of the physiological state. The cynomolgus monkey, but not mice, predicted for a parturition risk for women. Overall, this session demonstrated that biologics can be associated with significant adverse effects. While effects were mostly pharmacologically mediated, they were not necessarily always predicted, and identified previously unknown consequences of the respective pharmacologies.


8. Non-traditional mAbs – PK and safety implications


Non-traditional (non-canonical) mAbs like ADCs, but also bispecifics, engineered antibodies and antibody fragments are increasingly developed especially in indications like cancer. The canonical bivalent, monospecific, full-length IgG represents only about half the anticancer mAbs in development (Reichert and Dhimolea, 2012). Mathias Locher (Covagen) presented the FynomAb technology. FynomAbs are bispecific or trispecific IgG-Fynomer-fusion proteins where a set of Fynomers adds a second or third binding modality to the antibody. Fynomers are small (7 kD) fully human proteins,


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

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derived from the SH3 domain of the human Fyn kinase. The SH3 domain is the subunit of the Fyn-kinase that initiates the protein–protein interaction of the kinase. This protein–protein interaction is meditated by 2 loops within the SH3 domain. The fusion site of the Fynomers to a IgG molecule can be controlled (N terminal, C terminal, heavy chain, light chain) allowing the creation of all possible bispecific architectures. Bispecific or trispecific FynomAbs usually exhibit a different mode of action compared to the mono-specific IgG or even a combination of respective antibodies. Pharmacokinetics of FynomAbs where shown to be IgG-like and the functionality of the Fc-part of the antibody is always retained. COVA322, an anti-TNF/anti-IL17A bispecific FynomAb. was well tolerated in a 4 week cynomolgus monkey toxicology study and the clinical trial application to start a first into patient study early in 2014 has been filed. Benno Rattel (Amgen) presented new insights into the therapy of ALL patients with blinatumomab, a bispecific T-cell engager, commonly referred to as a BiTEÒ antibody. The molecule is comprises two different flexibly-linked single-chain antibodies, one directed against CD19 and one targeting CD3. Blinatumomab transiently links CD19-positive tumor cells with resting polyclonal T-cells for induction of a surface target antigen-dependent redirected lysis of tumor cells, closely mimicking a natural cytotoxic T-cell response. The non-clinical pharmacological and pharmacokinetic characterization was shown to be predictive for patients, and treatment with blinatumomab as a monotherapy has shown an 80% complete molecular response rate and prolonged leukemiafree survival in patients with minimal residual B-lineage acute lymphoblastic leukemia (MRD+B-ALL) Topp et al., 2011. After start of infusion, B-cell counts dropped to

New challenges and opportunities in nonclinical safety testing of biologics.

New challenges and opportunities in nonclinical safety testing of biologics were discussed at the 3rd European BioSafe Annual General Membership meeti...
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