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Non-clinical Safety Evaluation of Biotherapeutics – Challenges, Opportunities and new Insights
Accepted Manuscript Non-clinical safety evaluation of biotherapeutics – Challenges, opportunities and new insights Guenter Blaich, Andreas Baumann, Sven Kronenberg, Lolke de Haan, Peter Ulrich, Wolfgang F. Richter, Jay Tibbitts, Simon Chivers, Edit Tarcsa, Robert Caldwell, Flavio Crameri PII:
S0273-2300(16)30242-2
DOI:
10.1016/j.yrtph.2016.08.012
Reference:
YRTPH 3654
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 20 August 2016 Accepted Date: 25 August 2016
Please cite this article as: Blaich, G., Baumann, A., Kronenberg, S., de Haan, L., Ulrich, P., Richter, W.F., Tibbitts, J., Chivers, S., Tarcsa, E., Caldwell, R., Crameri, F., Non-clinical safety evaluation of biotherapeutics – Challenges, opportunities and new insights, Regulatory Toxicology and Pharmacology (2016), doi: 10.1016/j.yrtph.2016.08.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Workshop report
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Non-Clinical Safety Evaluation of Biotherapeutics – Challenges, Opportunities and New
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Insights
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Guenter Blaich a, Andreas Baumann b, Sven Kronenberg c, Lolke de Haan d, Peter Ulrich e, Wolfgang
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F. Richter c, Jay Tibbitts f, Simon Chivers g, Edit Tarcsa h, Robert Caldwell i, Flavio Crameri c
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a
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c
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Sciences, Roche Innovation Center Basel, Switzerland; dMedImmune, Cambridge,
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UK; eNovartis Pharma, Basel, Switzerland; fUCB Celltech, Slough, UK; gADC
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Therapeutics, SA; hAbbVie BioResearch, Worcester, USA.; iAbbVie Inc., North
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Chicago, USA.
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AbbVie GmbH, Ludwigshafen, Germany; bBayer Pharma AG, Berlin, Germany;
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Roche Pharmaceutical Research and Early Development, Pharmaceutical
12 Abstract
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New challenges and opportunities in nonclinical safety testing of biotherapeutics were presented and
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discussed at the 5th European BioSafe Annual General Membership meeting in November 2015 in
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Ludwigshafen. This article summarizes the presentations and discussions from both the main and the
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breakout sessions.
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The following topics were covered in six main sessions:
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(i)
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(ii)
Unexpected side effects of biotherapeutics
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(iii)
Safety testing of cell and gene therapies including vector safety and integration site
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analysis and setting up a GLP facility in this field (iv)
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Challenges around use of PEGylated biologics, results of BioSafe survey
Immunogenicity and PKPD including immunogenicity prediction or methodologies to prevent induction of anti-drug antibodies
(v)
Current approaches applied to antibody drug conjugate (ADC) development Page 1 of 46
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(vi)
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Approaches for non-clinical safety testing of human-selective biologics, including use of transgenic animals and MABEL
The following questions were discussed across 4 breakout sessions (i-iv) and a case-study based
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general discussion (v):
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(i)
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Do bi-specifics offer nonclinical and clinical development advantages over combinations
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? (ii)
Is the paradigm “SC is more immunogenic than IV” correct ” ?
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(iii)
Why do we need a 6-month toxicology study for a monoclonal antibody (mAb) when we
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already have a 13-week toxicology study ? (iv)
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How do we investigate immune complex formation pre-clinically and does it translate
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clinically ? (v)
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Adversity in nonclinical safety studies: STP and ESTP opinion, your opinion needed ?
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Key words: Biotherapeutics, PEGylated biologics, gene and cell therapy, pharmacokinetics,
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nonclinical safety, antibody-drug conjugate, immunogenicity, cross-reactive species.
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Introduction
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BioSafe is the Preclinical Safety expert group of the Biotechnology Industry Organization (BIO), with
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a mission to identify and respond to key scientific and regulatory issues and challenges related to the
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preclinical safety evaluation of biopharmaceuticals. In addition to the Annual BioSafe General
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Membership Meeting in the US, the 5th Annual BioSafe European General Membership meeting was
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hosted by AbbVie on November 4-5, 2015 in Ludwigshafen, Germany. Participants included 125
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scientists from the biopharmaceutical industry, small biotechnology companies and contract research
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organisations primarily from Europe and the UK, but also from the US, and representing expertise in
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pharmacology, toxicology, pathology, pharmacokinetics and bioanalytics. The focus of the meeting
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was to share experiences and insights into nonclinical safety assessment of biologics including
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ACCEPTED MANUSCRIPT monoclonal antibodies, recombinant proteins, and gene and cell therapies. The meeting covered
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various nonclinical safety topics including PEGylated biologics, unexpected side effects, gene and
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cell therapy, immunogenicity and PKPD, antibody drug conjugates (ADCs), and nonclinical safety
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strategies for human-selective biologics. In each session, case examples were presented followed by
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podium discussions. For the first time, a breakout session was introduced for discussion of four “hot
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topics” in smaller groups, with the feedback being presented to all attendees afterwards by the
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breakout session leads. Finally, a group discussion based on several case studies was stimulated
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around a fifth “hot topic”, that of adversity in nonclinical safety studies and determination of the
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NOAEL.
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Session 1: Challenges in developing PEGylated Biologics
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Andreas Baumann (Bayer) and Jenny Sims (Integrated Biologix) co-chaired the first main session
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which focused on the nonclinical development of PEGylated biologics. Conjugation of Polyethylene
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glycol (PEG) moieties to various types of biologics has become a standard method to improve their
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pharmacokinetic properties, specifically prolonging plasma half-life (Jevsevar et al, 2010; Nakaoka et
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al, 1997; Yang et al, 2004). PEG molecules themselves have been shown to be non toxic, non
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immunogenic, hydrophilic, uncharged and non degradable polymers (Webster et al, 2009). For many
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protein-conjugates, the primary mechanism to increase plasma half-life is through the reduction in the
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rate of clearance by the kidneys (Mehvar, 2000; Yamaoka et al, 1995; Baumann et al, 2014; Webster
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et al, 2009). Other mechanisms include shielding epitopes/surfaces of the target drug resulting in
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lower visibility to the immune system, and protecting the protein from proteolysis.
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comprehensive review of current experiences in non-clinical safety and pharmacokinetics of
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PEGylated biotherapeutics, see references Ivens et al, 2015 and Baumann et al, 2014. Twelve
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PEGylated biologics have received regulatory approval so far based on balanced risk/benefit
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evaluations. Cellular vacuolation has been seen in toxicology studies in about half of these products,
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however without any functional changes for organs and tissues (Rudmann et al, 2013; Ivens et al,
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2015, Turecek et al, 2016).
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For a
ACCEPTED MANUSCRIPT Jennifer Sims (Integrated Biologix) presented results on a pharmaceutical industry survey on
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nonclinical safety testing of PEGylated proteins, which was conducted by BioSafe in 2013 and
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published recently in more detail (Ivens et al, 2015). Ten companies provided information on 17
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PEGylated biopharmaceuticals including receptor or protein targeting products with molecular
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weights ranging from 25 kDa to 350 kDa and currently in various stages of nonclinical and clinical
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development. The molecular weight of the linear or branched PEG moieties ranged from 20 kDa to
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60 kDa. PEG-related tissue vacuolation was seen in toxicology studies for 10 of the 17 compounds in
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the survey. No other-PEG related effects were reported. For 5 of the 17 products there was some
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evidence for an impact on disposition related to receptor-mediated uptake of the PEGylated protein.
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For the remaining 12 products it is not known whether there was receptor-mediated uptake, or
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whether it was not evaluated. For 7 of 17 products no specific studies were conducted to assess the
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disposition of the PEG moiety, while information on pharmacokinetics and/or disposition was
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gathered from 10 products. For 11 products there no specific assays for measurement of anti-PEG
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antibodies were developed or employed in nonclinical studies.
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antibodies were detected in a single dose clinical study and others stated that clinical assays were
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available or planned.
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developed for 6 compounds, but either no positive responses were observed or there was little impact
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on the interpretation of the study. One company stated that the development of IgM and IgG anti-
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PEG antibodies was observed in mice and macaques, but this was not considered to have any impact
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on safety considerations in humans.
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Toxicity studies included in the survey were repeated IV or SC dose studies in mice, rats, dogs, and
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cynomolgus monkeys and ranged from 2 to 52-weeks duration. The dosing frequency varied from
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daily to weekly. For one product both the IV and intravitreal route of administration was used in
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toxicology studies. No cellular vacuolation was observed for 7 of the 17 compounds. For these
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compounds, the dose levels used were relatively low and duration of dosing was generally short (up to
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4-5 weeks). For 2 compounds studies of 26-39 weeks in duration were conducted. None of the 4
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products with a single 20 kDa conjugated PEG showed evidence of vacuolation. In general, the
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ACCEPTED MANUSCRIPT maximum study duration for these 4 products was only 5 weeks, although for one product there was a
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26-week study in cynomolgus monkeys. However, cellular vacuolation was observed in some of the
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4-week studies with 30 to 40 kDa PEG products.
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One of the survey questions related to whether or not any special methodology was used to evaluate
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potential adverse effects associated with PEG accumulation and/or vacuolation (e.g. IHC, electron
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microscopy, functional evaluations such as nerve conduction velocity measurements, or in vitro
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studies). No special investigations other than IHC were conducted for 16 of 17 compounds.
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In the next presentation, Hanne Kjær Offenberg and Inga Bjørnsdottir (Novo Nordisk) presented
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on the nonclinical safety and DMPK of N9-GP and N8-GP, 40 kDa pegylated human recombinant
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coagulation factors IX- and VIII, respectively. Nonclinical safety testing of new therapeutic drugs
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typically includes chronic toxicity testing of up to 26 or 52 weeks duration to support chronic dosing
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in the clinic. However, human coagulations factors are highly immunogenic in animals resulting in an
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anti-drug antibody response which reduces exposure considerably. Therefore, an alternative animal
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model involving immuno-deficient athymic rats (Rowett Nude), which are devoid of T-cells and do
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not mount T cell-dependent antibody responses, was used in 26 week studies for both compounds. In
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general both N8-GP and N9-GP were very well tolerated in these studies. The choroid plexus
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epithelial cells (CPE) have been described to develop vacuoles after dosing with PEGylated biologics
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(Ivens et al. 2015). Therefore, the choroid plexus was evaluated for presence for PEG. Using IHC
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staining, no PEG was detected in the CPE cells for N8-GP, whereas for N9-GP, PEG was detected in
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the CPE cells at all dose levels. PEG did not induce vacuole formation in any tissue in any of the
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studies. In the absence of any degenerative or inflammatory findings, presence of PEG in the CPE
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cells is not considered adverse. The No-Observed-Adverse-Effect Level for N9-GP or N8-GP in
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these studies was considered to be 1200 IU/kg every fifth day or 1200 IU/kg every fourth day,
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respectively. The immune-deficient rat model proved suitable for a chronic study of 26 weeks
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duration and could be considered in cases where a model is needed for the chronic testing of human
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proteins which are highly immunogenic in animals (Offenberg et al, 2015).
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The biologic fate of the [3H]PEG-moiety incorporated into N8-GP and N9-GP was evaluated in a
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single dose IV study in rats.
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investigated to assess if pharmacokinetics was dose-dependent (dose range: 0.6 mg/kg and up to 200
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mg/kg). For all compounds, plasma pharmacokinetics, distribution and excretion pathways were
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investigated, based on total radioactivity measurements. The plasma concentration of the intact N8-
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GP or N9-GP conjugate was also measured using an antibody-based analysis method. After single IV
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administration to rats, [³H]N8-GP, [³H]N9-GP as well as [³H]PEG were shown to be widely
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distributed, mainly in highly vascularized tissues, with the lowest levels of radioactivity found in the
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CNS. The protein part of N9-GP/N8-GP was degraded over time, with the PEG moiety circulating in
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plasma. The PEG was subsequently excreted as 40 kDa PEG and smaller sized PEGs. Biphasic
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elimination of the PEG moiety was observed from plasma with more than 50% of PEG excreted into
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urine and faeces within the 1st phase (t½, 1st phase ~2-3 days in rats, t½, 2nd phase ~ 13-18 days in
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rats). The [³H]PEG and metabolites were eliminated mainly via the kidney into urine but also via the
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liver into feces, with a larger fraction found in the feces for [³H]N8-GP and [³H]N9-GP compared to
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[³H]PEG. Elimination of the 40 kDa PEG-moiety was shown to be dose-dependent with faster
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elimination at lower dose levels.
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Finally, Andreas Baumann (Bayer) presented on the nonclinical safety and PK of a site-specific
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PEGylated-Factor VIII conjugate (BAY 94-9027). BAY 94-9027, a B-domain–deleted recombinant
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FVIII with one 60 kDa polyethylene glycol (PEG; 2 × 30 kDa branched) attached via a linker to
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cysteine 1804, is in clinical development for acute and prophylactic treatment of patients with
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hemophilia A. In vivo toxicology studies with BAY 94-9027 or PEG-60 alone involving single and
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repeated administration were conducted. Specifically, in a repeat-dose toxicity study, male Sprague
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Dawley rats were given PEG-60 IV at dose levels of up to 11.0 mg/kg every other day for 4 weeks
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which was in the range of the total expected cumulative life-time dose of PEG-60 in humans given
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BAY 94-9027. No adverse effects or histopathological changes were observed in these studies (Ivens
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et al, 2013). Whilst there is no clinical evidence to suggest that administration of PEGylated proteins
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is associated with long-term safety issues, questions have been raised regarding the pharmacokinetics
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(PK) including metabolism and distribution, in particular for PEGylated proteins used chronically
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and/or in the pediatric population.
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distribution of residual radioactivity (full length PEG-60 and radioactive metabolites) on Days 7–168
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after single IV administration of [14C] BAY 1025662, the cysteine-linker-PEG 60 kDa component of
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the molecule containing a covalently linked [14C] label in the linker, in male Wistar rats.
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Concentrations and amounts of radioactivity in organs and tissues were measured using whole body
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autoradiography 6 months after administration. Between Days 7–168 post-injection, radioactive
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residues in the body decreased steadily from 22.5% of the dose to 1.8%. A heterogeneous distribution
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of radioactive residues was observed, with apparent preferential distribution of radioactivity to
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endocrine and exocrine glands and lymphatic organs. No residual radioactivity was detected in brain
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and adipose tissue. Elimination was fairly complete by the end of the study, with a total recovery of
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radioactivity of 94% at Day 168. Terminal elimination was slow, but there was no indication of PEG
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retention in rats.
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Session 2: Unexpected side effects with biotherapeutics
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In the second main session, chaired by Sven Kronenberg (Roche) and Lolke de Haan
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(MedImmune), case examples describing the utility of nonclinical safety studies to detect unexpected
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side effects of biologics were presented.
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In the first presentation, Lolke de Haan, Simon Henderson and Mary McFarlane (All
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MedImmune) presented a retrospective portfolio-wide analysis of the utility of early, non-GLP, dose-
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range finding toxicity studies in the development of biologics. The analysis encompassed 53 drug
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candidates generated in support of the MedImmune drug portfolio between 2008-2014, with
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modalities including monoclonal antibodies, bispecifics, fusion proteins, antibody-drug conjugates,
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therapeutic proteins and peptides. Overall, the analysis showed that with 7/37 drug candidates that
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were tested in non-GLP toxicity studies, toxicity was observed, which for 4/37 lead to termination of
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further drug candidate development. Other factors influenced drug candidate attrition to a greater
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extent, with undesirable PK/PD properties, lack of disease rationale and/or portfolio fit being the
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The aim of the presented PK study was to investigate the
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ACCEPTED MANUSCRIPT reasons for attrition for 10/37 candidates. Of the 27 drug candidates that were taken forward in to
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Good Laboratory Practice (GLP) toxicity studies, with 5 candidates new toxicities were identified,
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with 4/5 of these toxicities becoming apparent after subchronic dosing, and development of 2/5
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candidates being halted because of these toxicities. Importantly, when toxicity was observed, it was
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consistent with the mode of action or pharmacology associated with the drug candidate. Furthermore,
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it appeared that the impact of conducting non-GLP dose-range finding toxicity studies on candidate
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drug progression was limited, with the majority of the observed toxicities in non-GLP studies either
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being acceptable based on safety margins, the intended clinical indication or the anticipated duration
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or frequency of clinical dosing. In addition, given that additional toxicities observed in GLP studies
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typically only became apparent in studies involving subchronic dosing, non-GLP dose range finding
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studies would be expected to have limited impact on progression the progression of these candidates
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to GLP toxicity studies. Based on this analysis, MedImmune has currently ceased to conduct non-
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GLP dose-range finding toxicity studies as a default, and conducts such studies only on a case-by-case
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basis, when there is a clear cause for concern based on modality (e.g. antibody-drug conjugates, which
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are inherently toxic) or based on an extensive review of the target biology and the likelihood of target
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modulation resulting in exaggerated pharmacology and toxicity in a naive animal model.
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In the second presentation, Sven Kronenberg (Roche) provided case examples of unexpected side
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effects with biotherapeutics that eventually contributed to the program termination. The first case
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example summarized findings from a single dose PK study in cynomolgus monkeys with a chimeric
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IgG4 mAb with abolished effector function targeting a receptor of the tetraspanin protein family. The
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antibody target is highly expressed by hepatocytes, endothelial and epithelial cells in various tissues
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and on hematopoietic cells. There were several key challenges with respect to the disposition and
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safety of the molecule: The ubiquitous receptor expression led to target-mediated drug disposition at
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lower doses. The huge antigen sink resulted in an unexpected and more than dose-proportional
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increase in exposure, i.e. a 3-fold increase of dose led to a 100-fold increase in drug concentration,
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making nonclinical safety testing and potential clinical studies challenging. Furthermore, safety-wise,
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two monkeys in the single-dose PK study became moribund within 2 hrs resulting in euthanasia of
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ACCEPTED MANUSCRIPT these animals; the monkeys showed symptoms of anaphylactoid shock and vascular leakage including
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edema and release of cytokines (IL-6, IL-2, MCP-1). There were no compelling in vitro alerts (on
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mast cell degranulation, vascular permeability, complement activation) except for cytokine release
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from whole blood. Cytokine release probably contributed to the fatalities, however, based on cell-
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based assays, no agonistic activity of the mAb could be demonstrated. Rather, the risk for cytokine
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release could be mitigated by modifications outside of the antigen-binding site of the mAb. In
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conclusion, while the exact mechanism of the fatalities could not be elucidated, modification of the Fc
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part reduced the risk of cytokine release but did not solve the steep “all or nothing” PK. In a 2nd case
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example, results from nonclinical safety testing with one of several pegylated peptides tested at Roche
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were presented. The peptide consisted of a 30 kDa polyethylene glycol (PEG) moiety and was dosed
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IV or SC to rats and cynomolgus monkeys. Accumulation of the PEG moiety in neurons of the
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hypothalamus and spinal cord was observed in 4-week rat and monkey toxicity studies. While these
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findings were also observed in the rat at the end of a 12-week recovery phase, no clinical signs were
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associated with the vacuolation. Neuronal uptake of PEG into neurons may have occurred via a
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specific transporter in the brain. Since neurons have only slow or no renewal, a 1-year toxicity study
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with weekly IV dosing was conducted in the monkey to investigate the potential for these effects to
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result in impairment of neuronal function. At the end of this study, PEG was present in all dose
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groups in all animals, including those of the 12-week recovery groups.
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vacuolation was evident in several organs and tissues, including the neurons of CNS and spinal cord.
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A reversible deficit in sural sensory and peroneal motor conduction velocities in animals at the high
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dose was noted in week 24 only, but not at the other time points. This was inconsistent with a
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progressive, degenerative peripheral neuropathy. Whether this finding at the high-dose at a single time
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point was spurious, related to accumulation of PEG, or a potential consequence of target engagement
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in the nervous system remains unclear. There were no electrophysiological effects seen in the CNS,
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nor were any neurobehavioral changes observed.
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Finally, Andreas Popp (AbbVie) reported on side effects of a mAb leading to the identification of a
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new clinical indication. Humanized mAbs ABT-207 and h5F9-AM8 directed against the Repulsive
Presence of PEG and
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ACCEPTED MANUSCRIPT Guidance Molecule a (RGMa) and with cross reactivity to hemojuvelin (RGMc) were described. In
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healthy animals (mice, rats, cynomolgus monkeys) both mAbs increased serum iron and decreased
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serum iron binding capacity. Histopathologically, iron was released from spleen macrophages
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(reduced iron in the spleen) and accumulates in periportal hepatocytes. The observed effects were
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dose-dependent and correlated negatively with hepcidin expression. Administration of a 20 mg/kg
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single dose of the more potent h5F9-AM8 antibody to female rats resulted in significantly reduced
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serum hepcidin levels compared to control, which lasted 6 weeks. In toxicology studies performed in
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rats and cynomolgus monkeys for up to 13 weeks with ABT-207, no adverse effects were observed, in
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particular no degeneration or inflammation due to the iron accumulation in the liver. The hepcidin-
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related effects on iron homeostasis showed complete or partial recovery after a 12 week treatment free
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period.
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In addition, ABT-207 and h5F9-AM8 were tested in animal models of chronically and genetically
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induced anemia and chronic inflammation. ABT-207 and h5F9-AM8 corrected anemia and shifted
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macrophage population in this inflammation model significantly from M1 to M2 by lowering hepcidin
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expression in the liver and serum hepcidin levels. In conclusion, targeting RGMc as a new concept to
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reduce hepcidin expression could be of high value for the causal treatment of conditions of chronic
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anemia.
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Session 3: Gene and Cell therapy
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The third main session of the meeting was chaired by Peter Ulrich (Novartis). First a short update
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on hot topics in the field of Advanced Therapy Medicinal Products was provided. This introduction
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was followed by two presentations on vector safety and integration site analysis by Manfred Schmidt
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and about the set-up of a GLP facility in the Gene Therapy field by Patrizia Cristofori.
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Ulrich summarized regulatory information from the meeting of the European Society for Gene and
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Cell Therapy (ESGCT) in Helsinki, September 2015. Paula Salmikangas, chair of the EMA
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Committee for Advanced Therapies (CAT), presented the CAT view on several ATMP topics in a
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regulatory session. She especially focused on the ongoing public consultation of Regulation (EC) No
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ACCEPTED MANUSCRIPT 1394/2007 “Good Manufacturing Practice for Advanced Therapy Medicinal Products”. A summary
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was published in Dec 2015 and can be downloaded from the webpage of the European Commission
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(European Commission>DG Health and Food Safety>Public health>Medicinal products for human
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use>Advanced Therapies>Developments). Another topic was the new clinical trial regulation (EC)
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536/2014, which asks for GLP compliance of pivotal non-clinical safety studies supporting first-in-
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man (FiM) clinical trials. The regulation would also include ATMP non-clinical studies, which raised
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many questions about the availability and the validation status of animal models in the heterogeneous
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ATMP area. Salmikangas pointed out that for ATMP a GLP-compliant pivotal non-clinical safety
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study may not be mandatory for FiM trials, however for Marketing Authorization Application (MAA)
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such a study may be required. For ATMPs without the possibility to utilize validated animal models in
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a GLP environment, the CAT would recommend to follow the instructions provided by the risk-based
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approach guideline (EMA/CAT/CPWP/686637/2011). In December 2015 the CAT position on the
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Application of GLP principles to ATMPs was adopted via a written procedure, which was forwarded
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to the national health authorities (see CAT meeting minutes 12/2015 on www.ema.europe.eu). With
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respect to hospital exemption (article 28 in EC 1394/2007) Salmikangas pointed out that the CAT
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favors revision of the respective regulation by the European Commission in order to provide more
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security for ATMP developing firms.
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The recent developments in integration site analysis of gene therapy vectors and genome editing
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approaches were also discussed (see also presentation by Manfred Schmidt below). The identification
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of off-target integration sites by genome editing methods like CRISPR or TALENs was subject of
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publications in 2015 as well as discussion in the ESGCT meeting (Helsinki 2015). All in all, the
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conclusion from these recent investigations and method validations suggest that each engineered
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nuclease has to be individually evaluated for their potential off-target activity and the conditions
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influencing this potential (Gabriel et al., 2015). Nevertheless, the same authors see a major
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methodological advancement of sensitive, genome-wide detection of nuclease activity.
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Another
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immunocompromised mouse model, where NSG mice were genetically engineered with human
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highlight from the ESGCT
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of a
new
ACCEPTED MANUSCRIPT transgenes coding for IL-3, GM-CSF and SCF. The resulting mouse strain, also called NSG-SGM3
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(JAX Datasheet – 013062), shows less xeno-induced graft-versus-host-disease (GvHD), when
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reconstituted with human haemopoietic stem cells (HSC). In addition, these mice, when reconstituted
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with HSC, develop human monocytes. Norelli et al. (2015) reported that mice reconstituted with
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human HSC and with CD44v6+ Acute Lymphoblastic Leukemia (ALL) semi-cell line, developed
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features consistent with a cytokine release syndrome after infusion with human autologous T cells
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genetically modified with a CD28-endocostimulated CD44v6 CAR. Leukemia clearance associated
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with transient malaise, high fevers and weight loss, appeared with CAR deletion of monocytes.
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Increased serum levels of IL-6 and IFNγ in relation to clinical observations were measured with
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monocytes as the main source of IL-6. The presented data suggest that this model may be suitable for
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assessing CRS potential of CARTs in a non-clinical setting.
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Viral vectors (Manfred Schmidt, NCT) have shown their efficacy in clinical studies of rare diseases.
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However, vector-mediated insertional side effects and malignant transformation have occurred in
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individual patients. These observations have prompted new vector designs, such as self-inactivating
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(SIN) LTR configurations and tissue-specific promoters that may improve safe and efficient viral
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vector-mediated gene-correction in patients. Now, with the advent of mammalian whole genome
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sequence availability and next generation sequencing (NGS), large-scale identification of vector
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integration profiles and vector persistence studies over time have become an essential part of
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pharmacokinetic studies for a gene therapeutics.
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mediated PCR) coupled to NGS technology for vector integration site analysis is subject to
310
continuous further development. The current state-of-the-art and future perspectives were presented,
311
which includes new technologies like capture-based quantitative sequencing of viral vector sequences
312
and integration sites as well as associated bioinformatical data mining tool suites.
313
Patrizia Cristofori (GSK) presented the GLP Principles in Gene Therapy and the Experience of
314
Academic facilities GLP certified (HSR-TIGET). Gene therapy (GT) is now emerging as a medical
315
reality with clinical efficacy demonstrated in a number of gene therapy trials and an increasing
316
number of products entering phase II and III trials each year. For the promise and potential of a gene
The current LAM-PCR (linear amplification-
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ACCEPTED MANUSCRIPT therapy medicinal product (GTMP) to be fully realized it is important to address regulatory
318
expectations. Guidelines for GTMP progression in clinical trials and marketing authorizations are
319
available to facilitate a harmonized approach in the EU and US. Non-clinical studies have the
320
primary objective of providing sufficient information for a proper risk assessment for the product’s
321
use in human subjects. The paradigm described in ICH M3 for safety evaluation of conventional
322
pharmaceuticals is not always appropriate or relevant to GTMPs. Non-clinical studies should be
323
designed on a case-by-case basis, understanding the relevant aspects of the science underpinning that
324
product and need for specific expertise beyond the traditional pharmaceutical field. The combination
325
of expertise of personnel trained in genetic therapy research, experimental pathology, safety
326
assessment and quality assurance has enabled setting up a GLP Test Facility in an academic
327
environment. The main objective was to i) maximise the early collection of proof-of concept data and
328
provide adequate preclinical information to support the safety and the scientific basis for the
329
administration of an investigational product in the target patient population, ii) provide data of high
330
regulatory standard with assurance of scientific integrity, validity and reliability, and iii) minimise the
331
use of animals (3Rs). Challenges to be addressed in designing non-standard study to investigate in
332
vivo fate of genetically modified cells (biodistribution) and toxicological and tumorigenicity potential
333
were discussed. Discussion focused on the definition of test item and its characterisation, the choice of
334
test system (animal model), sample traceability, duration of treatment and endpoints to be measured.
335
The key role of Quality Assurance (QA) was highlighted. QA must be aware of all non-standard
336
aspects and challenges of GT preclinical studies and knowledgeable in technical procedures and
337
methods working closely with technical personnel to share criticality and best practices. In
338
conclusion, due to the complexity of GTMPs, non-clinical safety testing can vary considerably and is
339
recommended to be handled on a case-by-case basis, understanding the product and areas of non-
340
compliance.
341
Session 4: Immunogenicity and PKPD – from Prediction to Interpretation
342
Formation of anti-drug antibodies (ADA) is one of the hallmarks of an immune response against
343
biotherapeutics. ADA may affect safety, efficacy and pharmacokinetics of a biotherapeutic. Thus,
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ACCEPTED MANUSCRIPT ADA often change the pharmacokinetic – pharmacodynamics (PKPD) of biotherapeutics. Therefore,
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presence of ADA needs to be considered in the interpretation of PKPD results. The fourth main
346
session, chaired by Wolfgang Richter (Roche) and Jay Tibbitts (UCB), gave overviews on PKPD
347
aspects of immunogenicity in toxicology studies, the immunogenicity of bispecific antibodies in
348
monkeys, and the current knowledge on in vitro tools for immunogenicity prediction. In addition,
349
approaches were discussed how to prevent immunogenicity in animal studies.
350
Olivier Petricoul (Novartis) presented the results of a survey conducted by the BioSafe PK/PD
351
working group on the analysis and reporting of toxicokinetic (TK) parameters. Toxicokinetic analysis
352
is a requirement for toxicology studies and, occasionally, in repeat-dose studies the test article
353
exposure in individual animals will change over the course of the study with increasing variability in
354
exposure in the TK assessment at the end of the study compared to Day 1 of the study. This
355
phenomenon may be due to the impact of ADA. The toxicokinetic analyst is then left to determine
356
how the TK data be reported as the effects of ADA formation on toxicokinetic/pharmacodynamic
357
parameters should be considered when interpreting the data (ICH S6).
358
It was highlighted that one of the main objectives of TK analysis is to show the validity of the
359
toxicology study, i.e. to confirm drug exposure at the start and end of the dosing period. Estimates of
360
the maximum observed concentration (Cmax) and area under the concentration-time curve (AUC) are
361
the most commonly generated TK parameters and usually the exposure multiple relative to human
362
exposure is based on the relevant mean AUC and Cmax values obtained in toxicology studies. As
363
ADA may impact on exposure (often a reduction, but in certain cases a prolonged exposure can be
364
observed) and consequently the estimates of Cmax and AUC, careful consideration should be given to
365
how mean values of these parameters are derived. Reporting of mean (and other summary statistics)
366
TK parameters in the context of ADA can be done in several ways. The mean TK parameters could
367
be calculated based on all animals independent of their ADA status. Alternatively, the mean TK
368
parameters of only ADA negative animals could be provided (Thway et al, 2013). The former could
369
be considered a more conservative estimate of drug exposure for the purposes of exposure margin
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ACCEPTED MANUSCRIPT calculations. Another approach would be to use TK parameters derived from Day 1 dosing (or a
371
similar early dosing period), where usually there is no impact of ADA.
372
A total of nine companies provided answers to the survey questions on how they calculate and report
373
TK parameters. All companies calculate TK parameters for all animals, regardless of ADA status.
374
Seven companies derive summary statistics using all animals, but four report, in addition, means for
375
ADA negative animals while the other three derive means for ADA negative animals only when ADA
376
appear to have affected the exposure. One company provides only one mean of TK parameters,
377
excluding ADA positive animals from the summary statistics when clear effects on exposure are
378
observed. Finally one company derives summary statistics using all animals, independent of the ADA
379
status. One example of a monoclonal antibody targeting a cell surface receptor was given to highlight
380
the effect of ADA on the TK (reduced exposure) associated with loss of PD effect. It shows the
381
importance to integrate the ADA status into the study summary to provide the appropriate
382
interpretation of the TK and PD results.
383
As noted above, one of the key elements of TK analysis is to confirm drug exposure in the study; and
384
as pointed out during the discussion on TK calculations some investigators choose to exclude animals
385
from summary TK parameters based on the degree of impact ADA has on exposure. Thus, one of the
386
major difficulties becomes how to define the «impact on exposure» of ADA? Several options were
387
discussed on how exclusion of ADA positive animals from mean TK parameter could be done, such
388
as scientific judgement, using a defined threshold (e.g. based on AUC decrease or increase from day
389
1), to report the median instead of the mean or to exclude time-points from profiles after animals
390
become ADA positive. Clearly, no established or mutually agreed-upon strategy exists for calculating
391
and reporting TK parameters in the presence of ADA. Good scientific judgement, clear explanation
392
and rationalization of the strategy used, and thoughtful consideration of the impact of the chosen
393
strategy on the exposure multiples and starting dose in humans are critical to appropriate risk
394
assessments under these circumstances.
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Finally, there was a discussion regarding the exposure data necessary to support a toxicology study
396
versus those required to provide suitable PK data. The conclusion was that both PK (for dose
397
predictions and modeling) and TK (to establish an exposure-toxicity relationship or an exposure
398
multiple) can be simultaneously obtained from toxicology studies.
399
employing the strategies described above to derive mean TK parameters for exposure multiples and
400
using data from ADA negative/not impacted by ADA for PK evaluations.
401
Eric Stefanich (Genentech) presented preclinical data assessing the immunogenicity of bispecific
402
therapeutic antibodies in cynomolgus monkeys. Some of the potential differences between bispecific
403
and monospecific antibodies that may contribute to differential immunogenicity were discussed
404
including increased “foreignness” in bispecific antibodies due to two engineered complementarity
405
determining regions (CDRs); unique product-related variants; the formation of different sizes of
406
circulating immune complexes with ADA; and asymmetry in the fragment antigen-binding (Fab)
407
region (e.g., charge differences) that could impact tertiary orientation at the hinge region. The
408
emerging data suggest there may be increased immunogenicity (both incidence and titer) with some
409
bispecific MAbs compared to what is typically encountered for monospecific MAbs. However there
410
are limited case studies for bispecific antibodies to date. The observed magnitude of ADA can impact
411
the ability to conduct long-term preclinical safety studies in monkeys.
412
immunogenic epitopes from cynomolgus monkey studies are mostly associated with CDRs of the
413
bispecific antibodies. Based on limitations of existing immunogenicity risk-prediction tools, we may
414
need to rely on clinical trial results to understand clinical risk.
415
Sebastian Spindeldreher (Novartis) presented four case studies to exemplify how currently
416
available tools and assays can be used to assess the immunogenicity risk of biotherapeutics. Before
417
introducing in vitro tools for immunogenicity risk assessment methods Sebastian briefly introduced
418
ABIRISK (Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to
419
minimize the RISK), which is a project of the Innovative Medicines Initiative (IMI) of the European
420
Union.
421
research groups and clinical centers. It is aimed at improving the understanding of patient- and
Where assessed, the
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This can be achieved by
ABIRISK is a public-private partnership between pharmaceutical companies, academic
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ACCEPTED MANUSCRIPT compound-related factors influencing immunogenicity in order to identify clinical biomarkers for
423
immunogenicity and to validate available, and develop new, assay tools to assess the immunogenicity
424
risk for a given compound.
425
In vitro tools have been developed that may allow immunogenicity risk assessment. One of these
426
tools is MHC-associated peptide proteomics (MAPPS), a technology that identifies sequences of
427
peptides bound to HLA-DR, i.e. potential T cell epitopes. Other commonly used in vitro tools are T
428
cell activation assays, which are applied in many different variations. More recently, dendritic cell
429
activation assays have been developed to understand the potential effects on the innate immune
430
system.
431
The first case study showed that the number of potential T cell epitopes identified with MAPPs and
432
the response rate in T cell activation assays conducted with several marketed monoclonal antibodies
433
matched roughly the ranking based on clinical immunogenicity incidence (Karle et al, 2016).
434
However, one of the key issues for clinical validation of immunogenicity prediction is the lack of
435
reliable clinical immunogenicity data that can be compared across studies. Published data are based
436
on different immunogenicity assays with different sensitivities and drug tolerances, different sampling
437
time points, different diseases and disease states, different concomitant treatments and potentially
438
further differences. A solid comparison of in vitro and clinical data will only be possible with reliable
439
clinical immunogenicity data that can be compared across studies.
440
In vitro tools have been developed that may allow immunogenicity risk assessment. One of these
441
tools is MHC-associated peptide proteomics (MAPPS), a technology that identifies sequences of
442
peptides bound to HLA-DR, i.e. potential T cell epitopes. Other commonly used in vitro tools are T
443
cell activation assays, which are applied in many different variations. More recently, dendritic cell
444
activation assays have been developed to understand the potential effects on the innate immune
445
system.
446
The second case study focused on unpublished data from the ABIRISK consortium
447
[www.abirisk.eu]. The work conducted in the labs of Bernard Maillère (CEA) and Anette Karle
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ACCEPTED MANUSCRIPT (Novartis) showed good agreement between HLA-DR-presented peptides identified by MAPPs, T cell
449
epitopes identified from drug-naïve healthy blood donors and T cell epitopes of memory T cells
450
recovered from patients who were treated with and developed immunogenicity to the tested
451
biotherapeutics. Those data nicely confirm that the biology of patients who develop ADA against
452
biotherapeutics is mimicked in in vitro assays that are based on drug-naïve healthy subjects.
453
The third case study looked at the effects of protein aggregation on dendritic cell activation, antigen
454
presentation and T cell activation (Rombach-Riegraf et al, 2014). A correlation was found between
455
the amount of subvisible particles as well as the protein amount contained in these particles and the
456
amount of peptides presented in the context of HLA-DR. This data was confirmed with four different
457
marketed monoclonal antibodies that were studied in the context of ABIRISK.
458
monoclonal antibodies reacted very differently to physical stress, leading to different extent of
459
aggregation, which was again linked to increased antigen presentation as determined by MAPPs.
460
The last case study indicated that transgenic mice which express a mini-repertoire of human IgG1
461
antibodies can mount ADA responses to foreign antigens but are tolerant to native monomeric human
462
antibodies. Chemical modification by UV but not process-related or pH-induced oligomers could
463
induce ADA responses, which led to the hypothesis that neo-epitopes are required for the break of the
464
tolerance in these mice (Bessa et al, 2015).
465
During the discussion the audience largely agreed to the overall conclusion that it is currently not
466
possible to predict clinical immunogenicity incidence or outcome of immunogenicity. However,
467
promising mathematical models are being developed (Chen et al, 2014). Data from the “prediction
468
tools” should be considered as an assessment of the potential for immunogenicity but not as an
469
absolute prediction of immunogenicity incidence and outcome in human. However, the currently
470
available methods can be used for investigative and mechanistic studies and can help candidate
471
selection and internal decision making.
472
Wolfgang Richter (Roche) gave an overview on approaches to prevent immunogenicity in non-
473
clinical studies. The conduct of non-clinical studies with human or humanized biotherapeutics can be
These four
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ACCEPTED MANUSCRIPT 474
hampered by an immune response against the biotherapeutic. As previously noted, the formation of
475
ADA can substantially affect biotherapeutic exposure.
476
immune response and formation of ADA usually leads to an accelerated clearance and loss of
477
biotherapeutic exposure. However, for small biotherapeutics ADA may result in increased clearance
478
but, in some instances, may result instead in an apparent reduction in clearance (“sustaining ADA”)
479
due to slowing of the often fast endogenous renal elimination by virtue of binding of the small
480
biotherapeutic to a slowly cleared IgG ADA. Beyond the biotherapeutic exposure implications,
481
potential safety sequelae of immune responses include deposition of immune complexes in some
482
organs (eg. lungs, kidney). Re-dosing after formation of ADA may lead to anaphylaxis and acute
483
death (Hattori et al, 2007). Therefore, conduct of long term studies in mice may benefit from
484
suppression of an immune response. Ways to prevent immunogenicity include general suppression of
485
the immune system or antigen-specific tolerance induction. General suppression of the immune
486
response via pre-treatment with an anti-CD4 antibody was successfully used to conduct long-term
487
pharmacology studies in mice with the human monoclonal antibody gantenerumab (Bohrmann et al,
488
2012). The anti-CD4 treatment acted by transient depletion of peripheral CD4+ T-helper cells one
489
day before the start of gantenerumab administration. Alternative approaches include e.g. treatment
490
with the immunosuppressant mycophenolate mofetil.
491
antigen-specific tolerance induction keeps the immune system fully functional. Antigen-specific
492
immune tolerance may be induced by administering a high loading dose of a biotherapeutic followed
493
by administration of a lower dose during the test period. High dose tolerance induction was used to
494
enable a low dose group in a mice juvenile toxicity study with the antibody MR16-1 without ADA
495
formation or ADA-related side effects (Sakurai et al, 2013). Immune tolerance can also be induced
496
by exposing neonates to the tolerogen. In a study with adalimumab in mice, neonatal immune
497
tolerance was induced by dosing of mother mice after delivery (Piccand et al, 2016). Adalimumab
498
was transferred to the suckling pups via milk, thus providing antigen that induced tolerance. After
499
reaching adulthood at 8 weeks of age the offspring was dosed with adalimumab. Offspring exposed
500
to adalimumab the first days after birth showed no indication for an immune response, while control
By contrast to the previous approaches,
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When the biotherapeutic is an IgG, the
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mice showed an immune-mediated accelerated adalimumab clearance starting 7 days after dosing and
502
formation of ADA.
503
Overall, the session showed multiple aspects of immunogenicity and its consequences for the PKPD
504
and safety assessment of biotherapeutics.
505
biotherapeutic exposure is an important component in the TK and PK assessment. Novel formats may
506
differ in their immunogenicity from canonical monoclonal antibodies. In vitro tools for
507
immunogenicity are useful for investigative and mechanistic studies and can help candidate selection
508
and internal decision making. Various approaches for immune tolerance induction have been reported
509
to enable long term studies in animals without immune response. Immune tolerance induction has
510
been mainly applied in mouse studies, while experience in other species is limited or lacking.
511
Session 5: Antibody Drug Conjugates (ADCs)
512
The fifth main session was chaired by Simon Chivers (ADC Therapeutics SA) and Edit Tarcsa
513
(AbbVie), and covered the current approaches applied to antibody drug conjugate (ADC)
514
development. ADCs comprise of a monoclonal antibody and a cytotoxic payload attached via a
515
chemical linker, designed with the notion that the specificity of the antibody will lead to exclusive
516
delivery of the toxic payload to the cancer cells expressing the antigen on the cell surface, at the same
517
time eliminating systemic toxicities typically observed when administering the unconjugated payload.
518
Most ADCs currently in development have been generated by randomly attaching the payload to
519
either lysine residues or to reduced interchain cysteine in the antibody. These processes generate
520
heterogeneous mixtures with varying drug to antibody ratios (DAR), where the different forms may
521
contribute differentially to efficacy and/or toxicity. Homogenous DAR ADC’s are also possible
522
through the introduction of engineered cysteins, such as with the Thiomab technology. In addition,
523
some ADCs are not stable in vivo, some may be prone to toxin loss via proteolytic cleavage of the
524
linker or unwanted migration of the payload to plasma proteins. Evidence is emerging that in addition
525
to targeted delivery of the payload to tumor cells, ADCs are taken up via a variety of mechanisms
526
(pinocytosis, phagocytosis, FcR-mediated, or pattern recognition receptor-mediated uptake, etc), often
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Knowledge of ADA formation and its impact on
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the ‘normal’ clearance route for antibodies that might contribute to the non-target related toxicities
528
observed in the clinic. Thus understanding the composition and in vivo fate of ADCs can be critical to
529
optimize their properties.
530
development, it is of outmost importance to understand how to optimize the therapeutic margin of
531
these complex therapeutic modalities.
532
The first presentation by Stephanie Fischmann (AbbVie) talked about what should be measured for
533
ADCs – examples and experiences. Today, 3-4 bioanalytical assays are required to support the
534
development of ADCs. Typically, un-conjugated or “free or toxin”, “total antibody” and “conjugated
535
antibody” concentration are used for this purpose. The toxin load of the antibody can also be
536
represented by the measuring the “conjugated toxin” levels. The latter is very valuable if cleavable
537
linkers were chosen as this enables the use of generic methods. From a bioanalytical perspective
538
Ligand Binding Assays (LBA) were the “state-of-the-art” platform over the years for any “total
539
antibody” or “conjugated antibody” assays run in support of an ADC projects. Meanwhile, more and
540
more Mass spectrometry based methods have been developed, i.e. High Resolution Mass
541
spectrometry, where the drug-antibody ratio can be elucidated either by way of full-size ADC
542
construct molecular weight measurement or after reduction for light chain and heavy chain and
543
determining their molecular weight separately. Hybrid LC-MS/MS assays are also evolving e.g. for
544
determination of conjugated toxin levels. Here, affinity pull-down followed by enzymatic cleavage of
545
the linker facilitates LC-MS/MS quantification of the released toxin as a small molecule. Another
546
approach follows unique peptides identified to represent total antibody and conjugated antibody levels
547
after tryptic digestion. Mass spectroscopy based methods in general offer more detailed information
548
on the ADC backbone (i.e. number and position of toxins) and on the potential biotransformation of
549
the payload. However, MS-based hybrid assays are more tedious and can be limited in throughput or
550
sensitivity. Hence, from a bioanalytical and scientific perspective LBA can serve as the method
551
platform of choice throughout the whole life cycle of an ADC project. However, additional mass spec
552
technology based methods are beneficial to elucidate the in vivo fate of the ADC constructs with more
553
granularity (esp. early stage). Today, many different factors influence the choice of assays in support
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With 2 ADCs currently approved and about 40 more in clinical
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ACCEPTED MANUSCRIPT of an ADC, including the development stage of the project, reagents availability, assay sensitivity,
555
assay development time and cost (cost per validation and cost per sample). For such reasons generic
556
platforms are often favorable at early stage but replaced by more specific assays later. Last, but not
557
least attempts are being made to simplify the bioanalytical efforts (i.e. the number of PK assays
558
undertaken in support of ADC projects). For example, multiplex technologies on LBA platforms and
559
multi-analyte strategies on LC-MS/MS platform are being evaluated.
560
In order to elucidate the immunogenicity of complex constructs like ADCs, all potential immunogenic
561
epitopes must be evaluated. Currently, a multistep strategy is recommended where after positive
562
screening assays the samples with potential ADAs are also tested in individual spike in experiments.
563
This so called “domain characterization” assay strategy employs unconjugated antibody spike as well
564
as full ADC spikes or spike-in experiments where only specific moieties of the ADC are spiked in,
565
thus the potential immunogenic epitopes can be identified.
566
Next, Matthias Machacek (LYO-X) presented on the PK, PD and toxicology modeling of IgG1
567
based ADCs and critical success factors. For ADCs three different PK levels can be identified: whole
568
body distribution, tumor penetration and uptake into the cell. Each of these three levels is critical for
569
successful toxin delivery to the tumor with a wide therapeutic margin. On a whole body level, it is the
570
IgG1 typical distribution and elimination processes that are relevant. IgG1 molecules remain
571
circulating in blood and interstitium until their uptake by cells and subsequent metabolism or
572
recycling via the FcRn mechanism. Thus, eventually all of the attached toxin will be delivered to
573
tissues that are active in IgG1 catabolism after internalization. For ADCs the IgG1 specificity to a
574
tumor target, a tumor specific cell surface molecule, will divert some of the ADC from the normal
575
clearance pathway. ADC binding to the tumor target will depend on the affinity and tumor target
576
abundance. Next, the tumor target bound ADC is internalized delivering the toxin into the cell.
577
Internalization depends on tumor target expression and the internalization rate. A back of the envelope
578
calculation showed that for a cell expressing 20’000 tumor targets and with a receptor internalization
579
half-life of about 2 hours intracellular toxin concentrations in the order of 10 nM are rapidly achieved.
580
A live ADC PK model simulation was then shown to illustrate the discussed concepts. The simulation
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ACCEPTED MANUSCRIPT demonstrated the impact of dose on how much toxin is delivered to the tumor versus to other tissues.
582
For high doses the fraction delivered to the tumor versus tissues was small. This was explained by the
583
saturation of the tumor up-take capacity in which case a large fraction of the toxin is delivered to IgG1
584
catabolizing tissues. By lowering the dose, the fraction delivered to the tumor could successively be
585
increased up to 80% for that specific case. Thus, for an optimal clinical dosing strategy the tumor
586
uptake capacity should be considered and the dose and regimen accordingly adjusted. Understanding
587
mechanistically and quantitatively the tumor target properties, ADC distribution and clearance as well
588
as the information drawn from the clinical PK profiles all help in better assessing the tumor uptake
589
capacity and hence the optimal dose and regimen.
590
Next, Andreas Pahl (Heidelberg Pharma) gave a talk about the toxicology of amanitin-based
591
ADCs. Payloads of today’s ADCs are exclusively based on compounds acting on microtubules or
592
DNA, are suffering from various limitations. New generations of payloads are entering the field
593
including Heidelberg Pharma’s amanitin, a highly effective inhibitor of the eukaryotic RNA
594
Polymerase II. Due to its unique mode of action and its hydrophilic nature this toxin differs
595
significantly from well-known payloads. Amanitin is the toxin of the green death cap mushroom.
596
Poisoning by amanitin leads to liver failure and subsequent death depending on the amount of toxin
597
ingested. Due to its hydrophilic nature amanitin cannot penetrate cell membranes passively. The
598
reason for its liver toxicity is a specific transporter OATB1B3, which is exclusively expressed in liver
599
cells. In vitro studies demonstrated that amanitin conjugated antibodies are not substrates for this
600
transporter indicating that amanitin-ADCs are not toxic to the liver. This was further explored in a
601
toxicological study in cynomolgus monkeys designed as an explorative study. Escalating doses of
602
Trastuzumab-Amanitin-ADCs from 0.3 to 10 mg/kg were administered to groups of three female
603
cynomolgus monkeys. In line with the in vitro findings no significant elevations of liver enzymes
604
were observed up to doses of 10 mg/kg. No other signs of liver toxicity or histological changes of the
605
liver were observed. LDH was the most sensitive parameter from clinical chemistry. A transient
606
increase at day 7 was observed. This was accompanied by hematological changes, namely elevated
607
levels of neutrophils. Dose-limiting toxicities were skin and ocular toxicity. These DLTs can be
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ACCEPTED MANUSCRIPT explained by target biology. Her2, the target of trastuzumab, is expressed on various epithelial cells.
609
Differences to T-DM1 toxicological findings can be explained by amanitin’s ability to kill also non-
610
dividing cells, whereas T-DM1 is only killing dividing cells. Next, different linker and conjugation
611
strategies for amanitin were compared. The MTD and the therapeutic index were significantly higher
612
for amanitin-ADCs based on site-specific conjugation to trastuzumab-thiomabs as compared to
613
random lysine conjugated ADCs. The therapeutic index for the amanitin-trastuzumab-thiomabs was
614
around 20 based on murine effective doses and monkey tolerable doses bridged by the AUC of the
615
ADC. This favorable therapeutic index will enable this amanitin-linker technology to move forward to
616
development.
617
In the final presentation for this session, Steve Alley (Seattle Genetics) described biotransformations
618
of auristatin ADCs. Biotransformations can be the intended effect of ADCs, such as release of
619
conjugated drug in the lysosomes of antigen positive cells, as well as amino acid modifications, loss
620
of drug-linker, differential clearance, or drug metabolism in the liver. As an example of the intent of
621
targeted delivery, the release of MMAE from brentuximab vedotin was demonstrated in vitro (Okeley
622
et al, 2010) and in mouse xenograft models. ADC modifications that occur in plasma were
623
demonstrated using native mass spectrometry of affinity-purified ADCs, showing that loss of drug-
624
linker as well as intact clearance of highly loaded ADCs contribute to the change in drug-load
625
distribution over time in vivo. Analyzing the samples under denaturing conditions allowed additional
626
modifications such as maleimide hydrolysis in the linker and cysteinylation of antibody cysteines to
627
be observed (Hengel et al, 2014). New drug-linker designs can lead to ADCs where loss of drug-
628
linker and differential clearance in plasma can be substantially reduced (Lyon et al, 2014). Finally,
629
MMAE released from brentuximab vedotin is excreted largely unchanged in feces, although several
630
metabolites due to demethylation, dehydrogenation, amide hydrolysis, and oxidation were observed
631
(Han et al, 2013).
632
Session 6: Lack of cross-reactive species – path forward to support FIH and clinical
633
development
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ACCEPTED MANUSCRIPT 634
The sixth and final main session was chaired by Rob Caldwell (AbbVie) and Flavio Crameri
635
(Roche).
636
justification as indicated by ICH S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived
637
Pharmaceuticals. The guidance indicates that species selection should be based upon a weight-of-
638
evidence utilizing in vitro and/or in vivo biologic activity of biologic agent in toxicology species, and
639
comparable binding of biologic to human and preclinical species tissues based upon
640
immunohistochemical staining of tissue panels (e.g. tissue cross-reactivity experiments). The totality
641
of these datasets can guide selection of a rodent and non-rodent species. Given the unique selectivity
642
of biologic agents, the guidance acknowledges that this weight of evident approach may also result in
643
lack of relevancy for routine toxicology species. Toxicology studies in non-relevant species was
644
considered misleading and was discouraged. Alternative non-traditional toxicology test strategies
645
could employ use of homologous species-specific proteins as biologic surrogate, species-specific
646
surrogate monoclonal antibody, use of human clinical candidate in transgenic species expressing the
647
human target, or evaluation of preclinical species exhibiting the intended pharmacology (i.e. knockout
648
mice for mAb sequestering soluble target). All of these strategies would again be justified using
649
weight-of-evidence approach following generation of relevant in vitro and vivo datasets. Scientific
650
advances provide additional tools to justify relevant as well as irrelevant species for safety testing
651
including greater understanding of active (i.e. neonatal Fc receptors) placental transfer of novel
652
biologic therapeutic, enhanced understanding of embryology across human and preclinical species,
653
and more sensitive analytical techniques to justify relevant pharmacology and tissue distribution
654
across human and preclinical species. Each of these alternative models have scientific advantages
655
and/or disadvantages including pharmacologic relevance to human biologic, reference range datasets
656
to evaluate the toxicologic significance of observed effects, and potential for neutralizing anti-drug
657
antibodies which may preclude use of the preferred model/species. Finally, alternative strategies also
658
have resource needs which impact the timing and efficiency of the selected toxicology model
659
including cost of unique animal colony maintenance and generation of reference range datasets, and
660
generation of regulatory compliance for surrogate proteins. The session provided cases studies of
661
species justification for novel biologic therapeutics.
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Rob Caldwell introduced the session by providing an overview of species selection
Page 25 of 46
ACCEPTED MANUSCRIPT Lolke de Haan (MedImmune) presented 2 case studies involving nonclinical safety testing of
663
human-selective monoclonal antibodies (mAbs) in transgenic mouse models. The first case study
664
described the challenges encountered in developing a transgenic mouse model for safety testing of
665
MEDI2843, a fully human monoclonal antibody directed against Toll-like receptor 4 (TLR4), the
666
innate immune receptor for bacterial lipopolysaccharide (LPS). MEDI2843 was isolated following
667
immunisation of a transgenic mouse strain expressing human Ig domains with human TLR4/MD-2
668
protein and/or TLR4/MD-2-expressing Human Endothelial Kidney (HEK) cells. None of the lead
669
panel of antibodies bound to rodent TLR4, and despite the fact that a number of lead antibodies bound
670
to cynomolgus monkey TLR4, none of these antibodies showed functional activity in cell-based
671
assays. By contrast, MEDI2843 was highly human selective and bound and inhibited functional
672
activity of human TLR4 in cell based assays very effectively.
673
pharmacologically relevant species for safety studies, attempts were made to generate human TLR4
674
transgenic mice. The approach followed involved deletion of the mouse TLR4 gene and insertion of a
675
mouse/human chimeric version of the TLR4 gene, comprised of gene fragments encoding the mouse
676
transmembrane and cytoplasmic TLR4 domains and the human TLR4 external domain. When this
677
gene construct was expressed recombinantly in a cell line, the resultant protein was shown to be fully
678
functional and mediate LPS signalling, with binding of MEDI2843 ablating LPS signalling. Given the
679
importance of MD-2 in LPS signalling, a second transgenic mouse was generated in which the mouse
680
MD-2 gene was replaced by the human MD-2 gene. A combined chimeric mouse/human TLR4 and
681
human MD-2 transgenic mouse strain could then be obtained by crossing these 2 transgenic strains.
682
Upon generation of the human/mouse chimeric TLR4 and combined human/mouse chimeric
683
TLR4/human MD-2 transgenic mice, bone marrow-derived macrophages were, however, shown to be
684
hyporesponsive to LPS. Furthermore, both transgenic mouse strains were hyporesponsive to LPS
685
challenge in vivo. Subsequent analyses showed that these defects were related to negligible
686
expression of both of the transgenic gene constructs. Hence, the transgenic animal approach failed to
687
produce a pharmacologically relevant transgenic mouse species that was suitable for pharmacology or
688
safety studies. In the second case study by Lolke de Haan, the development of a human C-X-C
689
chemokine receptor 2 (CXCR2) transgenic mouse model for safety testing of Hy29, a fully human
Given the apparent lack of
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ACCEPTED MANUSCRIPT monoclonal antibody directed against CXCR2 was presented. CXCR2 is a major chemotactic receptor
691
expressed on neutrophils with multiple chemokine ligands, including interleukin 8 (IL-8). Hy29 was
692
isolated after immunisation of a transgenic mouse strain expressing human Ig domains with human
693
CXCR2 overexpressing cells. Hy29 binds with high affinity to human CXCR2 and inhibits its
694
function in cell-based assays, whilst no or low affinity binding to rodent or cynomolgus monkey
695
CXCR2 was observed, and Hy29 did not inhibit rodent or cynomolgus monkey receptor function in
696
cell-based assays. In order to assess the nonclinical pharmacological activity and safety of Hy29, a
697
human CXCR2 transgenic mouse was generated in which the mouse CXCR2 gene was deleted or
698
disrupted and the human CXCR2 gene was inserted. Importantly, both in humans and mice CXCR2
699
has multiple ligands, not all of which are overlapping exactly with respect to function, and with some
700
ligands not having rodent counterparts in humans or vice versa.
701
precedence for the functionality of human CXCR2 transgenic mice, which provided additional
702
confidence in the approach (Mihara et al., 2005). Human CXCR2 transgenic mice were generated
703
successfully and functionality of the transgene was demonstrated in an intranasal LPS challenge
704
study, in which human CXCR2 transgenic mice showed similar lung neutrophil accumulation to wild
705
type mice 24 hours post challenge.
706
administration of Hy29 prior to LPS challenge in human CXCR2 transgenic mice, whilst the lung
707
neutrophil response to intranasal LPS was unchanged in wild type mice that received Hy29. The
708
human CXCR2 transgenic mouse model was then employed in a dose-range finding toxicity study, in
709
which animals received 3 doses of 4, 40 or 100 mg/kg Hy29 over a period of 14 days. In this study,
710
treatment with Hy29 was associated with dose-dependent reductions in circulating neutrophils as well
711
as dose-dependent increases in circulating mouse CXCR2 ligands, consistent with the anticipated
712
pharmacodynamic effects of Hy29.
713
anaphylactic reactions in individual animals following the second dose at 4 and 40 mg/kg Hy29, and
714
the third dose of 100 mg/kg Hy29. These effects correlated with a loss of exposure to Hy29 and
715
consequent pharmacodynamics effects, indicating that these reactions represented anti-drug antibody
716
(ADA)-mediated type III hypersensitivity reactions. Therefore, whilst the human CXCR2 mice
717
appeared fully functional and pharmacologically relevant, the immunogenicity of a fully human mAb
However, there is literature
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However, treatment with Hy29 was also associated with
Page 27 of 46
ACCEPTED MANUSCRIPT to mice prevented repeat dosing beyond 10 days, limiting the utility of the transgenic mice as a
719
species for toxicity studies. Together, these 2 case studies demonstrated that whilst transgenic mice
720
represent an option for assessing nonclinical safety of mAbs, their generation and utility for repeat-
721
dose nonclinical safety studies should be carefully considered prior to selecting transgenic animals as
722
a potential way forward for nonclinical safety testing.
723
Lise Loberg (AbbVie) presented a case study on a mAb for a disease-specific target with no cross-
724
reactivity to normal animal species commonly used in toxicology studies, focusing on study design
725
considerations. Selection of the specific transgenic (Tg) model was based upon several factors
726
including commercial availability, understanding of the pharmacology of mAb X in the particular Tg
727
model, and tissue expression of the transgene (i.e., expression primarily in CNS). Another
728
consideration was the optimal age of animals at the start of the toxicology study, whereby the
729
transgene was expressed at sufficiently high levels for mAb X to bind target, but before the
730
degenerative nature of the disease affected survivability or other toxicity endpoints. Target protein
731
expression was variable in animals at 2 and 4 months of age, was consistent with age-dependent
732
increases from 6 to 10 months of age, and reached a plateau at 10 and 13 months of age. mAb X
733
binding to target was first detectable at 6 months and considerably increased with age. Therefore, 6
734
months was selected as the optimal starting age for toxicology studies.
735
Pharmacology studies administered mAb X via IP administration, and were suggestive of anti-drug
736
antibody (ADA) development. To better understand potential for ADA formation, a pharmacokinetics
737
(PK) study was conducted in 6-month-old Tg mice, in which 6 mice/sex/group were dosed on Days 1
738
and 8 at 60 mg/kg IV or 100 mg/kg SC injection. There were no sex differences in PK parameters,
739
there was moderate SC bioavailability (approximately 60%), half-life was approximately 3 days, and
740
there were no indications for significant ADA development. Serum concentrations of mAb X were
741
maintained for up to one week after dosing. In addition, the PK study demonstrated feasibility for
742
sparse sampling to minimize animal numbers.
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ACCEPTED MANUSCRIPT An important consideration in the decision to evaluate potential toxicity in animal models of disease is
744
the lack of historical background knowledge of the model. This may be especially true for a Tg
745
animal model in which the transgene and its associated protein may be expressed very differently
746
from the normal protein and from the human disease of interest. For example, the animal model may
747
have alterations in behavior, clinical pathology parameters, or tissue histology. With that in mind,
748
toxicology studies in animal models are best conducted as hazard identification studies, with focus on
749
particular tissues or organ systems, rather than a comprehensive toxicological evaluation. In this case,
750
however, a full toxicological evaluation was intended and therefore, preliminary characterization of
751
background histopathological findings was conducted. To reduce and refine animal usage, tissues
752
were collected from Tg mice in a pharmacology study in which Tg mice were dosed with mAb X, one
753
of three alternate antibodies, a positive control or a negative control. Mice received 0.5 mg antibody
754
per week via IP administration for up to 18 weeks, starting at 6 months of age. Selected tissues (heart,
755
lung, kidney, liver, GI tract, and hindlimb joint and muscle) were evaluated from available animals in
756
the negative control group (N = 8), the mAb X group (N = 10), and two of the alternate antibody
757
groups (N = 7 and N = 11). There were no statistical differences in survival or cause of death (via
758
neoplasia or other causes), no changes in body weight, and no differences in types or incidence of
759
neoplasia between different experimental groups. Serum concentrations of mAb X were decreased by
760
6 weeks, suggestive of ADA development. Histopathological changes in kidney and degenerative
761
joint disease were common and attributable to aging, whereas skeletal muscle degeneration and
762
regeneration was severe in some animals and may have been a background change in the Tg model.
763
The preliminary PK and histopathologic studies informed study design for a GLP-compliant repeated
764
dose toxicology study. Tg mice were administered mAb X at 0 (vehicle control), 60 and 200
765
mg/kg/week IV (low and high doses), and 200 mg/kg/week SC (mid dose) for 4 weeks. Animal
766
numbers were increased to 18/sex/group to account for expected mortality up to 10%. ADA
767
development remained an unknown factor; therefore, the GLP study included two additional groups
768
dosed at 0 and 200 mg/kg/week IV for 13 weeks to evaluate PK and ADA, to inform feasibility for a
769
study longer than 4 weeks. In the 4- and 13-week GLP study, there was anticipated mortality (15%)
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ACCEPTED MANUSCRIPT during the study but also unanticipated mortality on the first day of dosing. The unanticipated deaths
771
were attributed to technical difficulty with IV injections into tail veins of dark-colored mice.
772
(Toxicology studies are often conducted in CD-1 mice, which are light-colored and tail veins are
773
easily observed on pink tails.) There was no relationship to dose and no clinical signs or
774
histopathologic changes suggestive of test article-related death; therefore the deaths on study were
775
attributed to procedure, aging and/or disease state. The mAb X half-life was < 2 days (IV), and 62%
776
of test article-dosed animals had detectable ADA. Only 1 (of 15) animals had detectable mAb X
777
concentrations after the 13th weekly dose of 200 mg/kg, confirming that a study longer than 4 weeks
778
would not be feasible. Histopathologic evaluation revealed anticipated effects (chronic nephropathy,
779
skeletal muscle degeneration/inflammation) that were common in control and test article-treated
780
animals and therefore considered age- and Tg-related but not mAb X-related.
781
In conclusion, the preliminary studies conducted to evaluate PK parameters after different routes of
782
administration and histopathological changes in the Tg mouse model of interest were instrumental to
783
the design of a 4-week GLP repeated-dose toxicology study and to the interpretation of study results.
784
Furthermore, the addition of dose groups administered control or mAb X for 13 weeks confirmed
785
development of ADA and associated decreased test article concentration, thereby providing evidence
786
that a study longer than 4 weeks would not be feasible.
787
Flavio Crameri (Roche) presented a combined surrogate and in vitro safety approach in support of
788
first-in-human dosing for CEA-IL2v (INN: cergutuzumab amunaleukin), a Roche proprietary tumor
789
antigen targeted immunocytokine currently in phase 1 clinical trials. CEA-IL2v is composed of a
790
monomeric human IL-2 with mutations (IL2v) to abolish binding to the high affinity IL-2 receptor
791
(IL-2Rαβγ). IL2v is fused to the c-terminal end of a bivalent anti-carcinoembryonic antigen (CEA)
792
human IgG1 with silenced effector functions. The anti-CEA component of the clinical candidate lacks
793
cross-reactivity with any toxicology species, while the IL2v component has been shown to be cross-
794
reactive in the cynomolgus monkey and the mouse. Given the lack of a fully cross-reactive species for
795
safety assessment it was decided to start the development of a cynomolgus monkey reactive anti
796
CEA-IL2v (cyCEA-IL2v) and at the same time to discuss and obtain feedback from a European health
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ACCEPTED MANUSCRIPT authority (HA) regarding the proposed preclinical safety strategy. The HA considered a surrogate
798
approach with cyCEA-IL2v appropriate for qualitative safety assessment once functional equivalence
799
of cyCEA-IL2v to CEA-IL2v had been shown. The surrogate approach however was to be combined
800
with an extensive quantitative in vitro safety assessment using CEA-IL2v in human cell based assays
801
to derive a minimum anticipated biological effect level (MABEL). The HA further commented that
802
CEA-IL2v may not be considered high risk based on the extensive previous clinical experience with
803
anti-CEA mAbs for imaging and with rhuIL-2 (Proleukin®) as well as the tumor targeted approach to
804
be used. In addition, the HA acknowledged that a starting dose based on a MABEL may be
805
unacceptably low and indicated that it was open to accept a combination of MABEL with other
806
approaches to determine a safe starting dose for CEA-IL2v. Based on the HA feedback received,
807
development of the surrogate cyCEA-IL2v was continued. Binding and functional equivalence of cy
808
CEA-IL2v when compared to CEA-IL2v was shown in several comparative in vitro assays; i.e.
809
affinity to recombinant CEA and IL-2Rβγ and IL2v-mediated functional activity using human and
810
cyno cells (cell signaling, cell activation and proliferation as well as cytokine release). The surrogate
811
cyCEA-IL2v was then tested in pilot and GLP studies in the cynomolgus monkey. The safety profile
812
obtained with cyCEA-IL2v in the cynomolgus monkey was consistent with the known safety profile
813
of wild type IL-2 (Proleukin®) thus validating the surrogate-IL2v for qualitative safety assessment.
814
No vascular leak syndrome, a major toxicity of wild type IL-2, was seen and there was no evidence
815
for any potential CEA-targeting related toxicity in tissues known to express CEA. The safe starting
816
dose for the FiH study was then selected based on a combination of the MABEL (IC20) obtained with
817
CEA-IL2v and published clinical data from competitor IL-2 immunocytokines for which safety and
818
exposure data were available. The resulting starting dose used for fist-in-human was approximately 3-
819
fold higher than the MABEL and was shown to be safe in the ongoing phase 1 clinical trial. This
820
approach helped to balance safety and number of patients without potential clinical benefit.
821
The second case study from Roche discussed a FiH that was supported exclusively by in vitro safety
822
data and a safe starting dose selection based on a MABEL only.
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ACCEPTED MANUSCRIPT Katharine Bray-French (Roche) presented a case study for CEA TCB which is a novel, Roche
824
proprietary T cell bispecific (TCB) antibody targeting carcinoembryonic antigen (CEA) expressed on
825
tumor cells and CD3 epsilon chain (CD3e) present on T cells that is currently in Phase 1 clinical trials
826
(NCT02324257) for the treatment of CEA positive solid tumors. Because the human CEA (hCEA)
827
binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents,
828
alternative nonclinical safety evaluation approaches were considered. These included the development
829
of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for
830
evaluation in cynomolgus monkey and the development of double transgenic mice, expressing hCEA
831
and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies.
832
However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the above
833
approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA
834
TCB. Therefore, an alternative approach, a MABEL based on an in vitro tumor lysis assay was used
835
to determine a safe starting dose for the first-in-human clinical study.
836
Benno Rattel (Amgen) presented three different nonclinical testing strategies for bispecific T-cell
837
engagers, commonly referred to as BiTE® antibody constructs. These molecules are comprised of two
838
different flexibly-linked single-chain antibodies, one directed against a tumor antigen and the other
839
targeting CD3. BITE® antibody constructs are designed to link transiently tumor cells with resting
840
polyclonal T-cells for induction of a surface target antigen-dependent re-directed lysis of tumor cells.
841
First-generation BiTE® antibodies cross-reacted only with respective antigens from chimpanzees
842
therefore, to facilitate in vivo safety testing, surrogate BiTE® antibodies were generated that are
843
cross-reactive with murine antigens. Firstly, the nonclinical safety package of Blincyto®, a CD19-
844
targeting BiTE® antibody construct, was presented (Nagorsen et. al, 2012; Topp et. al, 2015). It
845
included up to 3-month repeat-dose toxicity studies and an embryofetal toxicity study with the mouse
846
surrogate, which mimicked the parent compound`s pharmacological characteristics and successfully
847
supported the molecule`s recent approval both in the US and the EU (FDA, 2014; EMA, 2015) The
848
second case study described the determination of the clinical starting dose for a CEA-targeting BiTE®
849
antibody construct. For this tumor target, a surrogate molecule could not be developed that was
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Page 32 of 46
ACCEPTED MANUSCRIPT comparable to the parent molecule, since both the mouse and the cynomolgus surrogates evaluated
851
caused unspecific T cell activation. Therefore, the FIH dose was based on in vitro MABEL data, only.
852
Finally, the current nonclinical safety assessment paradigm for defining a safe clinical starting dose
853
was presented for the new generation of BiTE® antibody constructs that cross-react with cynomolgus
854
monkeys (Friedrich et al, 2012).
855
“Hot Topic” - Breakout Sessions
856
Breakout Session Question 1:
857
advantages over combinations ? For this breakout session Lolke de Haan (MedImmune) first
858
gave a brief presentation outlining the potential challenges in the nonclinical and clinical development
859
of bispecific antibodies and antibody combinations. Subsequent discussions focused on whether or
860
not bispecifics would provide drug molecules with additional, mechanistically differential, mode of
861
action. Or, alternatively, if bispecifics simply represent a more convenient way for the delivery of 2
862
pharmacodynamically active modalities, and an opportunity for replacing 2 separate injections with
863
monoclonal antibodies with 1. The consensus appeared to be that convenience was the main driver for
864
developing bispecifics. It was recognised that the ratio of the 2 bioactive components in a bispecific is
865
fixed, and that this could be a potential disadvantage. On the other hand, for combinations flexibility
866
could become a disadvantage, as with the number of variables available (antibody ratio and dosing
867
frequency and level) the number of combinations that could be tested are endless, and this could
868
complicate or hamper clinical development. With respect to targets suitable for bispecifics, there was
869
consensus that soluble targets are preferable, however, there was no expectation that both arms of the
870
bispecifics would engage 2 different soluble targets simultaneously. For membrane-bound targets,
871
binding 2 targets per bispecific molecule was generally not deemed likely, however, it would still be
872
possible to modulate 2 membrane-bound targets through monovalent interactions. Finally, the
873
bioanalytical challenges associated with bispecifics in the nonclinical and clinical setting were
874
discussed. For bispecifics, it was agreed that anti-idiotype antibodies against each bispecific arm to
875
determine the amount of free bispecific would be required. In addition, assays demonstrating stability
RI PT
850
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Page 33 of 46
ACCEPTED MANUSCRIPT 876
of the bispecific, e.g. capture with an anti-idiotype combined with an anti-Fc detection, were
877
considered desirable.
878
Breakout Session Question 2: “Is the paradigm “SC more immunogenic than IV” correct ?”
879
chaired by Benno Rattel (Amgen). Biotherapeutics have to be administered via a parenteral route,
880
because approaches to oral administration have failed so far.
881
administration, SC administration has the advantage of improved convenience and patient compliance
882
as well as prolonged half-life (Richter and Jacobsen , 2014). However, the SC route is perceived to be
883
problematic due to a greater potential for undesired immunogenicity (Braun et al, 1997; Ponce et al,
884
2009; FDA 2014). As there is generally a poor concordance between immunogenicity in animals and
885
humans, and, with few exceptions, human biologics are generally more immunogenic in animals than
886
in humans (Bugelski and Treacy, 2004; Ponce et al, 2009), ideally the validity of the above paradigm
887
should be verified with actual clinical experience of head-to-head comparisons of SC and IV
888
administrations of biotherapeutics.
889
There are only few published clinical examples directly comparing the two administration routes:
890
Rituximab (Bittner et al, 2014; Davies et al, 2014), Trastuzumab (Jakisch et al, 2015), Tocilizumab
891
(Ogata et al, 2014; Burmester et al, 2014), Belimumab (Shida et al, 2014), ATR-107 (Hua et al, 2014)
892
and Abatacept (Schiff, 2013). These clinical data as well as unpublished internal company data were
893
discussed, and the group agreed that these data do not support the above paradigm. The group argued
894
that a number of patient- and product-specific factors such as origin, post-translational modification,
895
aggregates, degradation products, impurities, formulation, container closure and, especially,
896
immunomodulatory properties (Parenky et al, 2014) are at least equally important for an unwanted
897
immune response as the administration route. Therefore, the group concluded that the generalization
898
that the SC administration bears a higher risk of immunogenicity compared to the IV route cannot be
899
universally upheld.
900
Breakout Session Question 3: For 3Rs considerations of NHP use “Why do we need a 6 month
901
toxicology study for a mAb when we already have a 13 week toxicology study?” chaired by Katja
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Page 34 of 46
ACCEPTED MANUSCRIPT Hempel (AbbVie) and Peter Ulrich (Novartis). Before the meeting a survey was send out by Peter
903
Ulrich to BioSafe members with the goal to collect the experience of the pharmaceutical industry with
904
toxicity studies supporting clinical development of monoclonal antibodies in order to evaluate
905
repeated dose toxicity strategy. This survey was limited to results generated with monoclonal
906
antibodies given via the intravenous or subcutaneous route at similar dose levels between the toxicity
907
studies and selected according to toxicology standard practice. Pure oncology mAb were not included
908
in this survey, since 26 week studies are not required for those mAbs. The findings asked for in the
909
survey were divided into pharmacodynamic (PD), target organ toxicity, and safety pharmacology
910
findings leading to a NOAEL.
911
Results: 26 case examples were provided for the survey by five BioSafe member companies. In none
912
of the 26 case examples were differences seen in the toxicological profile between 3 and 6 months. In
913
addition, one company informed verbally that they had 4 mAbs with again no difference in
914
toxicological profile. During the plenary session two additional interesting examples were mentioned
915
where toxicological effects were only seen in studies of longer duration. Taking into consideration the
916
results of the long study, and reevaluating the short term study, there was a hint of this toxicological
917
finding. With regard to the challenge as to when a 3 month stand-alone study might be sufficient,
918
there was agreement in panel that this needs to be a case by case decision also taking the target into
919
account. In addition, the use of sexually mature animals was recommended. A working group will be
920
formed to distribute the survey to companies that have not yet received the survey to include as many
921
case examples as possible, including especially the ones with a different outcome other than
922
mentioned in the plenum discussion. Additionally, it was suggested to collect data from marketed
923
products to update the list from former publications (Clarke et al, 2008). Goal is to publish the
924
current pharmaceutical expertise to leverage experience for mAbs with potential future extension to
925
more complex ADCs or DVDs, if possible.
926
Breakout Session Question 4:
927
Preclinically and does it Translate Clinically” chaired by Rajni Fagg (GSK). The breakout
928
session opened with an example from the literature (Heyden et. al, 2014) in which an investigative
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“How Do we Investigate Immune Complex Formation
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ACCEPTED MANUSCRIPT 929
study was conducted to characterise mAb induced immune complex (IC) formation in cynomolgus
930
monkeys. This preclinical investigative study was also designed to identify potential biomarkers that
931
could be indicative of immune complex formation and determine if the induced immune complex
932
formation was reversible. The discussions in the breakout session concluded: 1. Most likely, as antigen/mAb therapeutic ratios approach molar equivalence, the ICs are larger
934
and when clearance mechanisms are blocked or saturated, large ICs can deposit in tissue,
935
activate complement, and cause tissue damage.
937
2. Other factors that influence IC formation include high
antigen
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936
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933
concentration/density as well as antigen and mAb charge.
3. Although in the published example high anti-mAb antibody response in the monkeys
939
preceded the clinical pathology findings of thrombocytopenia, complement activation and
940
neutrophilia by several weeks, this was not considered to be always the case.
942
4. No clear correlation of preclinical findings of mAb therapeutic induced IC formation has been identified in the clinical setting.
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The fifth and final “hot topic” during the breakout session was a group discussion of “Adversity in
944
Nonclinical Safety Studies: STP and ESTP opinion, your opinion needed !”, chaired by John
945
Burkhardt (AbbVie) and Paul Germann (AbbVie). As an introduction John presented the STP (see
946
Figure 1, Kerlin et al, 2016; flow chart was developed for NCEs/small molecules but concept is also
947
relevant for biologics), and Paul the ESTP point of view (see Figure 2, Palazzi et al, 2015). Finally
948
several case studies from different companies were presented followed by a lively discussion on
949
whether a specific finding will be assessed as adverse and will therefore impact the NOAEL setting.
950
In essence, the tiered approach in histopathological adversity assessments, as depicted in Figure 2,
951
should guide younger colleagues towards a correct assessment of adversity. What still remains an
952
area of concern is the clear disconnect between the humoral reaction against biological products like
953
ADA formation and the histopathological findings which do not correlate in most of the cases.
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ACCEPTED MANUSCRIPT Figure 1: Schematic diagram delineating the hierarchy by which “adverse” test-article-related effects
956
are communicated (see Figure 2 from Kerlin et al, 2016)
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Conclusions
961
The opportunities for the application of biotherapeutics for the treatment or prevention of human
962
diseases continue to grow, and new molecular formats, including bi- or multi-specific molecules,
963
antibody fragments, and ADCs, are likely to reach the market in the near future.
964
complexity and unique properties of these novel modalities, standardized approaches for nonclinical
965
safety testing are becoming increasingly obsolete, and each molecule needs to be evaluated on case-
966
by-case basis, tailoring the nonclinical safety program and strategy to the molecule.
967
This meeting brought experts together from the nonclinical safety (toxicology, pathology, safety
968
pharmacology), pharmacokinetics and bioanalytical field to exchange knowledge and experience. In
969
addition to the presentations, podium discussions and breakout sessions, the meeting offered ample
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Given the
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opportunity for participants to share ideas, development strategies and case studies. This is becoming
971
increasingly important for the biopharmaceutical industry, as this often represent the only way in
972
which to share data on failed development programs, studies and strategies.
973
exchange of knowledge promotes the optimization of future biotherapeutics non-clinical development
974
programs, which will not only contribute to a responsible and justified use of animals, but hopefully
975
also to improved human risk assessment of innovative biotherapeutics.
976
Conflict of interest
977
Guenter Blaich, Rob Caldwell, and Edit Tarcsa are employees of AbbVie. Andreas Baumann is an
978
employee of Bayer Pharma AG; Sven Kronenberg, Wolfgang Richter and Flavio Crameri are
979
employees of Roche. Lolke de Haan is an employee of MedImmune; Peter Ulrich is an employee of
980
Novartis Pharma; Jay Tibbitts is an employee of UCB Celltech and Simon Chivers is an employee of
981
ADC Therapeutics. AbbVie and affiliates participated in the preparation of the workshop report,
982
interpretation of data, review, and approval of the manuscript.
983
Acknowledgements
984
The authors would like to thank all speakers for their contributions and members of the BioSafe
985
Leadership committee for valuable comments during the review of this manuscript.
986
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S0273-2300(16)30242-2
DOI:
10.1016/j.yrtph.2016.08.012
Reference:
YRTPH 3654
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 20 August 2016 Accepted Date: 25 August 2016
Please cite this article as: Blaich, G., Baumann, A., Kronenberg, S., de Haan, L., Ulrich, P., Richter, W.F., Tibbitts, J., Chivers, S., Tarcsa, E., Caldwell, R., Crameri, F., Non-clinical safety evaluation of biotherapeutics – Challenges, opportunities and new insights, Regulatory Toxicology and Pharmacology (2016), doi: 10.1016/j.yrtph.2016.08.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Workshop report
2
Non-Clinical Safety Evaluation of Biotherapeutics – Challenges, Opportunities and New
3
Insights
4
Guenter Blaich a, Andreas Baumann b, Sven Kronenberg c, Lolke de Haan d, Peter Ulrich e, Wolfgang
5
F. Richter c, Jay Tibbitts f, Simon Chivers g, Edit Tarcsa h, Robert Caldwell i, Flavio Crameri c
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a
7
c
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Sciences, Roche Innovation Center Basel, Switzerland; dMedImmune, Cambridge,
9
UK; eNovartis Pharma, Basel, Switzerland; fUCB Celltech, Slough, UK; gADC
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Therapeutics, SA; hAbbVie BioResearch, Worcester, USA.; iAbbVie Inc., North
11
Chicago, USA.
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AbbVie GmbH, Ludwigshafen, Germany; bBayer Pharma AG, Berlin, Germany;
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Roche Pharmaceutical Research and Early Development, Pharmaceutical
12 Abstract
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New challenges and opportunities in nonclinical safety testing of biotherapeutics were presented and
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discussed at the 5th European BioSafe Annual General Membership meeting in November 2015 in
16
Ludwigshafen. This article summarizes the presentations and discussions from both the main and the
17
breakout sessions.
18
The following topics were covered in six main sessions:
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(i)
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(ii)
Unexpected side effects of biotherapeutics
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(iii)
Safety testing of cell and gene therapies including vector safety and integration site
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analysis and setting up a GLP facility in this field (iv)
24 25
Challenges around use of PEGylated biologics, results of BioSafe survey
Immunogenicity and PKPD including immunogenicity prediction or methodologies to prevent induction of anti-drug antibodies
(v)
Current approaches applied to antibody drug conjugate (ADC) development Page 1 of 46
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(vi)
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Approaches for non-clinical safety testing of human-selective biologics, including use of transgenic animals and MABEL
The following questions were discussed across 4 breakout sessions (i-iv) and a case-study based
29
general discussion (v):
30
(i)
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Do bi-specifics offer nonclinical and clinical development advantages over combinations
31
? (ii)
Is the paradigm “SC is more immunogenic than IV” correct ” ?
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(iii)
Why do we need a 6-month toxicology study for a monoclonal antibody (mAb) when we
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already have a 13-week toxicology study ? (iv)
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How do we investigate immune complex formation pre-clinically and does it translate
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clinically ? (v)
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Adversity in nonclinical safety studies: STP and ESTP opinion, your opinion needed ?
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Key words: Biotherapeutics, PEGylated biologics, gene and cell therapy, pharmacokinetics,
40
nonclinical safety, antibody-drug conjugate, immunogenicity, cross-reactive species.
41
Introduction
42
BioSafe is the Preclinical Safety expert group of the Biotechnology Industry Organization (BIO), with
43
a mission to identify and respond to key scientific and regulatory issues and challenges related to the
44
preclinical safety evaluation of biopharmaceuticals. In addition to the Annual BioSafe General
45
Membership Meeting in the US, the 5th Annual BioSafe European General Membership meeting was
46
hosted by AbbVie on November 4-5, 2015 in Ludwigshafen, Germany. Participants included 125
47
scientists from the biopharmaceutical industry, small biotechnology companies and contract research
48
organisations primarily from Europe and the UK, but also from the US, and representing expertise in
49
pharmacology, toxicology, pathology, pharmacokinetics and bioanalytics. The focus of the meeting
50
was to share experiences and insights into nonclinical safety assessment of biologics including
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various nonclinical safety topics including PEGylated biologics, unexpected side effects, gene and
53
cell therapy, immunogenicity and PKPD, antibody drug conjugates (ADCs), and nonclinical safety
54
strategies for human-selective biologics. In each session, case examples were presented followed by
55
podium discussions. For the first time, a breakout session was introduced for discussion of four “hot
56
topics” in smaller groups, with the feedback being presented to all attendees afterwards by the
57
breakout session leads. Finally, a group discussion based on several case studies was stimulated
58
around a fifth “hot topic”, that of adversity in nonclinical safety studies and determination of the
59
NOAEL.
60
Session 1: Challenges in developing PEGylated Biologics
61
Andreas Baumann (Bayer) and Jenny Sims (Integrated Biologix) co-chaired the first main session
62
which focused on the nonclinical development of PEGylated biologics. Conjugation of Polyethylene
63
glycol (PEG) moieties to various types of biologics has become a standard method to improve their
64
pharmacokinetic properties, specifically prolonging plasma half-life (Jevsevar et al, 2010; Nakaoka et
65
al, 1997; Yang et al, 2004). PEG molecules themselves have been shown to be non toxic, non
66
immunogenic, hydrophilic, uncharged and non degradable polymers (Webster et al, 2009). For many
67
protein-conjugates, the primary mechanism to increase plasma half-life is through the reduction in the
68
rate of clearance by the kidneys (Mehvar, 2000; Yamaoka et al, 1995; Baumann et al, 2014; Webster
69
et al, 2009). Other mechanisms include shielding epitopes/surfaces of the target drug resulting in
70
lower visibility to the immune system, and protecting the protein from proteolysis.
71
comprehensive review of current experiences in non-clinical safety and pharmacokinetics of
72
PEGylated biotherapeutics, see references Ivens et al, 2015 and Baumann et al, 2014. Twelve
73
PEGylated biologics have received regulatory approval so far based on balanced risk/benefit
74
evaluations. Cellular vacuolation has been seen in toxicology studies in about half of these products,
75
however without any functional changes for organs and tissues (Rudmann et al, 2013; Ivens et al,
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2015, Turecek et al, 2016).
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ACCEPTED MANUSCRIPT Jennifer Sims (Integrated Biologix) presented results on a pharmaceutical industry survey on
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nonclinical safety testing of PEGylated proteins, which was conducted by BioSafe in 2013 and
79
published recently in more detail (Ivens et al, 2015). Ten companies provided information on 17
80
PEGylated biopharmaceuticals including receptor or protein targeting products with molecular
81
weights ranging from 25 kDa to 350 kDa and currently in various stages of nonclinical and clinical
82
development. The molecular weight of the linear or branched PEG moieties ranged from 20 kDa to
83
60 kDa. PEG-related tissue vacuolation was seen in toxicology studies for 10 of the 17 compounds in
84
the survey. No other-PEG related effects were reported. For 5 of the 17 products there was some
85
evidence for an impact on disposition related to receptor-mediated uptake of the PEGylated protein.
86
For the remaining 12 products it is not known whether there was receptor-mediated uptake, or
87
whether it was not evaluated. For 7 of 17 products no specific studies were conducted to assess the
88
disposition of the PEG moiety, while information on pharmacokinetics and/or disposition was
89
gathered from 10 products. For 11 products there no specific assays for measurement of anti-PEG
90
antibodies were developed or employed in nonclinical studies.
91
antibodies were detected in a single dose clinical study and others stated that clinical assays were
92
available or planned.
93
developed for 6 compounds, but either no positive responses were observed or there was little impact
94
on the interpretation of the study. One company stated that the development of IgM and IgG anti-
95
PEG antibodies was observed in mice and macaques, but this was not considered to have any impact
96
on safety considerations in humans.
97
Toxicity studies included in the survey were repeated IV or SC dose studies in mice, rats, dogs, and
98
cynomolgus monkeys and ranged from 2 to 52-weeks duration. The dosing frequency varied from
99
daily to weekly. For one product both the IV and intravitreal route of administration was used in
100
toxicology studies. No cellular vacuolation was observed for 7 of the 17 compounds. For these
101
compounds, the dose levels used were relatively low and duration of dosing was generally short (up to
102
4-5 weeks). For 2 compounds studies of 26-39 weeks in duration were conducted. None of the 4
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products with a single 20 kDa conjugated PEG showed evidence of vacuolation. In general, the
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ACCEPTED MANUSCRIPT maximum study duration for these 4 products was only 5 weeks, although for one product there was a
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26-week study in cynomolgus monkeys. However, cellular vacuolation was observed in some of the
106
4-week studies with 30 to 40 kDa PEG products.
107
One of the survey questions related to whether or not any special methodology was used to evaluate
108
potential adverse effects associated with PEG accumulation and/or vacuolation (e.g. IHC, electron
109
microscopy, functional evaluations such as nerve conduction velocity measurements, or in vitro
110
studies). No special investigations other than IHC were conducted for 16 of 17 compounds.
111
In the next presentation, Hanne Kjær Offenberg and Inga Bjørnsdottir (Novo Nordisk) presented
112
on the nonclinical safety and DMPK of N9-GP and N8-GP, 40 kDa pegylated human recombinant
113
coagulation factors IX- and VIII, respectively. Nonclinical safety testing of new therapeutic drugs
114
typically includes chronic toxicity testing of up to 26 or 52 weeks duration to support chronic dosing
115
in the clinic. However, human coagulations factors are highly immunogenic in animals resulting in an
116
anti-drug antibody response which reduces exposure considerably. Therefore, an alternative animal
117
model involving immuno-deficient athymic rats (Rowett Nude), which are devoid of T-cells and do
118
not mount T cell-dependent antibody responses, was used in 26 week studies for both compounds. In
119
general both N8-GP and N9-GP were very well tolerated in these studies. The choroid plexus
120
epithelial cells (CPE) have been described to develop vacuoles after dosing with PEGylated biologics
121
(Ivens et al. 2015). Therefore, the choroid plexus was evaluated for presence for PEG. Using IHC
122
staining, no PEG was detected in the CPE cells for N8-GP, whereas for N9-GP, PEG was detected in
123
the CPE cells at all dose levels. PEG did not induce vacuole formation in any tissue in any of the
124
studies. In the absence of any degenerative or inflammatory findings, presence of PEG in the CPE
125
cells is not considered adverse. The No-Observed-Adverse-Effect Level for N9-GP or N8-GP in
126
these studies was considered to be 1200 IU/kg every fifth day or 1200 IU/kg every fourth day,
127
respectively. The immune-deficient rat model proved suitable for a chronic study of 26 weeks
128
duration and could be considered in cases where a model is needed for the chronic testing of human
129
proteins which are highly immunogenic in animals (Offenberg et al, 2015).
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The biologic fate of the [3H]PEG-moiety incorporated into N8-GP and N9-GP was evaluated in a
131
single dose IV study in rats.
132
investigated to assess if pharmacokinetics was dose-dependent (dose range: 0.6 mg/kg and up to 200
133
mg/kg). For all compounds, plasma pharmacokinetics, distribution and excretion pathways were
134
investigated, based on total radioactivity measurements. The plasma concentration of the intact N8-
135
GP or N9-GP conjugate was also measured using an antibody-based analysis method. After single IV
136
administration to rats, [³H]N8-GP, [³H]N9-GP as well as [³H]PEG were shown to be widely
137
distributed, mainly in highly vascularized tissues, with the lowest levels of radioactivity found in the
138
CNS. The protein part of N9-GP/N8-GP was degraded over time, with the PEG moiety circulating in
139
plasma. The PEG was subsequently excreted as 40 kDa PEG and smaller sized PEGs. Biphasic
140
elimination of the PEG moiety was observed from plasma with more than 50% of PEG excreted into
141
urine and faeces within the 1st phase (t½, 1st phase ~2-3 days in rats, t½, 2nd phase ~ 13-18 days in
142
rats). The [³H]PEG and metabolites were eliminated mainly via the kidney into urine but also via the
143
liver into feces, with a larger fraction found in the feces for [³H]N8-GP and [³H]N9-GP compared to
144
[³H]PEG. Elimination of the 40 kDa PEG-moiety was shown to be dose-dependent with faster
145
elimination at lower dose levels.
146
Finally, Andreas Baumann (Bayer) presented on the nonclinical safety and PK of a site-specific
147
PEGylated-Factor VIII conjugate (BAY 94-9027). BAY 94-9027, a B-domain–deleted recombinant
148
FVIII with one 60 kDa polyethylene glycol (PEG; 2 × 30 kDa branched) attached via a linker to
149
cysteine 1804, is in clinical development for acute and prophylactic treatment of patients with
150
hemophilia A. In vivo toxicology studies with BAY 94-9027 or PEG-60 alone involving single and
151
repeated administration were conducted. Specifically, in a repeat-dose toxicity study, male Sprague
152
Dawley rats were given PEG-60 IV at dose levels of up to 11.0 mg/kg every other day for 4 weeks
153
which was in the range of the total expected cumulative life-time dose of PEG-60 in humans given
154
BAY 94-9027. No adverse effects or histopathological changes were observed in these studies (Ivens
155
et al, 2013). Whilst there is no clinical evidence to suggest that administration of PEGylated proteins
156
is associated with long-term safety issues, questions have been raised regarding the pharmacokinetics
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(PK) including metabolism and distribution, in particular for PEGylated proteins used chronically
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and/or in the pediatric population.
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distribution of residual radioactivity (full length PEG-60 and radioactive metabolites) on Days 7–168
160
after single IV administration of [14C] BAY 1025662, the cysteine-linker-PEG 60 kDa component of
161
the molecule containing a covalently linked [14C] label in the linker, in male Wistar rats.
162
Concentrations and amounts of radioactivity in organs and tissues were measured using whole body
163
autoradiography 6 months after administration. Between Days 7–168 post-injection, radioactive
164
residues in the body decreased steadily from 22.5% of the dose to 1.8%. A heterogeneous distribution
165
of radioactive residues was observed, with apparent preferential distribution of radioactivity to
166
endocrine and exocrine glands and lymphatic organs. No residual radioactivity was detected in brain
167
and adipose tissue. Elimination was fairly complete by the end of the study, with a total recovery of
168
radioactivity of 94% at Day 168. Terminal elimination was slow, but there was no indication of PEG
169
retention in rats.
170
Session 2: Unexpected side effects with biotherapeutics
171
In the second main session, chaired by Sven Kronenberg (Roche) and Lolke de Haan
172
(MedImmune), case examples describing the utility of nonclinical safety studies to detect unexpected
173
side effects of biologics were presented.
174
In the first presentation, Lolke de Haan, Simon Henderson and Mary McFarlane (All
175
MedImmune) presented a retrospective portfolio-wide analysis of the utility of early, non-GLP, dose-
176
range finding toxicity studies in the development of biologics. The analysis encompassed 53 drug
177
candidates generated in support of the MedImmune drug portfolio between 2008-2014, with
178
modalities including monoclonal antibodies, bispecifics, fusion proteins, antibody-drug conjugates,
179
therapeutic proteins and peptides. Overall, the analysis showed that with 7/37 drug candidates that
180
were tested in non-GLP toxicity studies, toxicity was observed, which for 4/37 lead to termination of
181
further drug candidate development. Other factors influenced drug candidate attrition to a greater
182
extent, with undesirable PK/PD properties, lack of disease rationale and/or portfolio fit being the
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The aim of the presented PK study was to investigate the
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ACCEPTED MANUSCRIPT reasons for attrition for 10/37 candidates. Of the 27 drug candidates that were taken forward in to
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Good Laboratory Practice (GLP) toxicity studies, with 5 candidates new toxicities were identified,
185
with 4/5 of these toxicities becoming apparent after subchronic dosing, and development of 2/5
186
candidates being halted because of these toxicities. Importantly, when toxicity was observed, it was
187
consistent with the mode of action or pharmacology associated with the drug candidate. Furthermore,
188
it appeared that the impact of conducting non-GLP dose-range finding toxicity studies on candidate
189
drug progression was limited, with the majority of the observed toxicities in non-GLP studies either
190
being acceptable based on safety margins, the intended clinical indication or the anticipated duration
191
or frequency of clinical dosing. In addition, given that additional toxicities observed in GLP studies
192
typically only became apparent in studies involving subchronic dosing, non-GLP dose range finding
193
studies would be expected to have limited impact on progression the progression of these candidates
194
to GLP toxicity studies. Based on this analysis, MedImmune has currently ceased to conduct non-
195
GLP dose-range finding toxicity studies as a default, and conducts such studies only on a case-by-case
196
basis, when there is a clear cause for concern based on modality (e.g. antibody-drug conjugates, which
197
are inherently toxic) or based on an extensive review of the target biology and the likelihood of target
198
modulation resulting in exaggerated pharmacology and toxicity in a naive animal model.
199
In the second presentation, Sven Kronenberg (Roche) provided case examples of unexpected side
200
effects with biotherapeutics that eventually contributed to the program termination. The first case
201
example summarized findings from a single dose PK study in cynomolgus monkeys with a chimeric
202
IgG4 mAb with abolished effector function targeting a receptor of the tetraspanin protein family. The
203
antibody target is highly expressed by hepatocytes, endothelial and epithelial cells in various tissues
204
and on hematopoietic cells. There were several key challenges with respect to the disposition and
205
safety of the molecule: The ubiquitous receptor expression led to target-mediated drug disposition at
206
lower doses. The huge antigen sink resulted in an unexpected and more than dose-proportional
207
increase in exposure, i.e. a 3-fold increase of dose led to a 100-fold increase in drug concentration,
208
making nonclinical safety testing and potential clinical studies challenging. Furthermore, safety-wise,
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two monkeys in the single-dose PK study became moribund within 2 hrs resulting in euthanasia of
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ACCEPTED MANUSCRIPT these animals; the monkeys showed symptoms of anaphylactoid shock and vascular leakage including
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edema and release of cytokines (IL-6, IL-2, MCP-1). There were no compelling in vitro alerts (on
212
mast cell degranulation, vascular permeability, complement activation) except for cytokine release
213
from whole blood. Cytokine release probably contributed to the fatalities, however, based on cell-
214
based assays, no agonistic activity of the mAb could be demonstrated. Rather, the risk for cytokine
215
release could be mitigated by modifications outside of the antigen-binding site of the mAb. In
216
conclusion, while the exact mechanism of the fatalities could not be elucidated, modification of the Fc
217
part reduced the risk of cytokine release but did not solve the steep “all or nothing” PK. In a 2nd case
218
example, results from nonclinical safety testing with one of several pegylated peptides tested at Roche
219
were presented. The peptide consisted of a 30 kDa polyethylene glycol (PEG) moiety and was dosed
220
IV or SC to rats and cynomolgus monkeys. Accumulation of the PEG moiety in neurons of the
221
hypothalamus and spinal cord was observed in 4-week rat and monkey toxicity studies. While these
222
findings were also observed in the rat at the end of a 12-week recovery phase, no clinical signs were
223
associated with the vacuolation. Neuronal uptake of PEG into neurons may have occurred via a
224
specific transporter in the brain. Since neurons have only slow or no renewal, a 1-year toxicity study
225
with weekly IV dosing was conducted in the monkey to investigate the potential for these effects to
226
result in impairment of neuronal function. At the end of this study, PEG was present in all dose
227
groups in all animals, including those of the 12-week recovery groups.
228
vacuolation was evident in several organs and tissues, including the neurons of CNS and spinal cord.
229
A reversible deficit in sural sensory and peroneal motor conduction velocities in animals at the high
230
dose was noted in week 24 only, but not at the other time points. This was inconsistent with a
231
progressive, degenerative peripheral neuropathy. Whether this finding at the high-dose at a single time
232
point was spurious, related to accumulation of PEG, or a potential consequence of target engagement
233
in the nervous system remains unclear. There were no electrophysiological effects seen in the CNS,
234
nor were any neurobehavioral changes observed.
235
Finally, Andreas Popp (AbbVie) reported on side effects of a mAb leading to the identification of a
236
new clinical indication. Humanized mAbs ABT-207 and h5F9-AM8 directed against the Repulsive
Presence of PEG and
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ACCEPTED MANUSCRIPT Guidance Molecule a (RGMa) and with cross reactivity to hemojuvelin (RGMc) were described. In
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healthy animals (mice, rats, cynomolgus monkeys) both mAbs increased serum iron and decreased
239
serum iron binding capacity. Histopathologically, iron was released from spleen macrophages
240
(reduced iron in the spleen) and accumulates in periportal hepatocytes. The observed effects were
241
dose-dependent and correlated negatively with hepcidin expression. Administration of a 20 mg/kg
242
single dose of the more potent h5F9-AM8 antibody to female rats resulted in significantly reduced
243
serum hepcidin levels compared to control, which lasted 6 weeks. In toxicology studies performed in
244
rats and cynomolgus monkeys for up to 13 weeks with ABT-207, no adverse effects were observed, in
245
particular no degeneration or inflammation due to the iron accumulation in the liver. The hepcidin-
246
related effects on iron homeostasis showed complete or partial recovery after a 12 week treatment free
247
period.
248
In addition, ABT-207 and h5F9-AM8 were tested in animal models of chronically and genetically
249
induced anemia and chronic inflammation. ABT-207 and h5F9-AM8 corrected anemia and shifted
250
macrophage population in this inflammation model significantly from M1 to M2 by lowering hepcidin
251
expression in the liver and serum hepcidin levels. In conclusion, targeting RGMc as a new concept to
252
reduce hepcidin expression could be of high value for the causal treatment of conditions of chronic
253
anemia.
254
Session 3: Gene and Cell therapy
255
The third main session of the meeting was chaired by Peter Ulrich (Novartis). First a short update
256
on hot topics in the field of Advanced Therapy Medicinal Products was provided. This introduction
257
was followed by two presentations on vector safety and integration site analysis by Manfred Schmidt
258
and about the set-up of a GLP facility in the Gene Therapy field by Patrizia Cristofori.
259
Ulrich summarized regulatory information from the meeting of the European Society for Gene and
260
Cell Therapy (ESGCT) in Helsinki, September 2015. Paula Salmikangas, chair of the EMA
261
Committee for Advanced Therapies (CAT), presented the CAT view on several ATMP topics in a
262
regulatory session. She especially focused on the ongoing public consultation of Regulation (EC) No
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ACCEPTED MANUSCRIPT 1394/2007 “Good Manufacturing Practice for Advanced Therapy Medicinal Products”. A summary
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was published in Dec 2015 and can be downloaded from the webpage of the European Commission
265
(European Commission>DG Health and Food Safety>Public health>Medicinal products for human
266
use>Advanced Therapies>Developments). Another topic was the new clinical trial regulation (EC)
267
536/2014, which asks for GLP compliance of pivotal non-clinical safety studies supporting first-in-
268
man (FiM) clinical trials. The regulation would also include ATMP non-clinical studies, which raised
269
many questions about the availability and the validation status of animal models in the heterogeneous
270
ATMP area. Salmikangas pointed out that for ATMP a GLP-compliant pivotal non-clinical safety
271
study may not be mandatory for FiM trials, however for Marketing Authorization Application (MAA)
272
such a study may be required. For ATMPs without the possibility to utilize validated animal models in
273
a GLP environment, the CAT would recommend to follow the instructions provided by the risk-based
274
approach guideline (EMA/CAT/CPWP/686637/2011). In December 2015 the CAT position on the
275
Application of GLP principles to ATMPs was adopted via a written procedure, which was forwarded
276
to the national health authorities (see CAT meeting minutes 12/2015 on www.ema.europe.eu). With
277
respect to hospital exemption (article 28 in EC 1394/2007) Salmikangas pointed out that the CAT
278
favors revision of the respective regulation by the European Commission in order to provide more
279
security for ATMP developing firms.
280
The recent developments in integration site analysis of gene therapy vectors and genome editing
281
approaches were also discussed (see also presentation by Manfred Schmidt below). The identification
282
of off-target integration sites by genome editing methods like CRISPR or TALENs was subject of
283
publications in 2015 as well as discussion in the ESGCT meeting (Helsinki 2015). All in all, the
284
conclusion from these recent investigations and method validations suggest that each engineered
285
nuclease has to be individually evaluated for their potential off-target activity and the conditions
286
influencing this potential (Gabriel et al., 2015). Nevertheless, the same authors see a major
287
methodological advancement of sensitive, genome-wide detection of nuclease activity.
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Another
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immunocompromised mouse model, where NSG mice were genetically engineered with human
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highlight from the ESGCT
2015
meeting was the presentation
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of a
new
ACCEPTED MANUSCRIPT transgenes coding for IL-3, GM-CSF and SCF. The resulting mouse strain, also called NSG-SGM3
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(JAX Datasheet – 013062), shows less xeno-induced graft-versus-host-disease (GvHD), when
292
reconstituted with human haemopoietic stem cells (HSC). In addition, these mice, when reconstituted
293
with HSC, develop human monocytes. Norelli et al. (2015) reported that mice reconstituted with
294
human HSC and with CD44v6+ Acute Lymphoblastic Leukemia (ALL) semi-cell line, developed
295
features consistent with a cytokine release syndrome after infusion with human autologous T cells
296
genetically modified with a CD28-endocostimulated CD44v6 CAR. Leukemia clearance associated
297
with transient malaise, high fevers and weight loss, appeared with CAR deletion of monocytes.
298
Increased serum levels of IL-6 and IFNγ in relation to clinical observations were measured with
299
monocytes as the main source of IL-6. The presented data suggest that this model may be suitable for
300
assessing CRS potential of CARTs in a non-clinical setting.
301
Viral vectors (Manfred Schmidt, NCT) have shown their efficacy in clinical studies of rare diseases.
302
However, vector-mediated insertional side effects and malignant transformation have occurred in
303
individual patients. These observations have prompted new vector designs, such as self-inactivating
304
(SIN) LTR configurations and tissue-specific promoters that may improve safe and efficient viral
305
vector-mediated gene-correction in patients. Now, with the advent of mammalian whole genome
306
sequence availability and next generation sequencing (NGS), large-scale identification of vector
307
integration profiles and vector persistence studies over time have become an essential part of
308
pharmacokinetic studies for a gene therapeutics.
309
mediated PCR) coupled to NGS technology for vector integration site analysis is subject to
310
continuous further development. The current state-of-the-art and future perspectives were presented,
311
which includes new technologies like capture-based quantitative sequencing of viral vector sequences
312
and integration sites as well as associated bioinformatical data mining tool suites.
313
Patrizia Cristofori (GSK) presented the GLP Principles in Gene Therapy and the Experience of
314
Academic facilities GLP certified (HSR-TIGET). Gene therapy (GT) is now emerging as a medical
315
reality with clinical efficacy demonstrated in a number of gene therapy trials and an increasing
316
number of products entering phase II and III trials each year. For the promise and potential of a gene
The current LAM-PCR (linear amplification-
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ACCEPTED MANUSCRIPT therapy medicinal product (GTMP) to be fully realized it is important to address regulatory
318
expectations. Guidelines for GTMP progression in clinical trials and marketing authorizations are
319
available to facilitate a harmonized approach in the EU and US. Non-clinical studies have the
320
primary objective of providing sufficient information for a proper risk assessment for the product’s
321
use in human subjects. The paradigm described in ICH M3 for safety evaluation of conventional
322
pharmaceuticals is not always appropriate or relevant to GTMPs. Non-clinical studies should be
323
designed on a case-by-case basis, understanding the relevant aspects of the science underpinning that
324
product and need for specific expertise beyond the traditional pharmaceutical field. The combination
325
of expertise of personnel trained in genetic therapy research, experimental pathology, safety
326
assessment and quality assurance has enabled setting up a GLP Test Facility in an academic
327
environment. The main objective was to i) maximise the early collection of proof-of concept data and
328
provide adequate preclinical information to support the safety and the scientific basis for the
329
administration of an investigational product in the target patient population, ii) provide data of high
330
regulatory standard with assurance of scientific integrity, validity and reliability, and iii) minimise the
331
use of animals (3Rs). Challenges to be addressed in designing non-standard study to investigate in
332
vivo fate of genetically modified cells (biodistribution) and toxicological and tumorigenicity potential
333
were discussed. Discussion focused on the definition of test item and its characterisation, the choice of
334
test system (animal model), sample traceability, duration of treatment and endpoints to be measured.
335
The key role of Quality Assurance (QA) was highlighted. QA must be aware of all non-standard
336
aspects and challenges of GT preclinical studies and knowledgeable in technical procedures and
337
methods working closely with technical personnel to share criticality and best practices. In
338
conclusion, due to the complexity of GTMPs, non-clinical safety testing can vary considerably and is
339
recommended to be handled on a case-by-case basis, understanding the product and areas of non-
340
compliance.
341
Session 4: Immunogenicity and PKPD – from Prediction to Interpretation
342
Formation of anti-drug antibodies (ADA) is one of the hallmarks of an immune response against
343
biotherapeutics. ADA may affect safety, efficacy and pharmacokinetics of a biotherapeutic. Thus,
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ACCEPTED MANUSCRIPT ADA often change the pharmacokinetic – pharmacodynamics (PKPD) of biotherapeutics. Therefore,
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presence of ADA needs to be considered in the interpretation of PKPD results. The fourth main
346
session, chaired by Wolfgang Richter (Roche) and Jay Tibbitts (UCB), gave overviews on PKPD
347
aspects of immunogenicity in toxicology studies, the immunogenicity of bispecific antibodies in
348
monkeys, and the current knowledge on in vitro tools for immunogenicity prediction. In addition,
349
approaches were discussed how to prevent immunogenicity in animal studies.
350
Olivier Petricoul (Novartis) presented the results of a survey conducted by the BioSafe PK/PD
351
working group on the analysis and reporting of toxicokinetic (TK) parameters. Toxicokinetic analysis
352
is a requirement for toxicology studies and, occasionally, in repeat-dose studies the test article
353
exposure in individual animals will change over the course of the study with increasing variability in
354
exposure in the TK assessment at the end of the study compared to Day 1 of the study. This
355
phenomenon may be due to the impact of ADA. The toxicokinetic analyst is then left to determine
356
how the TK data be reported as the effects of ADA formation on toxicokinetic/pharmacodynamic
357
parameters should be considered when interpreting the data (ICH S6).
358
It was highlighted that one of the main objectives of TK analysis is to show the validity of the
359
toxicology study, i.e. to confirm drug exposure at the start and end of the dosing period. Estimates of
360
the maximum observed concentration (Cmax) and area under the concentration-time curve (AUC) are
361
the most commonly generated TK parameters and usually the exposure multiple relative to human
362
exposure is based on the relevant mean AUC and Cmax values obtained in toxicology studies. As
363
ADA may impact on exposure (often a reduction, but in certain cases a prolonged exposure can be
364
observed) and consequently the estimates of Cmax and AUC, careful consideration should be given to
365
how mean values of these parameters are derived. Reporting of mean (and other summary statistics)
366
TK parameters in the context of ADA can be done in several ways. The mean TK parameters could
367
be calculated based on all animals independent of their ADA status. Alternatively, the mean TK
368
parameters of only ADA negative animals could be provided (Thway et al, 2013). The former could
369
be considered a more conservative estimate of drug exposure for the purposes of exposure margin
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ACCEPTED MANUSCRIPT calculations. Another approach would be to use TK parameters derived from Day 1 dosing (or a
371
similar early dosing period), where usually there is no impact of ADA.
372
A total of nine companies provided answers to the survey questions on how they calculate and report
373
TK parameters. All companies calculate TK parameters for all animals, regardless of ADA status.
374
Seven companies derive summary statistics using all animals, but four report, in addition, means for
375
ADA negative animals while the other three derive means for ADA negative animals only when ADA
376
appear to have affected the exposure. One company provides only one mean of TK parameters,
377
excluding ADA positive animals from the summary statistics when clear effects on exposure are
378
observed. Finally one company derives summary statistics using all animals, independent of the ADA
379
status. One example of a monoclonal antibody targeting a cell surface receptor was given to highlight
380
the effect of ADA on the TK (reduced exposure) associated with loss of PD effect. It shows the
381
importance to integrate the ADA status into the study summary to provide the appropriate
382
interpretation of the TK and PD results.
383
As noted above, one of the key elements of TK analysis is to confirm drug exposure in the study; and
384
as pointed out during the discussion on TK calculations some investigators choose to exclude animals
385
from summary TK parameters based on the degree of impact ADA has on exposure. Thus, one of the
386
major difficulties becomes how to define the «impact on exposure» of ADA? Several options were
387
discussed on how exclusion of ADA positive animals from mean TK parameter could be done, such
388
as scientific judgement, using a defined threshold (e.g. based on AUC decrease or increase from day
389
1), to report the median instead of the mean or to exclude time-points from profiles after animals
390
become ADA positive. Clearly, no established or mutually agreed-upon strategy exists for calculating
391
and reporting TK parameters in the presence of ADA. Good scientific judgement, clear explanation
392
and rationalization of the strategy used, and thoughtful consideration of the impact of the chosen
393
strategy on the exposure multiples and starting dose in humans are critical to appropriate risk
394
assessments under these circumstances.
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Finally, there was a discussion regarding the exposure data necessary to support a toxicology study
396
versus those required to provide suitable PK data. The conclusion was that both PK (for dose
397
predictions and modeling) and TK (to establish an exposure-toxicity relationship or an exposure
398
multiple) can be simultaneously obtained from toxicology studies.
399
employing the strategies described above to derive mean TK parameters for exposure multiples and
400
using data from ADA negative/not impacted by ADA for PK evaluations.
401
Eric Stefanich (Genentech) presented preclinical data assessing the immunogenicity of bispecific
402
therapeutic antibodies in cynomolgus monkeys. Some of the potential differences between bispecific
403
and monospecific antibodies that may contribute to differential immunogenicity were discussed
404
including increased “foreignness” in bispecific antibodies due to two engineered complementarity
405
determining regions (CDRs); unique product-related variants; the formation of different sizes of
406
circulating immune complexes with ADA; and asymmetry in the fragment antigen-binding (Fab)
407
region (e.g., charge differences) that could impact tertiary orientation at the hinge region. The
408
emerging data suggest there may be increased immunogenicity (both incidence and titer) with some
409
bispecific MAbs compared to what is typically encountered for monospecific MAbs. However there
410
are limited case studies for bispecific antibodies to date. The observed magnitude of ADA can impact
411
the ability to conduct long-term preclinical safety studies in monkeys.
412
immunogenic epitopes from cynomolgus monkey studies are mostly associated with CDRs of the
413
bispecific antibodies. Based on limitations of existing immunogenicity risk-prediction tools, we may
414
need to rely on clinical trial results to understand clinical risk.
415
Sebastian Spindeldreher (Novartis) presented four case studies to exemplify how currently
416
available tools and assays can be used to assess the immunogenicity risk of biotherapeutics. Before
417
introducing in vitro tools for immunogenicity risk assessment methods Sebastian briefly introduced
418
ABIRISK (Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to
419
minimize the RISK), which is a project of the Innovative Medicines Initiative (IMI) of the European
420
Union.
421
research groups and clinical centers. It is aimed at improving the understanding of patient- and
Where assessed, the
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This can be achieved by
ABIRISK is a public-private partnership between pharmaceutical companies, academic
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ACCEPTED MANUSCRIPT compound-related factors influencing immunogenicity in order to identify clinical biomarkers for
423
immunogenicity and to validate available, and develop new, assay tools to assess the immunogenicity
424
risk for a given compound.
425
In vitro tools have been developed that may allow immunogenicity risk assessment. One of these
426
tools is MHC-associated peptide proteomics (MAPPS), a technology that identifies sequences of
427
peptides bound to HLA-DR, i.e. potential T cell epitopes. Other commonly used in vitro tools are T
428
cell activation assays, which are applied in many different variations. More recently, dendritic cell
429
activation assays have been developed to understand the potential effects on the innate immune
430
system.
431
The first case study showed that the number of potential T cell epitopes identified with MAPPs and
432
the response rate in T cell activation assays conducted with several marketed monoclonal antibodies
433
matched roughly the ranking based on clinical immunogenicity incidence (Karle et al, 2016).
434
However, one of the key issues for clinical validation of immunogenicity prediction is the lack of
435
reliable clinical immunogenicity data that can be compared across studies. Published data are based
436
on different immunogenicity assays with different sensitivities and drug tolerances, different sampling
437
time points, different diseases and disease states, different concomitant treatments and potentially
438
further differences. A solid comparison of in vitro and clinical data will only be possible with reliable
439
clinical immunogenicity data that can be compared across studies.
440
In vitro tools have been developed that may allow immunogenicity risk assessment. One of these
441
tools is MHC-associated peptide proteomics (MAPPS), a technology that identifies sequences of
442
peptides bound to HLA-DR, i.e. potential T cell epitopes. Other commonly used in vitro tools are T
443
cell activation assays, which are applied in many different variations. More recently, dendritic cell
444
activation assays have been developed to understand the potential effects on the innate immune
445
system.
446
The second case study focused on unpublished data from the ABIRISK consortium
447
[www.abirisk.eu]. The work conducted in the labs of Bernard Maillère (CEA) and Anette Karle
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ACCEPTED MANUSCRIPT (Novartis) showed good agreement between HLA-DR-presented peptides identified by MAPPs, T cell
449
epitopes identified from drug-naïve healthy blood donors and T cell epitopes of memory T cells
450
recovered from patients who were treated with and developed immunogenicity to the tested
451
biotherapeutics. Those data nicely confirm that the biology of patients who develop ADA against
452
biotherapeutics is mimicked in in vitro assays that are based on drug-naïve healthy subjects.
453
The third case study looked at the effects of protein aggregation on dendritic cell activation, antigen
454
presentation and T cell activation (Rombach-Riegraf et al, 2014). A correlation was found between
455
the amount of subvisible particles as well as the protein amount contained in these particles and the
456
amount of peptides presented in the context of HLA-DR. This data was confirmed with four different
457
marketed monoclonal antibodies that were studied in the context of ABIRISK.
458
monoclonal antibodies reacted very differently to physical stress, leading to different extent of
459
aggregation, which was again linked to increased antigen presentation as determined by MAPPs.
460
The last case study indicated that transgenic mice which express a mini-repertoire of human IgG1
461
antibodies can mount ADA responses to foreign antigens but are tolerant to native monomeric human
462
antibodies. Chemical modification by UV but not process-related or pH-induced oligomers could
463
induce ADA responses, which led to the hypothesis that neo-epitopes are required for the break of the
464
tolerance in these mice (Bessa et al, 2015).
465
During the discussion the audience largely agreed to the overall conclusion that it is currently not
466
possible to predict clinical immunogenicity incidence or outcome of immunogenicity. However,
467
promising mathematical models are being developed (Chen et al, 2014). Data from the “prediction
468
tools” should be considered as an assessment of the potential for immunogenicity but not as an
469
absolute prediction of immunogenicity incidence and outcome in human. However, the currently
470
available methods can be used for investigative and mechanistic studies and can help candidate
471
selection and internal decision making.
472
Wolfgang Richter (Roche) gave an overview on approaches to prevent immunogenicity in non-
473
clinical studies. The conduct of non-clinical studies with human or humanized biotherapeutics can be
These four
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hampered by an immune response against the biotherapeutic. As previously noted, the formation of
475
ADA can substantially affect biotherapeutic exposure.
476
immune response and formation of ADA usually leads to an accelerated clearance and loss of
477
biotherapeutic exposure. However, for small biotherapeutics ADA may result in increased clearance
478
but, in some instances, may result instead in an apparent reduction in clearance (“sustaining ADA”)
479
due to slowing of the often fast endogenous renal elimination by virtue of binding of the small
480
biotherapeutic to a slowly cleared IgG ADA. Beyond the biotherapeutic exposure implications,
481
potential safety sequelae of immune responses include deposition of immune complexes in some
482
organs (eg. lungs, kidney). Re-dosing after formation of ADA may lead to anaphylaxis and acute
483
death (Hattori et al, 2007). Therefore, conduct of long term studies in mice may benefit from
484
suppression of an immune response. Ways to prevent immunogenicity include general suppression of
485
the immune system or antigen-specific tolerance induction. General suppression of the immune
486
response via pre-treatment with an anti-CD4 antibody was successfully used to conduct long-term
487
pharmacology studies in mice with the human monoclonal antibody gantenerumab (Bohrmann et al,
488
2012). The anti-CD4 treatment acted by transient depletion of peripheral CD4+ T-helper cells one
489
day before the start of gantenerumab administration. Alternative approaches include e.g. treatment
490
with the immunosuppressant mycophenolate mofetil.
491
antigen-specific tolerance induction keeps the immune system fully functional. Antigen-specific
492
immune tolerance may be induced by administering a high loading dose of a biotherapeutic followed
493
by administration of a lower dose during the test period. High dose tolerance induction was used to
494
enable a low dose group in a mice juvenile toxicity study with the antibody MR16-1 without ADA
495
formation or ADA-related side effects (Sakurai et al, 2013). Immune tolerance can also be induced
496
by exposing neonates to the tolerogen. In a study with adalimumab in mice, neonatal immune
497
tolerance was induced by dosing of mother mice after delivery (Piccand et al, 2016). Adalimumab
498
was transferred to the suckling pups via milk, thus providing antigen that induced tolerance. After
499
reaching adulthood at 8 weeks of age the offspring was dosed with adalimumab. Offspring exposed
500
to adalimumab the first days after birth showed no indication for an immune response, while control
By contrast to the previous approaches,
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When the biotherapeutic is an IgG, the
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mice showed an immune-mediated accelerated adalimumab clearance starting 7 days after dosing and
502
formation of ADA.
503
Overall, the session showed multiple aspects of immunogenicity and its consequences for the PKPD
504
and safety assessment of biotherapeutics.
505
biotherapeutic exposure is an important component in the TK and PK assessment. Novel formats may
506
differ in their immunogenicity from canonical monoclonal antibodies. In vitro tools for
507
immunogenicity are useful for investigative and mechanistic studies and can help candidate selection
508
and internal decision making. Various approaches for immune tolerance induction have been reported
509
to enable long term studies in animals without immune response. Immune tolerance induction has
510
been mainly applied in mouse studies, while experience in other species is limited or lacking.
511
Session 5: Antibody Drug Conjugates (ADCs)
512
The fifth main session was chaired by Simon Chivers (ADC Therapeutics SA) and Edit Tarcsa
513
(AbbVie), and covered the current approaches applied to antibody drug conjugate (ADC)
514
development. ADCs comprise of a monoclonal antibody and a cytotoxic payload attached via a
515
chemical linker, designed with the notion that the specificity of the antibody will lead to exclusive
516
delivery of the toxic payload to the cancer cells expressing the antigen on the cell surface, at the same
517
time eliminating systemic toxicities typically observed when administering the unconjugated payload.
518
Most ADCs currently in development have been generated by randomly attaching the payload to
519
either lysine residues or to reduced interchain cysteine in the antibody. These processes generate
520
heterogeneous mixtures with varying drug to antibody ratios (DAR), where the different forms may
521
contribute differentially to efficacy and/or toxicity. Homogenous DAR ADC’s are also possible
522
through the introduction of engineered cysteins, such as with the Thiomab technology. In addition,
523
some ADCs are not stable in vivo, some may be prone to toxin loss via proteolytic cleavage of the
524
linker or unwanted migration of the payload to plasma proteins. Evidence is emerging that in addition
525
to targeted delivery of the payload to tumor cells, ADCs are taken up via a variety of mechanisms
526
(pinocytosis, phagocytosis, FcR-mediated, or pattern recognition receptor-mediated uptake, etc), often
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Knowledge of ADA formation and its impact on
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ACCEPTED MANUSCRIPT 527
the ‘normal’ clearance route for antibodies that might contribute to the non-target related toxicities
528
observed in the clinic. Thus understanding the composition and in vivo fate of ADCs can be critical to
529
optimize their properties.
530
development, it is of outmost importance to understand how to optimize the therapeutic margin of
531
these complex therapeutic modalities.
532
The first presentation by Stephanie Fischmann (AbbVie) talked about what should be measured for
533
ADCs – examples and experiences. Today, 3-4 bioanalytical assays are required to support the
534
development of ADCs. Typically, un-conjugated or “free or toxin”, “total antibody” and “conjugated
535
antibody” concentration are used for this purpose. The toxin load of the antibody can also be
536
represented by the measuring the “conjugated toxin” levels. The latter is very valuable if cleavable
537
linkers were chosen as this enables the use of generic methods. From a bioanalytical perspective
538
Ligand Binding Assays (LBA) were the “state-of-the-art” platform over the years for any “total
539
antibody” or “conjugated antibody” assays run in support of an ADC projects. Meanwhile, more and
540
more Mass spectrometry based methods have been developed, i.e. High Resolution Mass
541
spectrometry, where the drug-antibody ratio can be elucidated either by way of full-size ADC
542
construct molecular weight measurement or after reduction for light chain and heavy chain and
543
determining their molecular weight separately. Hybrid LC-MS/MS assays are also evolving e.g. for
544
determination of conjugated toxin levels. Here, affinity pull-down followed by enzymatic cleavage of
545
the linker facilitates LC-MS/MS quantification of the released toxin as a small molecule. Another
546
approach follows unique peptides identified to represent total antibody and conjugated antibody levels
547
after tryptic digestion. Mass spectroscopy based methods in general offer more detailed information
548
on the ADC backbone (i.e. number and position of toxins) and on the potential biotransformation of
549
the payload. However, MS-based hybrid assays are more tedious and can be limited in throughput or
550
sensitivity. Hence, from a bioanalytical and scientific perspective LBA can serve as the method
551
platform of choice throughout the whole life cycle of an ADC project. However, additional mass spec
552
technology based methods are beneficial to elucidate the in vivo fate of the ADC constructs with more
553
granularity (esp. early stage). Today, many different factors influence the choice of assays in support
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With 2 ADCs currently approved and about 40 more in clinical
Page 21 of 46
ACCEPTED MANUSCRIPT of an ADC, including the development stage of the project, reagents availability, assay sensitivity,
555
assay development time and cost (cost per validation and cost per sample). For such reasons generic
556
platforms are often favorable at early stage but replaced by more specific assays later. Last, but not
557
least attempts are being made to simplify the bioanalytical efforts (i.e. the number of PK assays
558
undertaken in support of ADC projects). For example, multiplex technologies on LBA platforms and
559
multi-analyte strategies on LC-MS/MS platform are being evaluated.
560
In order to elucidate the immunogenicity of complex constructs like ADCs, all potential immunogenic
561
epitopes must be evaluated. Currently, a multistep strategy is recommended where after positive
562
screening assays the samples with potential ADAs are also tested in individual spike in experiments.
563
This so called “domain characterization” assay strategy employs unconjugated antibody spike as well
564
as full ADC spikes or spike-in experiments where only specific moieties of the ADC are spiked in,
565
thus the potential immunogenic epitopes can be identified.
566
Next, Matthias Machacek (LYO-X) presented on the PK, PD and toxicology modeling of IgG1
567
based ADCs and critical success factors. For ADCs three different PK levels can be identified: whole
568
body distribution, tumor penetration and uptake into the cell. Each of these three levels is critical for
569
successful toxin delivery to the tumor with a wide therapeutic margin. On a whole body level, it is the
570
IgG1 typical distribution and elimination processes that are relevant. IgG1 molecules remain
571
circulating in blood and interstitium until their uptake by cells and subsequent metabolism or
572
recycling via the FcRn mechanism. Thus, eventually all of the attached toxin will be delivered to
573
tissues that are active in IgG1 catabolism after internalization. For ADCs the IgG1 specificity to a
574
tumor target, a tumor specific cell surface molecule, will divert some of the ADC from the normal
575
clearance pathway. ADC binding to the tumor target will depend on the affinity and tumor target
576
abundance. Next, the tumor target bound ADC is internalized delivering the toxin into the cell.
577
Internalization depends on tumor target expression and the internalization rate. A back of the envelope
578
calculation showed that for a cell expressing 20’000 tumor targets and with a receptor internalization
579
half-life of about 2 hours intracellular toxin concentrations in the order of 10 nM are rapidly achieved.
580
A live ADC PK model simulation was then shown to illustrate the discussed concepts. The simulation
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ACCEPTED MANUSCRIPT demonstrated the impact of dose on how much toxin is delivered to the tumor versus to other tissues.
582
For high doses the fraction delivered to the tumor versus tissues was small. This was explained by the
583
saturation of the tumor up-take capacity in which case a large fraction of the toxin is delivered to IgG1
584
catabolizing tissues. By lowering the dose, the fraction delivered to the tumor could successively be
585
increased up to 80% for that specific case. Thus, for an optimal clinical dosing strategy the tumor
586
uptake capacity should be considered and the dose and regimen accordingly adjusted. Understanding
587
mechanistically and quantitatively the tumor target properties, ADC distribution and clearance as well
588
as the information drawn from the clinical PK profiles all help in better assessing the tumor uptake
589
capacity and hence the optimal dose and regimen.
590
Next, Andreas Pahl (Heidelberg Pharma) gave a talk about the toxicology of amanitin-based
591
ADCs. Payloads of today’s ADCs are exclusively based on compounds acting on microtubules or
592
DNA, are suffering from various limitations. New generations of payloads are entering the field
593
including Heidelberg Pharma’s amanitin, a highly effective inhibitor of the eukaryotic RNA
594
Polymerase II. Due to its unique mode of action and its hydrophilic nature this toxin differs
595
significantly from well-known payloads. Amanitin is the toxin of the green death cap mushroom.
596
Poisoning by amanitin leads to liver failure and subsequent death depending on the amount of toxin
597
ingested. Due to its hydrophilic nature amanitin cannot penetrate cell membranes passively. The
598
reason for its liver toxicity is a specific transporter OATB1B3, which is exclusively expressed in liver
599
cells. In vitro studies demonstrated that amanitin conjugated antibodies are not substrates for this
600
transporter indicating that amanitin-ADCs are not toxic to the liver. This was further explored in a
601
toxicological study in cynomolgus monkeys designed as an explorative study. Escalating doses of
602
Trastuzumab-Amanitin-ADCs from 0.3 to 10 mg/kg were administered to groups of three female
603
cynomolgus monkeys. In line with the in vitro findings no significant elevations of liver enzymes
604
were observed up to doses of 10 mg/kg. No other signs of liver toxicity or histological changes of the
605
liver were observed. LDH was the most sensitive parameter from clinical chemistry. A transient
606
increase at day 7 was observed. This was accompanied by hematological changes, namely elevated
607
levels of neutrophils. Dose-limiting toxicities were skin and ocular toxicity. These DLTs can be
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ACCEPTED MANUSCRIPT explained by target biology. Her2, the target of trastuzumab, is expressed on various epithelial cells.
609
Differences to T-DM1 toxicological findings can be explained by amanitin’s ability to kill also non-
610
dividing cells, whereas T-DM1 is only killing dividing cells. Next, different linker and conjugation
611
strategies for amanitin were compared. The MTD and the therapeutic index were significantly higher
612
for amanitin-ADCs based on site-specific conjugation to trastuzumab-thiomabs as compared to
613
random lysine conjugated ADCs. The therapeutic index for the amanitin-trastuzumab-thiomabs was
614
around 20 based on murine effective doses and monkey tolerable doses bridged by the AUC of the
615
ADC. This favorable therapeutic index will enable this amanitin-linker technology to move forward to
616
development.
617
In the final presentation for this session, Steve Alley (Seattle Genetics) described biotransformations
618
of auristatin ADCs. Biotransformations can be the intended effect of ADCs, such as release of
619
conjugated drug in the lysosomes of antigen positive cells, as well as amino acid modifications, loss
620
of drug-linker, differential clearance, or drug metabolism in the liver. As an example of the intent of
621
targeted delivery, the release of MMAE from brentuximab vedotin was demonstrated in vitro (Okeley
622
et al, 2010) and in mouse xenograft models. ADC modifications that occur in plasma were
623
demonstrated using native mass spectrometry of affinity-purified ADCs, showing that loss of drug-
624
linker as well as intact clearance of highly loaded ADCs contribute to the change in drug-load
625
distribution over time in vivo. Analyzing the samples under denaturing conditions allowed additional
626
modifications such as maleimide hydrolysis in the linker and cysteinylation of antibody cysteines to
627
be observed (Hengel et al, 2014). New drug-linker designs can lead to ADCs where loss of drug-
628
linker and differential clearance in plasma can be substantially reduced (Lyon et al, 2014). Finally,
629
MMAE released from brentuximab vedotin is excreted largely unchanged in feces, although several
630
metabolites due to demethylation, dehydrogenation, amide hydrolysis, and oxidation were observed
631
(Han et al, 2013).
632
Session 6: Lack of cross-reactive species – path forward to support FIH and clinical
633
development
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ACCEPTED MANUSCRIPT 634
The sixth and final main session was chaired by Rob Caldwell (AbbVie) and Flavio Crameri
635
(Roche).
636
justification as indicated by ICH S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived
637
Pharmaceuticals. The guidance indicates that species selection should be based upon a weight-of-
638
evidence utilizing in vitro and/or in vivo biologic activity of biologic agent in toxicology species, and
639
comparable binding of biologic to human and preclinical species tissues based upon
640
immunohistochemical staining of tissue panels (e.g. tissue cross-reactivity experiments). The totality
641
of these datasets can guide selection of a rodent and non-rodent species. Given the unique selectivity
642
of biologic agents, the guidance acknowledges that this weight of evident approach may also result in
643
lack of relevancy for routine toxicology species. Toxicology studies in non-relevant species was
644
considered misleading and was discouraged. Alternative non-traditional toxicology test strategies
645
could employ use of homologous species-specific proteins as biologic surrogate, species-specific
646
surrogate monoclonal antibody, use of human clinical candidate in transgenic species expressing the
647
human target, or evaluation of preclinical species exhibiting the intended pharmacology (i.e. knockout
648
mice for mAb sequestering soluble target). All of these strategies would again be justified using
649
weight-of-evidence approach following generation of relevant in vitro and vivo datasets. Scientific
650
advances provide additional tools to justify relevant as well as irrelevant species for safety testing
651
including greater understanding of active (i.e. neonatal Fc receptors) placental transfer of novel
652
biologic therapeutic, enhanced understanding of embryology across human and preclinical species,
653
and more sensitive analytical techniques to justify relevant pharmacology and tissue distribution
654
across human and preclinical species. Each of these alternative models have scientific advantages
655
and/or disadvantages including pharmacologic relevance to human biologic, reference range datasets
656
to evaluate the toxicologic significance of observed effects, and potential for neutralizing anti-drug
657
antibodies which may preclude use of the preferred model/species. Finally, alternative strategies also
658
have resource needs which impact the timing and efficiency of the selected toxicology model
659
including cost of unique animal colony maintenance and generation of reference range datasets, and
660
generation of regulatory compliance for surrogate proteins. The session provided cases studies of
661
species justification for novel biologic therapeutics.
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Rob Caldwell introduced the session by providing an overview of species selection
Page 25 of 46
ACCEPTED MANUSCRIPT Lolke de Haan (MedImmune) presented 2 case studies involving nonclinical safety testing of
663
human-selective monoclonal antibodies (mAbs) in transgenic mouse models. The first case study
664
described the challenges encountered in developing a transgenic mouse model for safety testing of
665
MEDI2843, a fully human monoclonal antibody directed against Toll-like receptor 4 (TLR4), the
666
innate immune receptor for bacterial lipopolysaccharide (LPS). MEDI2843 was isolated following
667
immunisation of a transgenic mouse strain expressing human Ig domains with human TLR4/MD-2
668
protein and/or TLR4/MD-2-expressing Human Endothelial Kidney (HEK) cells. None of the lead
669
panel of antibodies bound to rodent TLR4, and despite the fact that a number of lead antibodies bound
670
to cynomolgus monkey TLR4, none of these antibodies showed functional activity in cell-based
671
assays. By contrast, MEDI2843 was highly human selective and bound and inhibited functional
672
activity of human TLR4 in cell based assays very effectively.
673
pharmacologically relevant species for safety studies, attempts were made to generate human TLR4
674
transgenic mice. The approach followed involved deletion of the mouse TLR4 gene and insertion of a
675
mouse/human chimeric version of the TLR4 gene, comprised of gene fragments encoding the mouse
676
transmembrane and cytoplasmic TLR4 domains and the human TLR4 external domain. When this
677
gene construct was expressed recombinantly in a cell line, the resultant protein was shown to be fully
678
functional and mediate LPS signalling, with binding of MEDI2843 ablating LPS signalling. Given the
679
importance of MD-2 in LPS signalling, a second transgenic mouse was generated in which the mouse
680
MD-2 gene was replaced by the human MD-2 gene. A combined chimeric mouse/human TLR4 and
681
human MD-2 transgenic mouse strain could then be obtained by crossing these 2 transgenic strains.
682
Upon generation of the human/mouse chimeric TLR4 and combined human/mouse chimeric
683
TLR4/human MD-2 transgenic mice, bone marrow-derived macrophages were, however, shown to be
684
hyporesponsive to LPS. Furthermore, both transgenic mouse strains were hyporesponsive to LPS
685
challenge in vivo. Subsequent analyses showed that these defects were related to negligible
686
expression of both of the transgenic gene constructs. Hence, the transgenic animal approach failed to
687
produce a pharmacologically relevant transgenic mouse species that was suitable for pharmacology or
688
safety studies. In the second case study by Lolke de Haan, the development of a human C-X-C
689
chemokine receptor 2 (CXCR2) transgenic mouse model for safety testing of Hy29, a fully human
Given the apparent lack of
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ACCEPTED MANUSCRIPT monoclonal antibody directed against CXCR2 was presented. CXCR2 is a major chemotactic receptor
691
expressed on neutrophils with multiple chemokine ligands, including interleukin 8 (IL-8). Hy29 was
692
isolated after immunisation of a transgenic mouse strain expressing human Ig domains with human
693
CXCR2 overexpressing cells. Hy29 binds with high affinity to human CXCR2 and inhibits its
694
function in cell-based assays, whilst no or low affinity binding to rodent or cynomolgus monkey
695
CXCR2 was observed, and Hy29 did not inhibit rodent or cynomolgus monkey receptor function in
696
cell-based assays. In order to assess the nonclinical pharmacological activity and safety of Hy29, a
697
human CXCR2 transgenic mouse was generated in which the mouse CXCR2 gene was deleted or
698
disrupted and the human CXCR2 gene was inserted. Importantly, both in humans and mice CXCR2
699
has multiple ligands, not all of which are overlapping exactly with respect to function, and with some
700
ligands not having rodent counterparts in humans or vice versa.
701
precedence for the functionality of human CXCR2 transgenic mice, which provided additional
702
confidence in the approach (Mihara et al., 2005). Human CXCR2 transgenic mice were generated
703
successfully and functionality of the transgene was demonstrated in an intranasal LPS challenge
704
study, in which human CXCR2 transgenic mice showed similar lung neutrophil accumulation to wild
705
type mice 24 hours post challenge.
706
administration of Hy29 prior to LPS challenge in human CXCR2 transgenic mice, whilst the lung
707
neutrophil response to intranasal LPS was unchanged in wild type mice that received Hy29. The
708
human CXCR2 transgenic mouse model was then employed in a dose-range finding toxicity study, in
709
which animals received 3 doses of 4, 40 or 100 mg/kg Hy29 over a period of 14 days. In this study,
710
treatment with Hy29 was associated with dose-dependent reductions in circulating neutrophils as well
711
as dose-dependent increases in circulating mouse CXCR2 ligands, consistent with the anticipated
712
pharmacodynamic effects of Hy29.
713
anaphylactic reactions in individual animals following the second dose at 4 and 40 mg/kg Hy29, and
714
the third dose of 100 mg/kg Hy29. These effects correlated with a loss of exposure to Hy29 and
715
consequent pharmacodynamics effects, indicating that these reactions represented anti-drug antibody
716
(ADA)-mediated type III hypersensitivity reactions. Therefore, whilst the human CXCR2 mice
717
appeared fully functional and pharmacologically relevant, the immunogenicity of a fully human mAb
However, there is literature
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Moreover, this response could be ablated by intravenous
However, treatment with Hy29 was also associated with
Page 27 of 46
ACCEPTED MANUSCRIPT to mice prevented repeat dosing beyond 10 days, limiting the utility of the transgenic mice as a
719
species for toxicity studies. Together, these 2 case studies demonstrated that whilst transgenic mice
720
represent an option for assessing nonclinical safety of mAbs, their generation and utility for repeat-
721
dose nonclinical safety studies should be carefully considered prior to selecting transgenic animals as
722
a potential way forward for nonclinical safety testing.
723
Lise Loberg (AbbVie) presented a case study on a mAb for a disease-specific target with no cross-
724
reactivity to normal animal species commonly used in toxicology studies, focusing on study design
725
considerations. Selection of the specific transgenic (Tg) model was based upon several factors
726
including commercial availability, understanding of the pharmacology of mAb X in the particular Tg
727
model, and tissue expression of the transgene (i.e., expression primarily in CNS). Another
728
consideration was the optimal age of animals at the start of the toxicology study, whereby the
729
transgene was expressed at sufficiently high levels for mAb X to bind target, but before the
730
degenerative nature of the disease affected survivability or other toxicity endpoints. Target protein
731
expression was variable in animals at 2 and 4 months of age, was consistent with age-dependent
732
increases from 6 to 10 months of age, and reached a plateau at 10 and 13 months of age. mAb X
733
binding to target was first detectable at 6 months and considerably increased with age. Therefore, 6
734
months was selected as the optimal starting age for toxicology studies.
735
Pharmacology studies administered mAb X via IP administration, and were suggestive of anti-drug
736
antibody (ADA) development. To better understand potential for ADA formation, a pharmacokinetics
737
(PK) study was conducted in 6-month-old Tg mice, in which 6 mice/sex/group were dosed on Days 1
738
and 8 at 60 mg/kg IV or 100 mg/kg SC injection. There were no sex differences in PK parameters,
739
there was moderate SC bioavailability (approximately 60%), half-life was approximately 3 days, and
740
there were no indications for significant ADA development. Serum concentrations of mAb X were
741
maintained for up to one week after dosing. In addition, the PK study demonstrated feasibility for
742
sparse sampling to minimize animal numbers.
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ACCEPTED MANUSCRIPT An important consideration in the decision to evaluate potential toxicity in animal models of disease is
744
the lack of historical background knowledge of the model. This may be especially true for a Tg
745
animal model in which the transgene and its associated protein may be expressed very differently
746
from the normal protein and from the human disease of interest. For example, the animal model may
747
have alterations in behavior, clinical pathology parameters, or tissue histology. With that in mind,
748
toxicology studies in animal models are best conducted as hazard identification studies, with focus on
749
particular tissues or organ systems, rather than a comprehensive toxicological evaluation. In this case,
750
however, a full toxicological evaluation was intended and therefore, preliminary characterization of
751
background histopathological findings was conducted. To reduce and refine animal usage, tissues
752
were collected from Tg mice in a pharmacology study in which Tg mice were dosed with mAb X, one
753
of three alternate antibodies, a positive control or a negative control. Mice received 0.5 mg antibody
754
per week via IP administration for up to 18 weeks, starting at 6 months of age. Selected tissues (heart,
755
lung, kidney, liver, GI tract, and hindlimb joint and muscle) were evaluated from available animals in
756
the negative control group (N = 8), the mAb X group (N = 10), and two of the alternate antibody
757
groups (N = 7 and N = 11). There were no statistical differences in survival or cause of death (via
758
neoplasia or other causes), no changes in body weight, and no differences in types or incidence of
759
neoplasia between different experimental groups. Serum concentrations of mAb X were decreased by
760
6 weeks, suggestive of ADA development. Histopathological changes in kidney and degenerative
761
joint disease were common and attributable to aging, whereas skeletal muscle degeneration and
762
regeneration was severe in some animals and may have been a background change in the Tg model.
763
The preliminary PK and histopathologic studies informed study design for a GLP-compliant repeated
764
dose toxicology study. Tg mice were administered mAb X at 0 (vehicle control), 60 and 200
765
mg/kg/week IV (low and high doses), and 200 mg/kg/week SC (mid dose) for 4 weeks. Animal
766
numbers were increased to 18/sex/group to account for expected mortality up to 10%. ADA
767
development remained an unknown factor; therefore, the GLP study included two additional groups
768
dosed at 0 and 200 mg/kg/week IV for 13 weeks to evaluate PK and ADA, to inform feasibility for a
769
study longer than 4 weeks. In the 4- and 13-week GLP study, there was anticipated mortality (15%)
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ACCEPTED MANUSCRIPT during the study but also unanticipated mortality on the first day of dosing. The unanticipated deaths
771
were attributed to technical difficulty with IV injections into tail veins of dark-colored mice.
772
(Toxicology studies are often conducted in CD-1 mice, which are light-colored and tail veins are
773
easily observed on pink tails.) There was no relationship to dose and no clinical signs or
774
histopathologic changes suggestive of test article-related death; therefore the deaths on study were
775
attributed to procedure, aging and/or disease state. The mAb X half-life was < 2 days (IV), and 62%
776
of test article-dosed animals had detectable ADA. Only 1 (of 15) animals had detectable mAb X
777
concentrations after the 13th weekly dose of 200 mg/kg, confirming that a study longer than 4 weeks
778
would not be feasible. Histopathologic evaluation revealed anticipated effects (chronic nephropathy,
779
skeletal muscle degeneration/inflammation) that were common in control and test article-treated
780
animals and therefore considered age- and Tg-related but not mAb X-related.
781
In conclusion, the preliminary studies conducted to evaluate PK parameters after different routes of
782
administration and histopathological changes in the Tg mouse model of interest were instrumental to
783
the design of a 4-week GLP repeated-dose toxicology study and to the interpretation of study results.
784
Furthermore, the addition of dose groups administered control or mAb X for 13 weeks confirmed
785
development of ADA and associated decreased test article concentration, thereby providing evidence
786
that a study longer than 4 weeks would not be feasible.
787
Flavio Crameri (Roche) presented a combined surrogate and in vitro safety approach in support of
788
first-in-human dosing for CEA-IL2v (INN: cergutuzumab amunaleukin), a Roche proprietary tumor
789
antigen targeted immunocytokine currently in phase 1 clinical trials. CEA-IL2v is composed of a
790
monomeric human IL-2 with mutations (IL2v) to abolish binding to the high affinity IL-2 receptor
791
(IL-2Rαβγ). IL2v is fused to the c-terminal end of a bivalent anti-carcinoembryonic antigen (CEA)
792
human IgG1 with silenced effector functions. The anti-CEA component of the clinical candidate lacks
793
cross-reactivity with any toxicology species, while the IL2v component has been shown to be cross-
794
reactive in the cynomolgus monkey and the mouse. Given the lack of a fully cross-reactive species for
795
safety assessment it was decided to start the development of a cynomolgus monkey reactive anti
796
CEA-IL2v (cyCEA-IL2v) and at the same time to discuss and obtain feedback from a European health
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ACCEPTED MANUSCRIPT authority (HA) regarding the proposed preclinical safety strategy. The HA considered a surrogate
798
approach with cyCEA-IL2v appropriate for qualitative safety assessment once functional equivalence
799
of cyCEA-IL2v to CEA-IL2v had been shown. The surrogate approach however was to be combined
800
with an extensive quantitative in vitro safety assessment using CEA-IL2v in human cell based assays
801
to derive a minimum anticipated biological effect level (MABEL). The HA further commented that
802
CEA-IL2v may not be considered high risk based on the extensive previous clinical experience with
803
anti-CEA mAbs for imaging and with rhuIL-2 (Proleukin®) as well as the tumor targeted approach to
804
be used. In addition, the HA acknowledged that a starting dose based on a MABEL may be
805
unacceptably low and indicated that it was open to accept a combination of MABEL with other
806
approaches to determine a safe starting dose for CEA-IL2v. Based on the HA feedback received,
807
development of the surrogate cyCEA-IL2v was continued. Binding and functional equivalence of cy
808
CEA-IL2v when compared to CEA-IL2v was shown in several comparative in vitro assays; i.e.
809
affinity to recombinant CEA and IL-2Rβγ and IL2v-mediated functional activity using human and
810
cyno cells (cell signaling, cell activation and proliferation as well as cytokine release). The surrogate
811
cyCEA-IL2v was then tested in pilot and GLP studies in the cynomolgus monkey. The safety profile
812
obtained with cyCEA-IL2v in the cynomolgus monkey was consistent with the known safety profile
813
of wild type IL-2 (Proleukin®) thus validating the surrogate-IL2v for qualitative safety assessment.
814
No vascular leak syndrome, a major toxicity of wild type IL-2, was seen and there was no evidence
815
for any potential CEA-targeting related toxicity in tissues known to express CEA. The safe starting
816
dose for the FiH study was then selected based on a combination of the MABEL (IC20) obtained with
817
CEA-IL2v and published clinical data from competitor IL-2 immunocytokines for which safety and
818
exposure data were available. The resulting starting dose used for fist-in-human was approximately 3-
819
fold higher than the MABEL and was shown to be safe in the ongoing phase 1 clinical trial. This
820
approach helped to balance safety and number of patients without potential clinical benefit.
821
The second case study from Roche discussed a FiH that was supported exclusively by in vitro safety
822
data and a safe starting dose selection based on a MABEL only.
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ACCEPTED MANUSCRIPT Katharine Bray-French (Roche) presented a case study for CEA TCB which is a novel, Roche
824
proprietary T cell bispecific (TCB) antibody targeting carcinoembryonic antigen (CEA) expressed on
825
tumor cells and CD3 epsilon chain (CD3e) present on T cells that is currently in Phase 1 clinical trials
826
(NCT02324257) for the treatment of CEA positive solid tumors. Because the human CEA (hCEA)
827
binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents,
828
alternative nonclinical safety evaluation approaches were considered. These included the development
829
of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for
830
evaluation in cynomolgus monkey and the development of double transgenic mice, expressing hCEA
831
and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies.
832
However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the above
833
approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA
834
TCB. Therefore, an alternative approach, a MABEL based on an in vitro tumor lysis assay was used
835
to determine a safe starting dose for the first-in-human clinical study.
836
Benno Rattel (Amgen) presented three different nonclinical testing strategies for bispecific T-cell
837
engagers, commonly referred to as BiTE® antibody constructs. These molecules are comprised of two
838
different flexibly-linked single-chain antibodies, one directed against a tumor antigen and the other
839
targeting CD3. BITE® antibody constructs are designed to link transiently tumor cells with resting
840
polyclonal T-cells for induction of a surface target antigen-dependent re-directed lysis of tumor cells.
841
First-generation BiTE® antibodies cross-reacted only with respective antigens from chimpanzees
842
therefore, to facilitate in vivo safety testing, surrogate BiTE® antibodies were generated that are
843
cross-reactive with murine antigens. Firstly, the nonclinical safety package of Blincyto®, a CD19-
844
targeting BiTE® antibody construct, was presented (Nagorsen et. al, 2012; Topp et. al, 2015). It
845
included up to 3-month repeat-dose toxicity studies and an embryofetal toxicity study with the mouse
846
surrogate, which mimicked the parent compound`s pharmacological characteristics and successfully
847
supported the molecule`s recent approval both in the US and the EU (FDA, 2014; EMA, 2015) The
848
second case study described the determination of the clinical starting dose for a CEA-targeting BiTE®
849
antibody construct. For this tumor target, a surrogate molecule could not be developed that was
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ACCEPTED MANUSCRIPT comparable to the parent molecule, since both the mouse and the cynomolgus surrogates evaluated
851
caused unspecific T cell activation. Therefore, the FIH dose was based on in vitro MABEL data, only.
852
Finally, the current nonclinical safety assessment paradigm for defining a safe clinical starting dose
853
was presented for the new generation of BiTE® antibody constructs that cross-react with cynomolgus
854
monkeys (Friedrich et al, 2012).
855
“Hot Topic” - Breakout Sessions
856
Breakout Session Question 1:
857
advantages over combinations ? For this breakout session Lolke de Haan (MedImmune) first
858
gave a brief presentation outlining the potential challenges in the nonclinical and clinical development
859
of bispecific antibodies and antibody combinations. Subsequent discussions focused on whether or
860
not bispecifics would provide drug molecules with additional, mechanistically differential, mode of
861
action. Or, alternatively, if bispecifics simply represent a more convenient way for the delivery of 2
862
pharmacodynamically active modalities, and an opportunity for replacing 2 separate injections with
863
monoclonal antibodies with 1. The consensus appeared to be that convenience was the main driver for
864
developing bispecifics. It was recognised that the ratio of the 2 bioactive components in a bispecific is
865
fixed, and that this could be a potential disadvantage. On the other hand, for combinations flexibility
866
could become a disadvantage, as with the number of variables available (antibody ratio and dosing
867
frequency and level) the number of combinations that could be tested are endless, and this could
868
complicate or hamper clinical development. With respect to targets suitable for bispecifics, there was
869
consensus that soluble targets are preferable, however, there was no expectation that both arms of the
870
bispecifics would engage 2 different soluble targets simultaneously. For membrane-bound targets,
871
binding 2 targets per bispecific molecule was generally not deemed likely, however, it would still be
872
possible to modulate 2 membrane-bound targets through monovalent interactions. Finally, the
873
bioanalytical challenges associated with bispecifics in the nonclinical and clinical setting were
874
discussed. For bispecifics, it was agreed that anti-idiotype antibodies against each bispecific arm to
875
determine the amount of free bispecific would be required. In addition, assays demonstrating stability
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of the bispecific, e.g. capture with an anti-idiotype combined with an anti-Fc detection, were
877
considered desirable.
878
Breakout Session Question 2: “Is the paradigm “SC more immunogenic than IV” correct ?”
879
chaired by Benno Rattel (Amgen). Biotherapeutics have to be administered via a parenteral route,
880
because approaches to oral administration have failed so far.
881
administration, SC administration has the advantage of improved convenience and patient compliance
882
as well as prolonged half-life (Richter and Jacobsen , 2014). However, the SC route is perceived to be
883
problematic due to a greater potential for undesired immunogenicity (Braun et al, 1997; Ponce et al,
884
2009; FDA 2014). As there is generally a poor concordance between immunogenicity in animals and
885
humans, and, with few exceptions, human biologics are generally more immunogenic in animals than
886
in humans (Bugelski and Treacy, 2004; Ponce et al, 2009), ideally the validity of the above paradigm
887
should be verified with actual clinical experience of head-to-head comparisons of SC and IV
888
administrations of biotherapeutics.
889
There are only few published clinical examples directly comparing the two administration routes:
890
Rituximab (Bittner et al, 2014; Davies et al, 2014), Trastuzumab (Jakisch et al, 2015), Tocilizumab
891
(Ogata et al, 2014; Burmester et al, 2014), Belimumab (Shida et al, 2014), ATR-107 (Hua et al, 2014)
892
and Abatacept (Schiff, 2013). These clinical data as well as unpublished internal company data were
893
discussed, and the group agreed that these data do not support the above paradigm. The group argued
894
that a number of patient- and product-specific factors such as origin, post-translational modification,
895
aggregates, degradation products, impurities, formulation, container closure and, especially,
896
immunomodulatory properties (Parenky et al, 2014) are at least equally important for an unwanted
897
immune response as the administration route. Therefore, the group concluded that the generalization
898
that the SC administration bears a higher risk of immunogenicity compared to the IV route cannot be
899
universally upheld.
900
Breakout Session Question 3: For 3Rs considerations of NHP use “Why do we need a 6 month
901
toxicology study for a mAb when we already have a 13 week toxicology study?” chaired by Katja
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Page 34 of 46
ACCEPTED MANUSCRIPT Hempel (AbbVie) and Peter Ulrich (Novartis). Before the meeting a survey was send out by Peter
903
Ulrich to BioSafe members with the goal to collect the experience of the pharmaceutical industry with
904
toxicity studies supporting clinical development of monoclonal antibodies in order to evaluate
905
repeated dose toxicity strategy. This survey was limited to results generated with monoclonal
906
antibodies given via the intravenous or subcutaneous route at similar dose levels between the toxicity
907
studies and selected according to toxicology standard practice. Pure oncology mAb were not included
908
in this survey, since 26 week studies are not required for those mAbs. The findings asked for in the
909
survey were divided into pharmacodynamic (PD), target organ toxicity, and safety pharmacology
910
findings leading to a NOAEL.
911
Results: 26 case examples were provided for the survey by five BioSafe member companies. In none
912
of the 26 case examples were differences seen in the toxicological profile between 3 and 6 months. In
913
addition, one company informed verbally that they had 4 mAbs with again no difference in
914
toxicological profile. During the plenary session two additional interesting examples were mentioned
915
where toxicological effects were only seen in studies of longer duration. Taking into consideration the
916
results of the long study, and reevaluating the short term study, there was a hint of this toxicological
917
finding. With regard to the challenge as to when a 3 month stand-alone study might be sufficient,
918
there was agreement in panel that this needs to be a case by case decision also taking the target into
919
account. In addition, the use of sexually mature animals was recommended. A working group will be
920
formed to distribute the survey to companies that have not yet received the survey to include as many
921
case examples as possible, including especially the ones with a different outcome other than
922
mentioned in the plenum discussion. Additionally, it was suggested to collect data from marketed
923
products to update the list from former publications (Clarke et al, 2008). Goal is to publish the
924
current pharmaceutical expertise to leverage experience for mAbs with potential future extension to
925
more complex ADCs or DVDs, if possible.
926
Breakout Session Question 4:
927
Preclinically and does it Translate Clinically” chaired by Rajni Fagg (GSK). The breakout
928
session opened with an example from the literature (Heyden et. al, 2014) in which an investigative
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“How Do we Investigate Immune Complex Formation
Page 35 of 46
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study was conducted to characterise mAb induced immune complex (IC) formation in cynomolgus
930
monkeys. This preclinical investigative study was also designed to identify potential biomarkers that
931
could be indicative of immune complex formation and determine if the induced immune complex
932
formation was reversible. The discussions in the breakout session concluded: 1. Most likely, as antigen/mAb therapeutic ratios approach molar equivalence, the ICs are larger
934
and when clearance mechanisms are blocked or saturated, large ICs can deposit in tissue,
935
activate complement, and cause tissue damage.
937
2. Other factors that influence IC formation include high
antigen
and
antibody
SC
936
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933
concentration/density as well as antigen and mAb charge.
3. Although in the published example high anti-mAb antibody response in the monkeys
939
preceded the clinical pathology findings of thrombocytopenia, complement activation and
940
neutrophilia by several weeks, this was not considered to be always the case.
942
4. No clear correlation of preclinical findings of mAb therapeutic induced IC formation has been identified in the clinical setting.
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938
The fifth and final “hot topic” during the breakout session was a group discussion of “Adversity in
944
Nonclinical Safety Studies: STP and ESTP opinion, your opinion needed !”, chaired by John
945
Burkhardt (AbbVie) and Paul Germann (AbbVie). As an introduction John presented the STP (see
946
Figure 1, Kerlin et al, 2016; flow chart was developed for NCEs/small molecules but concept is also
947
relevant for biologics), and Paul the ESTP point of view (see Figure 2, Palazzi et al, 2015). Finally
948
several case studies from different companies were presented followed by a lively discussion on
949
whether a specific finding will be assessed as adverse and will therefore impact the NOAEL setting.
950
In essence, the tiered approach in histopathological adversity assessments, as depicted in Figure 2,
951
should guide younger colleagues towards a correct assessment of adversity. What still remains an
952
area of concern is the clear disconnect between the humoral reaction against biological products like
953
ADA formation and the histopathological findings which do not correlate in most of the cases.
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954
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ACCEPTED MANUSCRIPT Figure 1: Schematic diagram delineating the hierarchy by which “adverse” test-article-related effects
956
are communicated (see Figure 2 from Kerlin et al, 2016)
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ACCEPTED MANUSCRIPT Figure 2: Tiered approach for evaluating adversity (see figure 1 from Palazzi et al, 2015)
959
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958
Conclusions
961
The opportunities for the application of biotherapeutics for the treatment or prevention of human
962
diseases continue to grow, and new molecular formats, including bi- or multi-specific molecules,
963
antibody fragments, and ADCs, are likely to reach the market in the near future.
964
complexity and unique properties of these novel modalities, standardized approaches for nonclinical
965
safety testing are becoming increasingly obsolete, and each molecule needs to be evaluated on case-
966
by-case basis, tailoring the nonclinical safety program and strategy to the molecule.
967
This meeting brought experts together from the nonclinical safety (toxicology, pathology, safety
968
pharmacology), pharmacokinetics and bioanalytical field to exchange knowledge and experience. In
969
addition to the presentations, podium discussions and breakout sessions, the meeting offered ample
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Page 38 of 46
Given the
ACCEPTED MANUSCRIPT 970
opportunity for participants to share ideas, development strategies and case studies. This is becoming
971
increasingly important for the biopharmaceutical industry, as this often represent the only way in
972
which to share data on failed development programs, studies and strategies.
973
exchange of knowledge promotes the optimization of future biotherapeutics non-clinical development
974
programs, which will not only contribute to a responsible and justified use of animals, but hopefully
975
also to improved human risk assessment of innovative biotherapeutics.
976
Conflict of interest
977
Guenter Blaich, Rob Caldwell, and Edit Tarcsa are employees of AbbVie. Andreas Baumann is an
978
employee of Bayer Pharma AG; Sven Kronenberg, Wolfgang Richter and Flavio Crameri are
979
employees of Roche. Lolke de Haan is an employee of MedImmune; Peter Ulrich is an employee of
980
Novartis Pharma; Jay Tibbitts is an employee of UCB Celltech and Simon Chivers is an employee of
981
ADC Therapeutics. AbbVie and affiliates participated in the preparation of the workshop report,
982
interpretation of data, review, and approval of the manuscript.
983
Acknowledgements
984
The authors would like to thank all speakers for their contributions and members of the BioSafe
985
Leadership committee for valuable comments during the review of this manuscript.
986
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