Toxicologic Pathology, XX: 1-25, 2015 Copyright # 2015 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623315591171
PEGylated Biopharmaceuticals: Current Experience and Considerations for Nonclinical Development INGE A. IVENS1, WILLIAM ACHANZAR2, ANDREAS BAUMANN3, ANNAMARIA BRA¨NDLI-BAIOCCO4, JOY CAVAGNARO5, MAGGIE DEMPSTER6, B. OLYMPE DEPELCHIN7, ARMANDO R. IRIZARRY ROVIRA8, LAURA DILL-MORTON9, JOAN H. LANE10, BIRGIT M. REIPERT11, THEODORA SALCEDO12, BECKY SCHWEIGHARDT13, LAURIE S. TSURUDA13, PETER L. TURECEK11, AND JENNIFER SIMS14 1
Bayer HealthCare, San Francisco, California, USA Bristol-Myers Squibb Co., New Brunswick, New Jersey, USA 3 Bayer Pharma AG, Berlin, Germany 4 Roche Innovation Center Basel, Basel, Switzerland 5 Access BIO, Boyce, Virginia, USA 6 GlaxoSmithKline, LLC, King of Prussia, Pennsylvania, USA 7 UCB BioPharma, Braine L’Alleud, Belgium 8 Lilly Research Laboratories, Indianapolis, Indiana, USA 9 Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA 10 Biogen, Inc., Cambridge, Massachusetts, USA 11 Baxalta Innovations GmbH, Vienna, Austria 12 Janssen R&D, LLC, Spring House, Pennsylvania, USA 13 BioMarin Pharmaceutical Inc., Novato, California, USA 14 Integrated Biologix GmbH, Basel, Switzerland 2
ABSTRACT PEGylation (the covalent binding of one or more polyethylene glycol molecule to another molecule) is a technology frequently used to improve the half-life and other pharmaceutical or pharmacological properties of proteins, peptides, and aptamers. To date, 11 PEGylated biopharmaceuticals have been approved and there is indication that many more are in nonclinical or clinical development. Adverse effects seen with those in toxicology studies are mostly related to the active part of the drug molecule and not to polyethylene glycol (PEG). In 5 of the 11 approved and 10 of the 17 PEGylated biopharmaceuticals in a 2013 industry survey presented here, cellular vacuolation is histologically observed in toxicology studies in certain organs and tissues. No other effects attributed to PEG alone have been reported. Importantly, vacuolation, which occurs mainly in phagocytes, has not been linked with changes in organ function in these toxicology studies. This article was authored through collaborative efforts of industry toxicologists/nonclinical scientists to address the nonclinical safety of large PEG molecules (>10 kilo Dalton) in PEGylated biopharmaceuticals. The impact of the PEG molecule on overall nonclinical safety assessments of PEGylated biopharmaceuticals is discussed, and toxicological information from a 2013 industry survey on PEGylated biopharmaceuticals under development is summarized. Results will contribute to the database of toxicological information publicly available for PEG and PEGylated biopharmaceuticals. Keywords:
PEG; polyethylene glycol; biopharmaceutical; PEGylation; nonclinical; toxicology; vacuolation.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The author(s) received no financial support for the research, authorship, and/or publication of this article. Address correspondence to: Inge A. Ivens, Bayer HealthCare, 455 Mission Bay Bld. South, San Francisco, CA 94158, USA; e-mail:
[email protected]. Abbreviations: BBB, blood–brain barrier; BCSF, blood-cerebrospinal fluid barrier; BIW, twice per week; BUN, blood urea nitrogen; CHMP, Committee for Medicinal Products for Human Use; CNS, central nervous system; CSF, cerebrospinal fluid; Cynomolgus, cynomolgus macaque; D, days; DRG, dorsal root ganglia; EFD study, embryo-fetal development study; EMA, European Medicinal Agency; EOD, end-of-dose; EPARs, European public assessment reports; Fab, antibody fragment; FDA, Food and Drug Administration (United States); FOB, functional observational battery; H&E, hematoxylin and eosin histological stain; HD, high dose; ICH, International Conference on Harmonization; IGF-1, insulin-like growth factor 1; IHC, immunohistochemistry; IU, international units; Iv, intravenous; kDa, kilo Dalton (molecular weight); LPS, lipopolysaccharide; MD, mid-dose; mo, month; mPEG, monomethoxy PEG (methoxy group at end of PEG chain); mPEG, methoxypoly (ethylene glycol); MW, molecular weight; NHP, nonhuman primate (genus not specified); NOAEL, no adverse effect level; nm, nanometer; NOEL, no effect level; NP, information not provided; PEG, polyethylene glycol; PEGylation, covalent binding of one or more polyethylene glycol molecules to another molecule; PK, pharmacokinetics; QOD, every other day; REMS, risk evaluation management plan; RES, reticuloendothelial system; RIA, radio immune assay; SBA, FDA’s summary basis of approval; sc, subcutaneous; SCID, severe combined immunodeficiency; TNF, tumor necrosis factor; TNF-bp, tumor necrosis factor binding protein; wk(s), week(s); mmol, micromol. 1
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IVENS ET AL.
INTRODUCTION Since the 1982 introduction of the first recombinant insulin, biotechnologically derived drugs (biopharmaceuticals) have become essential in the treatment of serious life-threatening and chronic diseases. These drugs are often recombinant forms of endogenous molecules. Suitable methods to modify these biotechnologically derived molecules, in particular their half-life are limited, since some properties of peptides or proteins may be changed and result in either loss of efficacy or increased immunogenicity. To date, PEGylation (the covalent binding of one or more polyethylene glycol [PEG] molecule to another molecule) has been the most successful strategy to chemically modify these biopharmaceuticals. The addition of PEG can extend their half-life, increase stability, and in some cases, ameliorate the immunogenicity of highly immunogenic proteins such as enzymes. There are currently 11 approved PEGylated biopharmaceuticals in the United States and Europe (10 peptides/proteins and 1 aptamer as of 2015) and many more are in nonclinical and clinical development. PEG has a simple repetitive structure, is chemically inert, and has low toxicity (Webster, Didier et al. 2007; Schellekens, Hennink, and Brinks 2013). Findings including adverse effects observed in nonclinical studies of approved PEGylated biopharmaceuticals are usually related to the intended pharmacological actions of the active drug and not to the PEG moiety (Webster, Didier et al. 2007; Kang, Deluca, and Lee 2009; Jevsevar, Kunstelj, and Porekar 2010; Ivens et al. 2013). However, PEG-related histologic changes, characterized as cellular vacuolation in certain tissues and cell types, have been observed in nonclinical toxicology studies for approximately half of the approved PEGylated drugs (Tables 1 and 2). These changes were not associated with pathologic effects such as tissue degeneration, necrosis, and cellular distortion or changes in study end points including hematology, clinical chemistry, urinalysis, or organ weight. Assessment and characterization of tissue vacuolation continues to be an important component of the toxicology evaluation of PEGylated biopharmaceuticals. Many factors may impact the occurrence and degree of cellular vacuolation in toxicology studies of PEGylated biopharmaceuticals (Yamaoka, Tabata, and Ikada 1994, 1995; Webster, Didier et al. 2007) including:
Pharmacological activity of the protein, Distribution of drug target, Drug dose, Dose frequency and duration of treatment, Distribution of the drug, Amount of nonspecific uptake versus specific drug receptor mediated uptake, Potential immunogenicity, Overall molecular weight of the drug, The molecular weight of PEG itself, and Types of clearance/removal mechanisms for drug or PEG.
Location, severity, and reversibility of vacuolation, as well as the presence or absence of any secondary morphologic changes
TOXICOLOGIC PATHOLOGY
such as tissue degeneration, cellular distortion, and inflammation or necrosis, should be carefully considered in risk assessment along with the dose/dose frequency at which such changes were observed in relation to the human therapeutic dose and dosing frequency. While common risk assessment strategies can be applied to effects related to the pharmacologically active drug component(s) of the molecule, determination of the potential adversity (or lack thereof) of cellular vacuolation attributed to PEG and extrapolation of potential risks for the patient poses a challenge since much about the mechanism and functional consequences of tissue vacuolation remains unknown. This article summarizes experience with 11 approved PEGylated biopharmaceuticals and 17 PEGylated biopharmaceuticals in development and the impact of cellular vacuolation on development and product approval. Another emerging area of concern with PEGylated biopharmaceuticals is the potential for generation of antibodies to PEG, the potential for anti-PEG antibodies to cross-react with other PEGylated biopharmaceuticals, and the existence of preexisting anti-PEG antibodies of unknown origin in humans (Schellekens, Hennink, and Brinks 2013; Verhoef et al. 2014). The following points will be addressed specifically:
Regulatory guidance and health authority recommendations to support clinical development, PEG disposition/metabolism, and excretion; Immunogenicity of PEG; Brief review of publicly available toxicology information for 11 approved PEGylated biopharmaceuticals; Toxicological relevance of cellular vacuolation observed in nonclinical toxicology studies and their potential risk for the patient population; 2013 BioSafe survey of pharmaceutical industry of 17 products from 10 companies before approval, including specific nonclinical case studies; and Considerations for nonclinical safety evaluation of PEGylated biopharmaceuticals.
REGULATORY GUIDANCE
FOR
PEGYLATED BIOPHARMACEUTICALS
The current guidance for nonclinical safety evaluation of biopharmaceuticals is ICHS6 R1 (International Conference on Harmonization [ICH] S6 R1, 2011). It also applies to PEGylated biopharmaceuticals but no specific guidance regarding the assessment of PEGylated biopharmaceuticals or observations related to systemic PEG exposure, such as cellular vacuolation, is included. Two additional guidance documents, which specifically address PEGylated drug products, have been published recently: Food and Drug Administration (FDA) Guidance for Industry: Immunogenicity Assessment of Therapeutic Protein Products (Part V.B.4 Glycosylation/ PEGylation [FDA 2014]) and the Committee for Medicinal Products for Human Use (CHMP) Safety Working Party’s response to the European medicinal agency (EMA) Pediatric
Vol. XX, No. X, 2015
NON-CLINICAL DEVELOPMENT OF PEGYLATED BIOPHARMACEUTICALS
Committee regarding the Use of PEGylated drug products in the pediatric population intended for long-term treatment. The CHMP response acknowledged that, while vacuolation in cells of the reticuloendothelial system (RES) may represent the body’s normal response to remove a foreign body, there was a concern for other cells and tissues such as the renal tubular epithelium or the choroid plexus epithelium (ependymal cells). The CHMP summarized that vacuolation in these cells was observed following certain conditions including PEG molecular weight 30 kilo dalton (kDa), toxicology study duration of at least 4 weeks, and a cumulative PEG dose of 0.4 mmol/kg/month. It was concluded: ‘‘until more data are available, the risk for ependymal cell vacuolation needs to be addressed before conducting longer term clinical trials in the paediatric population.’’ However, neither the definition of what constitutes an adverse finding nor the magnitude of the safety margin was defined in the CHMP response. It is also not known whether demonstrating evidence of partial or full reversibility of vacuolation may be sufficient for progression of development of the PEGylated drug candidate. Currently, there is no clear understanding if vacuoles that occur with prolonged exposure to PEG may lead to any functional consequences. However, the body of evidence available for approved PEGylated biopharmaceuticals has not identified any clinically reported functional consequences for compounds where vacuolation was observed in toxicology studies. This is reflected in the lack of significant impact on the risk evaluation management (REMS) plans following PEGylated product approvals. PEG DISPOSITION, METABOLISM,
AND
EXCRETION
The aspects of biodistribution, metabolism, and excretion of PEG, relevant for nonclinical toxicology assessment of PEGylated biopharmaceuticals, are briefly summarized below. More detailed reviews are available in the literature (Yamaoka, Tabata, and Ikada 1994, 1995; Webster, Didier et al. 2007; Webster, Elliott et al. 2009; Cheng et al. 2012; Ivens et al. 2013; Longley et al. 2013; Rudmann et al. 2013; Baumann et al. 2014). A core assumption for PEGylated biopharmaceuticals that are associated with the presence of tissue vacuolation attributed to the presence of intracellular PEG is that at some point after repeated administration, a steady state will be achieved, in which the rate of tissue distribution is equilibrated with the rate of catabolism and excretion. Demonstrating this, however, may be difficult. Limited data are available on distribution, metabolism, and excretion of conjugated PEG, particularly for PEGs with molecular weights 20 kDa. One reason for this may be that the amount of PEG linked to biopharmaceutical drugs is usually relatively low (Webster, Didier et al. 2007), requiring sensitive methods to quantify free and/or conjugated PEG in tissues and body fluids. Such methods have advanced only recently (Cheng et al. 2012), although there are no published studies to the best of our knowledge of their application in PEG detection in tissue
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beyond immunohistochemistry (IHC), radioactive labeling, or ELISA methods. The bioanalytical detection of PEG (conjugated or unconjugated) is challenging because it has repetitive subunits; therefore, chromophore and radiolabeling may be confined to the terminal structure, which is more available to metabolism. For a review of PEG pharmacokinetics (PK), metabolism, and distribution, see Baumann et al. (2014). Even with the availability of recent, more sensitive analytical methods such as nuclear magnetic resonance (NMR), it is still difficult to distinguish between different PEG sizes that might result from (albeit limited) metabolism of the PEG chain (Cheng et al. 2012) or from the inherent polydispersity of the PEG products used in PEGylation. Method development for PEG localization in cellular structures like vacuoles beyond IHC may pose even greater challenges. Using 125I-labeled PEG of different molecular weights administered intravenously (iv) to mice, Yamaoka, Tabata, and Ikada (1994, 1995) concluded that a two-compartment model could describe the distribution of PEG in the body. The time dependence of tissue accumulation was based on vascular permeability, with larger PEG translocating slowly from the circulation. The half-life increased from 18 min for a 3-kDa PEG to 24 hr for a 190-kDa PEG. Liver clearance, at least in part due to Kupffer cell uptake, was enhanced at higher molecular weights. Using 14C labeling, Wang et al. (2012) reported a new labeling approach to assess tissue distribution of a PEG-40 kDa protein in mice. Their results show that the lowest amount of PEG-related radioactivity is seen in the brain relative to the other tissues (tissues evaluated in mice: blood, tumor, brain, bone marrow, heart, liver, lung, kidney, and muscle). There are few literature reports of the use of IHC to identify PEG in tissue (Ton et al. 2007; Sundy et al. 2011; Rudmann et al. 2013). Rudmann et al. showed that the molecular weight of the PEG influenced both the tissue distribution and the vacuolation observed with high iv doses of unconjugated PEG (10, 20, or 40-kDa, 100 mg/kg once daily, every other day, and twice weekly, respectively, for 3 months). With 10- and 20kDa PEG, PEG immunoreactivity was most prominent in the renal tubule epithelium and in alveolar macrophages, and hepatic Kupffer cells, while cellular vacuolation was absent. In contrast, rats given 40-kDa PEG had strong PEG immunoreactivity in splenic subcapsular red pulp macrophages, renal interstitial macrophages, and choroid plexus epithelial cells frequently associated with cellular vacuolation. These studies with PEG alone also demonstrated that PEG can be found immunohistochemically in cells without vacuolation. It is not known how far these data can be accurately extrapolated to PEGylated biopharmaceuticals or how much their distribution patterns depend on PEG dose versus drug receptor–mediated uptake and/or ligand-specific transport mechanisms. Although antiPEG antibodies can be used to evaluate specific cellular and subcellular locations of PEG in tissues by IHC and may identify a correlation between presence of PEG and morphological changes, IHC is not quantitative and cannot show the entire sequence of events in catabolism of PEGylated biopharmaceuticals.
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There are very few published studies specifically addressing potential mechanisms of cellular uptake of PEG, either alone or conjugated to other molecules (Yu et al. 2004). The predominant mode of cellular uptake (nonspecific vs. drug target mediated) and tissue distribution of PEGylated biopharmaceuticals and/or their free PEG catabolites likely depends on many factors, including dose, dose frequency and duration of treatment, the product’s pharmacological activity, distribution of relevant receptors, potential immunogenicity, overall molecular weight, PEG molecular weight, and types of clearance/ removal mechanisms for drug or PEG (Yamaoka, Tabata, and Ikada 1994, 1995; Webster, Didier et al. 2007; Webster, Elliott et al. 2009; Baumann et al. 2014; Bendele et al. 1998). The frequent presence of PEG and PEG-associated vacuoles in Kupffer cells and macrophages as reported in the BioSafe survey (Tables 1, 2 and Table S2 in the Online Appendix) suggests that mechanisms of cellular uptake unique to phagocytic cells are major determinants of tissue reactions to high molecular weight PEG. As has been pointed out by the CHMP working party’s paper (CHMP Safety Working Party of the European Medicinal Agency 2012), this is likely related to the normal removal functions of macrophages. It can be assumed that irrespective of the cellular uptake mechanisms of a biopharmaceutical due to nonspecific removal or in relation to its biological drug target or function, the protein part of the PEGylated biopharmaceutical will be catabolized into amino acids, and the PEG molecule or parts will be separated from the active drug part since there are few PEG metabolizing possibilities (Webster, Didier et al. 2007; Ivens et al. 2013; Baumann et al. 2014). Renal clearance appears to be the most common route of excretion for small and large unconjugated PEG molecules. For large PEG molecules, there is evidence that long linear PEG molecules with molecular weights greater than the glomerular filtration rate cutoff for globular proteins can still pass through the glomerulus and can be excreted by the kidney, as seen in studies in mice (Yamaoka, Tabata, and Ikada 1994, 1995; Caliceti and Veronese 2003). This shape-changing behavior is called reptation (Nakaoka et al. 1997; Caliceti and Veronese 2003). The mechanism allows PEG molecules that exceed the glomerular filtration rate cutoff for proteins still to be excreted by the kidney. PEG has been localized within the proximal convoluted tubules of the kidney (Bendele et al. 1998; Webster, Didier et al. 2007; Rudmann et al. 2013), which is consistent with renal excretion followed by reuptake via mechanisms (such as pinocytosis) that are common to other solutes that are freely filtered at the level of the glomerulus. IMMUNOGENICITY
OF
PEG
PEG has been regarded as a nonantigenic, nonimmunogenic moiety and has been used in some settings to protect proteins from the host’s immune system by reducing their immunogenicity (Dreborg and Akerblom 1990; Roberts, Bentley, and Harris 2002; Saenger et al. 2011). Historically, most PEGylated
TOXICOLOGIC PATHOLOGY
biopharmaceuticals have proven to be safe and efficacious and have not been associated with the development of anti-PEG antibodies. Recently, a number of reports have documented the development of anti-PEG antibodies following treatment with PEGylated therapeutics, which have been associated with reduced efficacy and hypersensitivity (Ganson et al. 2006; Judge et al. 2006; Armstrong et al. 2007; Hershfield et al. 2014). Some of these reports have evoked controversial discussion (Schellekens, Hennink, and Brinks 2013). It is not yet understood why some PEGylated biopharmaceuticals induce a clinically relevant anti-PEG antibody response, whereas others do not. Some important factors may involve the molecular weight of the PEG moiety, linker-type, number of attached PEG strands, and the immunogenicity of the protein part of the PEGylated biopharmaceutical. One additional variable may also be the assay format and assay sensitivity to detect antiPEG antibodies (Schellekens, Hennink, and Brinks 2013). Antibodies against PEG were first documented in animal studies following immunization with PEGylated proteins (Richter and Akerblom 1983). In these studies, PEG was found to be non-immunogenic when administered alone, but acquired immunogenic properties when conjugated to an immunogenic protein. The immunogenic potential of PEG was described as being directly related to the immunogenic nature of the protein. In these situations, PEG was considered to be a polyvalent hapten, a small molecule that can elicit an immune response only when attached to a large carrier molecule, such as a protein. Similar data were recently reported for PEGylated factor VIII (van Helden et al. 2011; Reipert et al. 2012). Two different mouse models were used to assess the immunogenicity of PEGylated factor VIII: a transgenic mouse model that expresses human factor VIII, and therefore recognizes it as self-protein, and a conventional hemophilic mouse model that recognizes human factor VIII as foreign protein. Only those mice that recognized factor VIII as a foreign protein developed anti-PEG antibodies. These data support the hapten concept for the immunogenicity of PEG. The immunological principles of anti-hapten antibody responses were first described by Mitchison (1971) and more extensively studied by Rock, Benacerraf, and Abbas (1984). An additional explanation for the development of anti-PEG antibodies could be cross-linking of B cell receptors that recognize repetitive epitopes within the PEG moiety. This type of antibody response requires multiple regularly spaced identical epitopes to allow simultaneous engagement of multiple B cell receptors with each epitope. The collective signal is sufficient to induce T cell–independent antibody responses (Mond, Lees, and Snapper 1995; Bachmann and Zinkernagel 1997). IgM is the major isotype resulting from T-cell independent antibody responses, although antibody class switching can also occur. IgM immune complexes are efficient activators of the complement system and can rapidly clear antigen from the blood stream, as well as induce hypersensitivity through buildup of the complement anaphylatoxins C3a, C4a, and C5a (Kindt, Osborne, and Goldsby 2006). T cell–independent mechanisms might also be responsible for the induction of anti-PEG antibodies observed in patients
20
20 Linear
20 Linear (mPEG) 20 (mPEG) 20 branched
30 Linear
30 Linear (mPEG)
1/1
8/1
9
4/2
6/2
5 7
PEG MW (kDa), Linearity
Company/ Product
2.4
11
25 70–350 protein þ PEG
12
19.6
5
Protein MW (kDa)
sc
sc
sc iv
Intravitreal
sc
sc
Route
Cyno: 0.5, 1.5, or 7.5 mg/kg/week for 52 wks; 12-wk recovery NOEL (for vacuoles):
