Biopharmaceuticals

C H A P T E R

25 Biopharmaceuticals Wendy Halpern1, David Hutto2 1

Genentech, Inc., South San Francisco, CA, USA, 2Eisai, Inc., Andover, MA, USA

O U T L I N E 1. Introduction 1.1. Brief History of Biopharmaceuticals 1.2. Biopharmaceuticals Currently Approved for Use 1.3. General Safety Concerns when Developing Biopharmaceuticals

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2. Biopharmaceuticals 2.1. What Makes Biopharmaceuticals Different From Other Drugs? 2.2. Classes of Biopharmaceuticals Recombinant Native Peptides and Proteins Monoclonal Antibodies (mAbs) Antibody-Like Molecules Combined Products (mAb Conjugates) Nucleic Acid-Based Therapies

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3. Safety Evaluation Strategies for Biopharmaceuticals

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1. INTRODUCTION 1.1. Brief History of Biopharmaceuticals Originally a niche category within the global pharmacopeia, the biopharmaceutical industry has expanded tremendously over the past three decades. Over 125 biopharmaceutical products have been registered in the United States and Europe, with hundreds more under clinical investigation at the current time. By some estimates, about 30% of the global pharmaceutical pipeline today consists of biopharmaceuticals. The bulk of biopharmaceutical products and therapeutic candidates are protein- and peptidebased, typically manufactured using cell culture Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Third Edition. http://dx.doi.org/10.1016/B978-0-12-415759-0.00025-X

3.1. Regulatory Resources 3.2. General Toxicity Studies for Biologics Single-Dose Studies Repeat-Dose Studies Safety Pharmacology Studies Developmental and Reproductive Toxicity (DART) Studies Assessment of Carcinogenic Potential Studies to Support Pediatric Use Tissue Cross-Reactivity Studies 3.3. Toxicologic Pathology Findings with Biopharmaceuticals Discussion of Exaggerated Pharmacology vs Toxicity

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4. Summary and Conclusions

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Suggested Reading

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systems, so this line of molecules will be the focus of this chapter. Marketed products of this sort include recombinant growth factors, monoclonal antibody (mAb) products, and enzyme therapies. Vaccines utilizing engineered antigen sources, nucleic acid-based therapeutics, and cell-based therapeutics are also included in a broad definition of biopharmaceuticals, and will be dealt with briefly. In the 1970s and 1980s, advances in molecular biology made it possible to generate large quantities of recombinant proteins in vitro using bacterial, insect cell, or mammalian cell systems. The same molecular biology breakthroughs that propelled the biotechnology industry as a whole

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also provided major innovations in manufacturing tools for protein purification which influenced and advanced the production processes for non-recombinant protein therapeutics isolated from microbial, plant, and animal sources. These same tools enhanced and accelerated the development of novel diagnostic assays; some of these are currently used to evaluate whether or not patients have a disease amenable to treatment with particular protein-based therapies. In particular, the invention of mAbs and polymerase chain reaction (PCR) amplification techniques in the 1980s and 1990s now permits the routine use of molecular pathology methods to phenotype tumors and other disease conditions, identify and characterize biomarkers, and more rapidly and accurately detect and type microbial infections. In 1982, recombinant human (rh) insulin was the first manufactured protein approved for human therapeutic use. Today there are 35 recombinant human insulin products approved for use in humans by the US Food and Drug Administration (FDA). In the remaining 1980s and early 1990s, several other protein therapeutics were developed and approved, and the biotechnology age was born. The first mAb approved for therapeutic use in 1985 was muromonab-CD3 (Orthoclone OKTÒ 3), a murine antihuman CD3 antibody intended to attenuate graft versus host (GVH) disease after organ transplantation. This murine antibody would be highly immunogenic if given to a healthy person, but was functional as a therapeutic agent when given to the immunosuppressed post-transplant patient population. Most approved mAb products today are chimeric (part mouse and part human), humanized (all human except for the antigen-specific variable regions), or fully human proteins by design, and thus are far less immunogenic in humans as compared to the early generation of fully murine antibodies. These first cases served as the baseline for the development of regulatory guidelines that have come to establish a path for the safety assessment of future biopharmaceutical products. The initial marketed products were primarily limited to molecules of known function, often participating in well-defined signaling pathways, and with biochemical characteristics identical to or closely modeled after those of native molecules. For the most part this strategy is still employed,

although engineering advances have challenged this paradigm. High-throughput screening of gene and protein expression profiles as well as targeted analyses of protein or nucleic acid activities in particular tissues have now opened the path for the more rapid development and approval of newly identified molecules from less well-defined pathways. These profiles have also informed the definition of patient subsets within a given disease diagnosis, allowing the tracking and even the utilization of individual genetic signatures and disease qualities to direct these new therapies more efficiently to patients who will benefit from them. Therefore, modern biopharmaceutical investigations have turned the promise of personalized medicine into a reality. From a pathology perspective, knowledge of the targeted biological pathway underpinning a novel therapeutic candidate is the cornerstone for interpreting study results. Biopharmaceuticals provide unique problems as candidates for drug development in that the specific pharmacology may well extend beyond the range of expected pharmacology and into the toxicity profile. As with small molecules, the communal experience gained by evaluating hundreds of candidate biopharmaceutical therapeutics has demonstrated important class effects for types of molecules and/or targets that need to be considered as part of the pharmacology and toxicologic spectrum. Numerous examples of exaggerated pharmacology have been identified for various protein products that have manifested in unexpected ways, or which have defined a novel biologic activity of the targeted protein or cell population. Species or strain differences in pathway modulation may also contribute to the spectrum of toxicities identified, and understanding these differences may be critical in developing a solid risk assessment translating non-clinical data to the clinical setting. With the development of multiple molecules – either multiple biomolecules or a mixture or biomolecules and conventional small molecules – engaging the same target, it has become apparent that agents with the same anticipated pharmacology based on their common target do not always behave identically in vivo. Small differences in core structure, post-translational modification, binding kinetics, pharmacodynamics (PD), and pharmacokinetics (PK) can result in

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1. INTRODUCTION

unique pharmacology and toxicology profiles despite their shared target. This fact has significant ramifications in the current efforts to define a path for registering biosimilar products (i.e., socalled generic biopharmaceuticals). It is a difficult task to set an appropriate bar for a safety assessment program that is intended to avoid unnecessarily redundant non-clinical or clinical studies for such biosimilar products, but which will also provide adequate safety and efficacy information to inform a product-specific label for what is in many respects a new molecule. Ultimately, in evaluating any novel biopharmaceutical candidate, it is important to consider the expected pharmacology, the known toxicities associated with the targeted activity, and any known toxicities of the molecule class, in addition to empirically evaluating the molecule itself. It is also important to track and understand the spectrum of background lesions that may be seen commonly or sporadically in test systems used for the non-clinical safety evaluation of biopharmaceuticals. In these respects, the development pathway for biopharmaceuticals is consistent with that employed for small molecules.

1.2. Biopharmaceuticals Currently Approved for Use As demonstrated by the examples in Table 25.1, a wide range of biopharmaceuticals are in active use today. Established products include recombinant human replacement therapies such as peptides (e.g., the hormones insulin and somatotropin) and proteins (coagulation factors and enzymes), antineoplastic or immunomodulatory mAbs (chimeric, humanized, or human) and fusion proteins (e.g., part of an antibody linked with part of a receptor protein), and vaccine components. As of August 2011, more than 100 FDAapproved biopharmaceutical products were on the market. These included 27 FDA-approved biopharmaceutical vaccine and blood product therapeutics, of which 3 were live-cell therapeutics, as well as 64 non-blood product recombinant proteins for therapeutic use and 38 mAb therapeutics (i.e., “drugs”). For each of these categories, there are numerous additional products currently under evaluation. Several

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approved products have been marketed for more than a decade. Many of these products are marketed globally, while others are marketed only within, or only outside of, the US. The biopharmaceutical field has now been in existence for a sufficient length of time to allow, in some instances, the development and registration of multiple biomolecules directed against a single condition or specific target. For example, five FDA-approved vaccines currently on the market utilize recombinant protein antigens to stimulate protection against human hepatitis B virus infection. Similarly, numerous recombinant blood proteins, such as clotting factors, are approved for the treatment of hemophilia. While blood products may also be produced by extraction and purification from natural sources, and vaccines may include live or modified-live infectious agents, recombinant DNA technologies often are preferred to generate these therapeutic proteins or antigens due to manufacturing consistency and the decreased potential for transmission of infectious diseases. There is also mature experience with protein therapeutics that are not directly based on a single naturally occurring protein or peptide. For example, multiple mAbs that target the human epidermal growth factor receptor (EGFR) or the CD20 Blymphocyte antigen have been approved for oncology indications, and multiple protein therapeutics that inhibit tumor necrosis factor alpha (TNF-a) signaling have been approved for rheumatology indications. With different products competing in the same therapeutic space, similarities in their pharmacology can be identified, leading to a better understanding of class effects among biomolecules, or shared by biomolecules and small molecules targeting a common entity. Furthermore, key attributes that distinguish individual products can be identified, and these can enable more refined use of specific agents and/ or can guide engineering of future therapeutic candidates.

1.3. General Safety Concerns when Developing Biopharmaceuticals For many years biopharmaceuticals were considered to be “safer” than other drugs due to their origin as naturally occurring proteins or peptides, and, in the case of antibodies, their

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Clinical Applications of Biopharmaceutical Products

Therapeutic goal

Selected examplesa

Replacement of essential endogenous enzymes, peptides or proteins that are lacking due to genetic or acquired deficiency or insufficiency

• FabrazymeÒ (agalsidase alfa) recombinant human a-galactosidase for Fabry’s disease • MyozymeÒ (alglucosidase alfa) recombinant human acid a-glucosidase for type II glycogen storage disease (Pompe disease) • NovoSevenÒ RT (coagulation factor VIIa [recombinant]) human FVIIa for hemophilia A and B • XynthaÒ (antihemophilic factor [recombinant], plasma/albumin free) human recombinant coagulation factor VIII (rFVIII) for hemophilia A • BeneFIXÒ (recombinant coagulation factor IX; rFIX) for hemophilia B • NovoLogÒ (insulin aspart [rDNA origin] injection) rapid-acting recombinant insulin for diabetes (type I and II) • LantusÒ (insulin glargine [rDNA origin] injection) long-acting insulin for diabetes (types I and II) • NutropinÒ (somatropin [rDNA origin] for injection) recombinant human growth hormone for growth hormone insufficiency

Supplementation of endogenous enzymes, peptides or proteins to manage disease course

• ActivaseÒ (alteplase) recombinant human tissue plasminogen activator for acute ischemic stroke • PulmozymeÒ (dornase alfa) recombinant human deoxyribonuclease to break down airway secretions in patients with cystic fibrosis • EpogenÒ (epoietin alfa injection) recombinant human erythropoietin for anemia • AranespÒ (darbepoietin alpha) hyperglycosylated recombinant human erythropoietin analog for anemia • NeupogenÒ (filgrastim) recombinant human granulocyte colony stimulating factor (G-CSF) to stimulate myelopoiesis before transplant or during chemotherapy • NeulastaÒ (peg-filgrastim) pegylated recombinant human G-CSF for long-acting stimulation of myelopoiesis • ElsparÒ (asparaginase) enzyme used as an antileukemic for pediatric acute lymphocytic leukemia

Targeted cancer therapies, with mechanisms such as inhibiting soluble growth factors, cancer cell-specific targeting through surface receptors, blockade of receptor signaling, and immunostimulation

• AvastinÒ (bevacizumab) humanized antivascular endothelial growth factor (VEGF) antibody • BexxarÒ (tositumomab) 131I-conjugated mouse anti-human CD20 radioimmunoconjugate • RituxanÒ (rituximab) chimeric anti-CD20 antibody • CampathÒ (alemtuzumab) humanized anti-CD52 antibody • ErbituxÒ (cetuximab) chimeric anti-epidermal growth factor receptor (EGFR) antibody • VectibixÒ (panitumumab) human anti-epidermal growth factor receptor (EGFR) antibody • HerceptinÒ (trastuzumab) humanized anti-HER2 antibody

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TABLE 25.1

• PegasysÒ (peg-interferon alfa-2a) pegylated recombinant human IFN-a for chronic human hepatitis virus infection (hepatitis B and hepatitis C viruses) • Recombivax HBÒ (hepatitis B vaccine, recombinant) • SynagisÒ (palivizumab) humanized antibody specific for the F protein of respiratory syncytial virus (RSV)

Targeted immunomodulation to specifically treat various immunemediated diseases

Indications

Selected Examplesa (with targets and therapeutic class)

Rheumatoid arthritis Multiple sclerosis Other

• RemicadeÒ (infliximab) chimeric anti-tumor necrosis factor-alpha (TNF-a) antibody • HumiraÒ (adalimumab) human anti-TNF-a antibody • EnbrelÒ (etanercept) TNF-receptor:Fc fusion protein • ActemraÒ (tocilizumab) humanized anti-interleukin (IL)-6 receptor antibody • AvonexÒ (interferon beta-1a) human interferon b-1a • TysabriÒ (natalizumab) humanized anti-a4 integrin antibody • ZenapaxÒ (daclizumab) humanized antibody specific for the alpha subunit of the IL-2 receptor (CD25) (belimumab) human anti-B-lymphocyte • BenlystaÒ stimulator antibody for systemic lupus erythematosus • StelaraÒ (ustekinumab) human antibody specific for the p40 subunit of IL-12 and IL-23, for use against plaque psoriasis • XolairÒ (omalizumab) humanized anti-IgE antibody for asthma • ActimmuneÒ (interferon [INF] gamma-1b) recombinant human INF-g for chronic granulomatous disease

1. INTRODUCTION

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Antimicrobial therapies

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This is not a comprehensive list of approved biopharmaceuticals, but provides major examples of clinical utility for many currently approved biopharmaceuticals. Some products are also approved for indications beyond those listed here.

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highly targeted activity. That said, over time some non-clinical studies with biomolecules identified potential toxicities. Some of these programs were discontinued entirely based on non-clinical toxicity, and others required the development of expanded clinical monitoring to mitigate potential pharmacologic liabilities discovered in early testing. Then, in 2006, a hallmark incident occurred that, for many, forever changed the manner in which risk assessment for first-in-human (FIH) trials with biopharmaceuticals was approached. A small biotechnology company, TeGenero, conducted a FIH study in healthy volunteers in the United Kingdom with their “superagonist” T-cell targeted antibody TGN1412. This antibody had been shown in cynomolgus monkeys to directly induce T-cell signaling without ligation of the T-cell receptor, but it had also been well tolerated by the monkeys at very high doses. The human response was strikingly different. Within hours of administration, all six healthy volunteers developed severe toxicity, in some cases leading to multi-organ failure or amputation. Ultimately, this unexpected outcome was indexed in part to the difference in specific subpopulations of lymphocytes expressing the molecular target, which differ between cynomolgus monkeys and humans. In this case, the known and intended pharmacology highlighted a potential safety concern, but the “clean” data from the toxicity study in monkeys led to a false sense of security regarding TGN1412’s safety profile. As illustrated by the TeGenero incident, drugs with known or desired T-lymphocyte stimulatory properties have an inherent risk of triggering a “cytokine storm.” Such storms represent the rapid and acutely escalating release of multiple cytokines and acute phase reactant proteins, ultimately resulting in severe clinical signs and widespread organ damage and/or failure. These potentially catastrophic events can be difficult to predict from non-clinical studies as there are substantial differences in T-lymphocyte activation and regulation among species, even between non-human primates (NHP) and humans. Once triggered, these storms represent acute and life-threatening reactions that can be difficult to manage clinically. Accordingly, it is currently recommended that clinical investigators for biomolecules follow a very conservative initial dose selection and

escalation approach, based on minimal biological activity, for first-in-human (FIH) testing of products considered at risk of initiating this explosive cytokine release. Another example of a pharmacologicallymediated safety concern is the tumor lysis syndrome sometimes seen following the initial administration of some antineoplastic biopharmaceuticals. As with targeted small molecule therapeutics, cytolytic therapies can induce acute death of massive numbers of tumor cells that subsequently leads to cytokine release, progressive inflammation, and electrolyte imbalances. This syndrome may manifest with administration of the first dose, when the largest population of susceptible tumor cells are present, and may not recur with subsequent doses despite continued potential for antitumor effects. Biopharmaceuticals that are intended to negatively modulate immune responses may predispose to an increase in opportunistic or atypical infections. For instance, reactivation of latent tuberculosis has been described with mAbs and soluble receptors directed against TNF-a. In like manner, reactivation of latent polyoma (JC) virus infections in the brain leading to progressive multifocal leukoencephalomalacia (PML) has been described following administration of immunomodulatory mAbs that bind certain cell adhesion molecules and result in decreased immune surveillance of the central nervous system. The risk of infections may not be wellmodeled during safety testing due to routine health screening and husbandry practices that decrease the chance of viral infections (active or latent) in well-controlled non-clinical studies. Although the specific agents causing infections may not be directly paralleled in animals and humans, the non-clinical studies can still help in identifying the potential general risk of such infections by revealing the phenotype of immunosuppression. Decreased immune activity following treatment with immunosuppressive biomolecules is also postulated to impact innate tumor surveillance. The main example of this effect is the increased incidence of lymphomas that has been attributed to treatment with anti-TNF agents. Once again, verification of this potential effect is difficult to attain from conventional non-clinical studies as spontaneous tumors are generally rare in studies with relatively young

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and healthy non-human primates, and are also unusual in rodent studies with non-aged animals. In general, conventional rodent lifetime (2-year) carcinogenicity bioassays with protein products that act via immunomodulation have not been shown to reproducibly predict an increased risk for developing cancer (see Carcinogenicity Assessment, Chapter 27). Therefore, clinical tumor registries may be required for immunomodulatory biopharmaceutical therapies during the post-approval (Phase 4) period. Finally, although the overall safety concerns of biopharmaceuticals can generally be indexed to their pharmacology, predicting the full spectrum of pharmacologic activity across species can be difficult even when the target is well-conserved. For example, replacement enzyme therapeutics will not be recognized as “self” by a genetically deficient host, and may initiate a neutralizing immune response in the target patient population. For protein therapeutics, monitoring the immune response to the therapeutic in the intended patient population is a key component of product characterization. When developing mAb therapeutics, additional assessments on effector function that are conferred by antibody glycosylation and isotype should also be considered, as well as the possibility that immune complex formation and deposition may initiate or exacerbate systemic inflammatory disease. In vitro assessment of antibody-dependent cellmediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), although not always feasible, can contribute to an understanding of the overall safety profile of antibody products, and also aid in the interpretation of non-clinical and/or clinical toxicities. These examples highlight some of the typical and more serious safety concerns that have been identified for biopharmaceutical candidates over the past decades. These adverse effects bridge multiple targets and apply to many product types. Early on, biopharmaceuticals as a group were perceived to have less toxicity than corresponding small molecule drugs directed against the same target. However, the past two decades have also highlighted the need for a thoughtful and informed risk–benefit analysis for each candidate biopharmaceutical depending on its pharmacology and related safety profile. An important indicator of the growing impact of safety considerations with

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respect to biopharmaceuticals is the temporary or permanent recall of products for adverse events that are first identified after licensure. While not all products with such late-stage safety concerns are withdrawn, their labels may be modified to carry extensive “black box” warnings to guide appropriate and informed use. Finally, many biopharmaceutical products, especially those intended for indications that are not immediately life-threatening, must clear a higher safety bar today to be considered for approval, with many dropping out of development (due to issues evident in the non-clinical and/or clinical data sets) that formerly would have received relatively rapid approval. In this regard, the non-clinical pathology data are often critical in providing context around potential safety concerns early in a development program, thereby enabling more scientifically-driven development decisions to be made before a clear assessment of potential clinical benefits and risks can be assessed in human patients.

2. BIOPHARMACEUTICALS 2.1. What Makes Biopharmaceuticals Different From Other Drugs? High-level comparisons between biopharmaceuticals and small molecule drugs generally focus on differences in size, PK/PD properties, and the potential for “off-target” effects. Most small molecule drugs have a molecular weight of less than 1 kilodalton (kD), while protein therapeutics tend to be much larger (dozens to several hundred kD). For example, a typical immunoglobulin G (IgG) therapeutic antibody is about 150 kD. This massive size difference directly affects other parameters such as the volume of distribution. Most biopharmaceuticals do not penetrate cells but instead engage circulating proteins (e.g., soluble binding proteins or receptors) or cell surface proteins, particularly receptors. As such, biomolecules have little potential for direct interactions with DNA, and, by extension, low potential for direct genotoxicity. Most antibodies do not have direct access to sequestered antigens in immune-privileged compartments of the body (e.g., brain, eye, testis) in the absence of preexisting damage that has disrupted physical

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and molecular obstacles to entry (e.g., the blood– brain barrier). If such biomolecules do gain entrance to these privileged sites in the absence of barrier compromise, their concentrations usually will be negligible. Depending on the activity, though, even relatively low concentrations may still be pharmacologically relevant. The absorption, distribution, metabolism, and excretion (ADME) profile of any given drug, whether a biomolecule or a small molecule, typically governs the optimal route and schedule for its administration. Many small molecule drugs are chemically stable and can be administered orally, which is convenient for patients. However, oral administration is not feasible for proteins and peptides as they would be digested into small peptide fragments or their composite amino acids before they could be absorbed, let alone reach and act upon the target. Therefore, most protein therapeutics are administered parenterally, with the intravenous and subcutaneous routes generally being most favored. Alternate routes, such as inhaled aerosol suspensions, intrathecal or intravitreal injections, and isolated limb perfusions have also been utilized. For subcutaneous delivery, at-home use has become an achievable target for patient convenience, and needle-less (i.e., pressure-based) injections are also under investigation to avoid pain (typically minor) at the injection site. Once systemic exposure is achieved, small molecule drugs are often partially bound to proteins while in circulation, and they tend to undergo fairly rapid metabolism and elimination once they are released – whether or not they have had a chance to encounter their target. In this scenario, species differences in the complement of metabolizing enzymes like cytochromes P450 (CYPs) are important for the assessment of toxicity. In particular, consideration must be given to the therapeutic and toxic properties of both the administered form of the drug and its major metabolites. In contrast, most biopharmaceuticals circulate in an intact, unbound form among the circulating pool of endogenous proteins, with more gradual clearance predominantly through uptake by the liver and kidneys. Proteins then are degraded to peptides and ultimately their constituent amino acids, which may be reused in protein synthesis. Smaller circulating peptides may be eliminated directly in the urine. Thus the metabolism and

excretion components are less critical in establishing a toxicity profile for biopharmaceuticals as compared to small molecule therapeutics. The PK–PD properties of individual biopharmaceuticals are also influenced by the target-specific and physical properties of the therapeutic molecule. For example, a mAb targeting a widely expressed antigen may demonstrate non-linear PK as the dose is escalated until the circulating concentration becomes high enough to saturate the available antigen pool. Likewise, proteins with little or no glycosylation may be rapidly removed from circulation by the hepatic asialoglycoprotein receptor. Numerous covalent modifications (e.g., conjugation to a polymer like polyethylene glycol [PEG], via a process termed PEGylation) have been developed to try to slow the removal and breakdown, and thus extend the half-life, of smaller therapeutic proteins. Finally, as has been noted above, most therapeutic proteins principally exert their pharmacologic effects through their targeted pathway. In other words, biotransformation events are rare, metabolism is limited, and the general PK–PD profile is fairly predictable. Although this does not mean that all effects identified in vivo will have been predicted by the outcome of nonclinical studies, it does mean that when unexpected findings arise with a biopharmaceutical, a careful exploration of potential relationship to the pharmacology is warranted. Such investigations will usually find that the adverse effect is the result of exaggerated pharmacology mediated via on-target activity.

2.2. Classes of Biopharmaceuticals Biopharmaceuticals can be categorized many ways, including by their chemical structure, mode of action (MOA), site of action, and others. This section considers the properties of biopharmaceuticals from several structural categories: (1) recombinant native peptides and proteins; (2) mAbs; (3) antibody-like molecules; (4) combined products; (5) nucleic acid-based therapies; and (6) cell-based therapies. Some of these categories, such as mAbs and native proteins, include numerous approved products. Other categories, such as nucleic acid- and cellbased therapies, are still emerging as indicated

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by active research at numerous institutions but few or no currently approved products. Recombinant Native Peptides and Proteins Many marketed biopharmaceuticals are identical or very nearly identical to endogenous human proteins or peptides. These proteins find clinical use as replacement therapies in individuals who lack sufficient endogenous quantities of such substances due to genetic deficiencies or disease states. They may also be used to amplify endogenous hormonal or immunologic systems even when endogenous levels of the native substances are within physiologic ranges. This category of biopharmaceuticals includes many agents: • Interferons (INF). Alpha, beta, and gamma INFs are used to bolster antimicrobial responses (e.g., INF-a for viral hepatitis) and redirect non-specific stimulation of the immune system (e.g., INF-b for multiple sclerosis). • Interleukins (IL). These molecules activate or sustain activity in specific arms of the immune and hematopoietic systems for many therapeutic indications (e.g., IL-2 to attack melanoma). • Hormones. Agents are given as replacement therapy (insulin, growth hormone) or to control the reproductive cycle (gonadotropinreleasing hormone [GnRH], folliclestimulating hormone [FSH]). • Hematopoiesis-stimulating factors. These molecules promote expansion of specific subsets of hematopoietic progenitor cells, often to minimize or prevent bone marrow suppression in individuals receiving cytotoxic chemotherapeutics in oncology (granulocytic and monocytic-colony stimulating factor [GMCSF], granulocytic colony stimulating factor [G-CSF]). • Growth factors. These proteins stimulate growth or enhance survival of endangered cell populations (e.g., somatotropin for growth hormone deficiency, or keratinocyte growth factor (KGF) to attenuate mucositis expected with hematopoietic stem cell therapy protocols for hematologic malignancies). • Enzymes. These molecules are given to individuals with genetic defects resulting in insufficient or absent levels of the affected

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enzymes (e.g., glucocerebrosidase for Gaucher’s disease), or as therapeutic products (e.g., DNAse used to break down thick secretions in the lungs of cystic fibrosis patients, or asparaginase as an antineoplastic agent). Even when these endogenous recombinant proteins are identical in amino acid sequence to the native proteins, they may be recognized in human recipients as immunologically “nonself,” resulting in production of an immune response against the recombinant protein. This immunogenicity may, but does not always, result in three clinically important safety problems: (1) decrease in the desired efficacy due to antibodymediated neutralization or clearance of the recombinant (therapeutic) protein; (2) toxicities due to antigen–antibody complex deposition (type III hypersensitivity); and (3) deficiency of the host-produced endogenous factor itself, due to extension of the same mechanism of antibody-mediated clearance to the native molecule. A well-known example of the latter is the recombinant erythropoietins (EPOs) and their potential to initiate aplastic anemia due to induction of anti-erythropoietin antibodies. Therefore, extensive efforts to minimize the clinical immunogenic potential and to be able to monitor immunogenicity in clinical studies are important components in the development program for protein therapeutics, regardless of whether the molecule is the native form or a slightly modified recombinant version. However, it is also generally accepted that the degree of immunogenicity in animal species used for non-clinical toxicity studies is not predictive of a protein’s immunogenic potential in humans. It should also be noted that fusion proteins are an active area of study. Combinations of peptide or hormone therapeutics with larger carrier protein molecules are desirable for their potential to extend circulating residence time for the bioactive component. In addition to fusion proteins with antibody components, one non-antibody example is the fusion of small peptides or hormones to the large and abundant serum protein albumin. The potential to introduce novel antigens at the junction of the two protein components of a fusion protein is a general point of concern. However, collective experience with many fusion proteins and the individual

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products in isolation will be needed to define the actual risk, if any, beyond that of other recombinant protein therapeutics. Monoclonal Antibodies (mAbs) The chemical structure, creation, engineering, and production of mAbs have been extensively reviewed in other publications. Some basic points will be recapitulated here with reference to the basic immunoglobulin (Ig) structure as depicted in Figure 25.1. A high degree of similarity or homology exists across mammalian species in the basic structure of antibodies, particularly with regard to the arrangement of the functional antibody domains. Thus, Y-shaped antibodies from mouse to human will contain an Fc (“Fraction crystallizable” or

“constant”) domain at the tail and twin CDR (“complementarity determining region” or “variable”) domains on the arms. These individual units may exist under normal conditions as stand-alone units (as for IgG) or as multi-unit aggregates (as for IgM, which includes five Ig components arranged in pentagonal fashion). The Fc domain has important roles in mediating effector function through its ability to engage isotype-specific Fc receptors (i.e., FcgRI, FcgRII, FcgRIII, FcεR), as well as the FcRn receptormediated recycling and clearance that accounts for the relatively long elimination half-life of most antibodies in circulation. The specificity of an antibody resides in the CDR, which is the portion that interacts with the cognate molecular target (i.e., antigen). The CDR is determined by

FIGURE 25.1 Schematic illustrating the components of a various protein biotherapeutics based on the immunoglobulin G (IgG) antibody class. The amino acid sequence of a complete monoclonal antibody (mAbs [enclosed in the circle]) typically has one of four origins: fully murine, chimeric (human Fc [fraction-crystallizable domain] connected to murine Fv [fraction-variable domains, i.e., “arms”]); humanized (human except for mouse CDR [complementarity determining region]), and fully human. Other synthetic biomolecules illustrated here, each produced using different antibody domains, include scFv (single-chain Fv), F(ab)2 ¼ two-armed antigen-binding antibody fragment typical of pepsin digestion, F(ab)0 ¼ single-arm antigen-binding antibody fragment typical of papain digestion, and mixed molecules such as diabodies (bispecific molecules comprised of two linked F(ab)0 domains) and Fc-fusion proteins (a combination of the Fc domain and one or two functional entities [orange lines], typically non-antibody peptides or protein receptors). Antibody conjugates are not specifically illustrated here, but have radioisotope or toxin moieties added via a linker molecule that forms a bond through amino acid side chains of the mAb framework.

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contributions from both the heavy-chain and light-chain components, and variability in the respective gene loci dictating the amino acid sequence in the antigen-binding site defines the antigenic site to which any given antibody will home. The spectrum of potential CDRs within an individual allows the body to select the mature antibody response to immune stimulation that has the best specificity, affinity, and avidity to address an external challenge. However, even though a high degree of functional homology exists among species, there is considerable lack of species homology with regard to the precise amino acid sequences that comprise the individual domains and antibodies as a whole. Engineered therapeutic mAbs may take advantage of these differences to make proteins with heightened binding properties that would not be generated under natural conditions. This ability gives biopharmaceutical engineers an increased opportunity for fabricating a therapeutic with optimal efficiency. Possible conformations of bioengineered mAbs may contain: • only murine amino acid sequences (assigned generic product names ending in “-momab”); • murine and human sequences (“chimeric,” which are given generic product names ending in “-uximab”); • mostly human sequences with some residual murine sequences in the antigen-binding site of the variable domains (“humanized,” assigned generic names ending in “-izumab”); • only human amino acid sequences (“fully human,” assigned generic names ending in “-umab”). As the technology for producing mAbs has become increasingly sophisticated, it is more common to see fully human antibodies, as these molecules are anticipated to have minimal immunogenicity potential in humans relative to therapeutic proteins that contain murine sequences. It should be noted that the possibility for immunogenicity remains a hypothetical possibility even for fully human mAbs, as there may be subtle amino acid sequence variations in the protein backbones among individuals with different genetic backgrounds. Specific amino acid sequences in the Fc and CDR domains of antibodies engage or bind to particular epitopes on other molecules, where the epitope is likely to consist of defined amino

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acid (AA) sequences but may also include conformational specificity driven by nucleic acid (NA), carbohydrate, or lipid elements. This feature accounts for both the desired therapeutic efficacy and potential adverse properties of mAbs that are used as therapeutic agents. Typically, each CDR binds or recognizes a specific short (approximately eight acids) sequence that will often be found only in a single peptide or protein. Accordingly, antibodies are molecules with an exquisite degree of binding specificity, theoretically locking onto only one molecular target, yet also including the potential for crossreactivity to other molecules with structurally similar sequences. The Fc domain of mAbs also demonstrates binding specificity in that the Fc for a given antibody isotype (e.g., IgG, IgE, IgA) will only bind to the Fc receptor that is specific to that isotype (i.e., FcgRs for IgG, FcεRs for IgE, or a single FcaR for IgA). Once bound, however, it is important to recognize that therapeutic mAbs are not little daggers that directly kill or injure foreign invaders or diseased cells. Instead, therapeutic mAbs act by their inhibitory or stimulatory effects on signal transduction pathways through CDR binding, by activating other effector molecules or cells through Fc binding, or by sequestering other ligands away from their receptors. In addition to their desired pharmacologic properties, therapeutic mAbs may induce undesirable effects (“toxicity”) through any of the four classic mechanisms of hypersensitivity. Acute infusion reactions are not uncommon with intravenously administered mAbs. Such responses may be due to type I hypersensitivities (especially upon repeated exposures) or, more commonly, to anaphylactoid reactions due to the liberation of bioactive complement fragments. Type II hypersensitivities are based on the effector role of the Fc domain in complement fixation, cytotoxicity and subsequent downstream innate immune events. These type II reactions are often the desired pharmacologic effect when cytoxicity is the desired outcome and the CDR is used to target antibodies to cells bearing a specific cell surface protein (e.g., a tumor antigen). However, type II reactions obviously can cause undesired consequences if they occur in an uncontrolled manner or in an undesired anatomic or physiologic compartment. Type III hypersensitivities (“immune complex disease”)

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may occur when a mAb targets a soluble antigen and the immune complexes become deposited in the small tortuous vessels (e.g., glomeruli). Type III reactions can also occur when the antibody binds to a target antigen in those locations, or as a consequence of interactions between a therapeutic mAb and an “anti-antibody antibody,” as previously discussed. Once these type III immune complexes are deposited, type II mediated mechanisms are incurred in which complement- and cell-mediated mechanisms of immunologic injury become predominate. Delayed (type IV) hypersensitivities appear to be relatively uncommon outcomes of mAb exposure, perhaps with the exception of repeat-dose subcutaneous injection of antibodies (or other protein therapeutics). In this instance, the slowly cleared protein may persist locally and serve as a depot for gradual antigen release, leading to a cell-mediated immune response against the protein depot. Importantly, for the many mAbs approved for therapeutic use these hypersensitivity reactions occur clinically at a low level that is considered to represent an acceptable risk profile given the great therapeutic benefits of these targeted drugs. Antibody-Like Molecules The unique and desirable properties of the two principle functional regions of antibodies (Fc and CDR) have resulted in a growing trend toward engineering these domains, or portions thereof, to achieve therapeutic goals that are not possible with conventional mAbs, isolated CDR or Fc domains, or the other peptide or protein alone. The typical approach is to artificially recombine the desired antibody domain with another peptide or protein molecules (e.g., the combination of a short, single-chain peptide to an Fc region to create a “peptibody”). Perhaps the most desirable property of antibody-based fusion proteins is that the interaction between the Fc domain and the FcRn receptor provides a relatively long elimination half-life for IgG antibodies; thus, an engineered antibody-like molecule containing a Fc domain will most likely have the elimination characteristics closer to those of an antibody (typically about 7–21 days for IgG-based mAbs, assuming linear and doseproportional clearance). Thus, fusion proteins with Fc domains as one component may be given less frequently than the protein alone. Without an

Fc domain, the half-life of many engineered molecules will be relatively short (2–3 days or less), due to mass-based renal clearance. Because most recombinant protein therapeutics are expensive, some development programs for synthetic peptide therapeutics focus instead on low molecular weight antibody-like molecules that lack an Fc domain but which have other biochemical modifications intended to increase the molecular mass, reduce mass-based renal clearance, and hence prolong the treatment interval. A common example of this latter strategy is the conjugation of small molecular weight therapeutic proteins to PEG, a relatively inert polymer. Examples of PEGylated products include peginterferon alfa-2a (PegasysÒ ) and pegfilgrastim (NeulastaÒ ). The number and arrangement of PEG molecules can be modified to adjust the circulating half-life. Such adjustments are also commonly undertaken to reduce the likelihood that the agent will be removed by tissue phagocytes and secretory cells (e.g., choroid plexus epithelium, renal tubular epithelium), as over time such cells develop a vacuolated appearance as PEG accumulates following chronic intermittent exposure. To date, no adverse function has been shown to be a consequence of PEG accumulation. Fusion proteins contain two unrelated, functionally distinct peptide or protein domains that are chemically joined (fused) such that the properties of each distinct domain influence the overall properties of the entire molecule, leading to optimized pharmacology. While fusion proteins are used for many investigative purposes in molecular and cell biology, one common therapeutic approach is to fuse the antibody Fc domain to a low molecular-weight protein of pharmacologic interest (e.g., there are clinical trials currently investigating the safety and efficacy of Fc combined with Factor IX, Factor VIII, and glucagon-like protein 1 [GLP1]). As described above, the lower molecular-weight protein would ordinarily undergo rapid mass-based clearance by the kidneys, but the presence of the Fc receptor reduces the clearance and prolongs the efficacy of the fusion protein due both to its increased molecular weight and to the ability of the Fc–FcRn interaction to permit fusion protein recycling. Thus, this simple engineering feat prolongs both the elimination phase and the pharmacologic half-lives of

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the fusion protein. This approach may also be used in the case where a receptor protein normally expressed on the surface of the target cell is fused to the Fc immunoglobulin domain. The soluble Fc : receptor fusion protein that is in circulation binds the soluble cognate ligand for the receptor, thus preventing the free ligand from interacting with the intact functional receptors that remain on the target cell surface (i.e., the basis for the so-called “molecular sponge” or “decoy receptor” strategy). Fusion proteins containing the Fc domain observe the convention of adding “-cept” as the terminal syllable of the generic name (e.g., etancercept (EnbrelÒ ), a soluble recombinant molecule linking TNF receptor, type I to the Fc domain of IgG). Other molecules may be considered to be “antibody-like” in principle because they utilize constructs with highly specific CDR targeting, like an antibody, even though they often do not include the Fc domain. These subgroups include various isolated components or artificial assemblies of CDR-containing molecules, again optimizing for the desired pharmacology and pharmacokinetics along with manufacturing feasibility. Some of these molecules include: • Fab or di-Fab fragments. These contain the variable regions of both heavy and light chains from one or both arms, respectively, of the parent antibody. Fabs are essentially what is left when the Fc region is removed from an antibody molecule, thus they have a short elimination half-life. • ScFv, diabodies, tribodies, etc. These single chain (Sc) or multimeric CDRs are engineered by using synthesized linkers to artificially join just the variable (Fv) regions of the light chain and heavy chain, which are linked together to form a functional CDR. Depending on the length of the linker used, scFv may be polymerized into defined multimers, indicated by their name. Like the Fabs, these molecules have fairly short elimination half-lives. • Minibodies. These are built upon the scFv approach, by the addition of domain 3 of the heavy chain to a CDR-containing scFv molecule. The added domain increases the elimination half-life due to the increase in molecular mass, and retains a basic physical resemblance to a mAb despite the lack of the Fc region.

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• Bispecific antibodies. These are antibodies with a normal Y-shaped conformation, but where each arm contains a unique CDR or CDR-like region directed against a different target peptide sequence. The choice of target peptides is determined by the therapeutic objective, but typically the targets are cell surface-associated proteins found on the same cell or on cells that would already be or are desired to be in close proximity to each other. The presence of the bi-specific antibody permits anchoring (“crosslinking”) of the two different targets on or between cells. • T-cell engaging dual-specificity molecules. These molecules may be constructed based on further extending the approaches used to make the bispecific antibody, minibody, or scFv. The concept is engineering molecules where there is an antigen-specific CDR region, and a second molecular component, which may be CDR-like, that engages an effector cell such as a T lymphocyte. This crosslinking between cells can then lead to localized T-cell activation. Combined Products (mAb Conjugates) Combined products consist of a protein therapeutic component, such as an antibody, and a non-protein component, such as a toxin, a chemotherapeutic drug, or a radioactive moiety. These products are intended to selectively deliver a highly potent cytotoxic agent to a target cell of interest, such as a tumor, in order to minimize collateral damage to normal tissues. However, the non-clinical strategy for developing such products can be challenging. For example, the pharmacological relevance of the test system should be established for the targeting component (e.g., the antibody). To this end, some characterization of the naked antibody (i.e., without the addition of the cytotoxic moiety) may need to be performed if the properties of this “delivery” element have not already been characterized. The “payload” component (drug, toxin, or radioisotope) must also be characterized in its native state (i.e., unbound to the antibody) so that toxicities specific to that component can be recognized. However, in some instances the toxicity profile of the “payload” component may already be well-established, so evaluation of toxicity may be limited to effects produced

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by the protein conjugate. A “linker” component – the bridge between the delivery element and the payload – is often included in building such proteins, and it is intended to be pharmacologically inert. However, the toxicity of the linker must also be considered for testing, especially with regard to the immunogenic potential of the complete molecule. Finally, the PK and elimination of the intact molecule and its composite parts should be described as part of characterizing the fusion product, and must be considered with the toxicity profile. This expansive testing for the effects of the combined molecule as well as its constituent parts is required because in some instances the payload is designed to be released following enzymatic cleavage of the linker molecule only after delivery to the target. To date, most approved antibody–cytotoxic protein conjugates have been used in oncology indications. One example is brentuximab vedotin (AdcetrisÔ ), an antibody–drug conjugate (ADC) between the antimitotic agent monomethyl auristatin E (MMAE, a tubulin-binding toxin) and an anti-human CD30 antibody that is used in the treatment of Hodgkin’s lymphoma. For this product, the antibody binds CD30 on the target tumor cells, and the Ab : Ag complex is internalized. After internalization, the linker is proteolytically cleaved, releasing MMAE intracellularly to bind tubulin in the microtubule network of the cell. The resulting cytoskeletal disruption is fatal to the targeted tumor cells, which express CD30 at much higher levels than other cells in the body. Another example is gemtuzumab ozogamicin (MylotargÒ ). This is an anti-human CD33 antibody conjugated to another tubulin binding toxin, calecheamicin. Approved in 2000 for the treatment of acute myeloid leukemia (AML) in older patients, this product was withdrawn from the market in 2010 due to accumulated evidence that the survival benefit was small compared to the risk of toxicity with this therapy. Examples of approved radioimmunoconjugates include tositumomab (BexxarÔ ) and ibritumomab tiuxetan (ZevalinÔ ), both of which are mouse anti-human CD20 mAbs bound to isotopes. The fully murine CD20 antibodies were specifically chosen for these products due to their relatively short half-lives as compared to rituximab, which is an unconjugated murine–human chimeric antibody specific for human CD20. Tositumomab is linked with 131I,

which emits both beta and gamma radiation but has rapid elimination. In contrast, ibritumomab tiuxetan is conjugated to 90Y, a beta-emitter with a short half-life but a longer elimination phase. These examples show that conceptually similar biomolecules can have complex and divergent individual profiles. From a biopharmaceutical perspective, these radioimmunoconjugates also have high potential to induce a human anti-mouse antibody response, which might be expected to reduce their efficacy over time. However, for these particular cases, such responses may be attenuated by targeting CD20-expressing B lymphocytes, which are responsible for initiating the immunogenic response to the conjugate. In addition, patients receiving these CD20-targeted radioimmunoconjugate therapies have had or are receiving cytotoxic chemotherapy which may likewise attenuate the immune response to the conjugate via general immunosuppression. Overall, these combined products utilize many of the clinical advantages of both protein and small molecule therapeutics, but the non-clinical assessment can be challenging. The pathology is likely to reflect aspects of the toxic moiety – although this assumption cannot be taken for granted without testing – but with antibodylike distribution and half-life. Target organ toxicities may reflect cell populations that express the intended target, but additional off-target effects may be unveiled in tissues that have a particular sensitivity to the “payload” or that are involved in the distribution or processing of the ADC (and therefore have more concentrated exposure to the toxic moiety than would occur based on the quantity of the combined protein measured in the circulation). As more of these combined products are evaluated, it is likely that the list of indications for which they are approved as therapies will expand to include nonlife-threatening chronic diseases, or microbial targets. In this event, the approach to non-clinical safety assessment and clinical translatability of effects for combined products will be informed by the accumulated information from these early approvals in this area. Nucleic Acid-Based Therapies GENE THERAPY

Gene therapy is based on the transfer of genetic information to targeted host/patient cells

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to provide a durable treatment effect. Such approaches are attractive for genetic deficiencybased diseases in which a single protein is absent or minimally produced (e.g., enzyme deficiencies). However, this strategy has been difficult to implement. Most vectors that provide persistent, high-efficiency gene transfer are viral-based, and are often based on viruses selected or modified to limit replication competence and/or host cell integration potential. Early examples included adenovirus and herpesvirus, which elicit some degree of cytotoxicity and/or immunogenicity that results in clearance of the viral vector in relatively short order. Recent advances have focused on less immunogenic viruses like adeno-associated virus (AAV) and lentivirus. Even so, safety concerns remain with respect to the therapeutic use of viral vectors for gene therapy, especially regarding potential recombination events that either could allow replication-competent viruses to regain their infectivity or permit germ-line integration (i.e., insertional mutation) of the viral components. Although technically not a genetic transfer, the therapeutic use of oncolytic viruses to selectively target and lyse tumor cells is similar enough to gene therapy that it also can be considered here. There are similar safety concerns regarding potential viral integration as with other virallymediated forms of gene transfer. In addition, these viruses may be minimally modified or even unmodified, and thus are typically replication competent. Therefore, oncolytic viral therapies also pose a risk to non-cancerous host cells, although this risk is relatively trivial when measured against the likelihood that malignant cells will escape the lytic zone. A more problematic possibility is that these replication-competent viruses pose a risk of viral shedding from the patient, with subsequent infection of other individuals in contact with the host. The International Conference for Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use has published three monographs as “Considerations” documents from the Gene Therapy Working Group. Although not fully endorsed as formal ICH guidelines, these draft documents represent the current thinking of this expert panel regarding some of the most common challenges posed by gene therapy programs: germline integration, virus and vector shedding,

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and oncolytic viruses. In addition, an ICH guideline on gene therapy has been drafted and is currently under review. DNA-BASED VACCINES

One interesting veterinary product approved by the US Department of Agriculture (USDA) is a xenogeneic DNA vaccine delivered via needlefree injection for the treatment of canine oral melanoma (OnceptÔ ). This immunoactivating antitumor vaccine is a landmark biopharmaceutical on many fronts. It relies on injection of DNA encoding a xenoprotein that is similar to one expressed naturally by the tumor cells. The DNA is then taken up by resident cutaneous macrophages, transcribed, and translated into an immunoreactive xenoprotein, after which the host immune response to the xenoprotein crossreacts with the endogenous antigen on tumor cells. The antitumor response can eliminate or slow tumor progression, resulting in substantially prolonged survival times for many canine patients. A similar approach is under investigation as a human melanoma vaccine. ANTISENSE OLIGONUCLEOTIDES, SIRNAS, AND APTAMERS

Numerous antisense oligonucleotide and small interfering RNA (siRNA) candidate therapeutics have been investigated or are currently under investigation, but to date only one has been approved for use. This agent fomiversen (VitraveneÒ ) was approved by the FDA in 1998 as an intravitreal injection for treating cytomegalovirus (CMV) retinitis in immunocompromised patients. Like many other oligonucleotide-based therapies under investigation, it is fairly short (21 bases) and has a phosphorothioate (sulfurbased) backbone, where a P–S linkage replaces the P–O linkage found in natural RNAs. The substitution of sulfur for oxygen is designed to be resistant to endogenous ribonuclease activity, and thus to prolong the half-life of the agent. These agents generally act by being taken into cells where they bind to nuclear DNA sequences to prevent transcription or to cytoplasmic RNA sequences to block translation. For fomiversen, the target is the major immediate-early gene of the human CMV virus. The non-target specific toxicity profile for oligonucleotide-based therapeutics typically includes several class effects. Most notable are

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complement activation in non-human primates, inhibition of the intrinsic coagulation pathway resulting in prolonged activated partial thromboplastin times (APTT) in multiple species, and general immunostimulation. The latter changes are most prominent in rodents, where oligonucleotides can produce marked splenomegaly, lymphoid follicular hypertrophy in lymphoid organs, and reversible accumulation of large, pleomorphic lymphoplasmacytic aggregates in non-lymphoid tissues (prominently including liver and submandibular salivary gland). A dose-related accumulation of basophilic granular material in hepatic sinusoidal macrophages (Kupffer cells), lymph node macrophages, and renal proximal tubular epithelial cells has also been described, and is generally considered to reflect accumulation of the modified oligonucleotide in these cells. In general, these effects seem to be more pronounced for oligonucleotides with sulfur-based rather than oxygen-based backbones. Another related class of nucleic acid therapeutics is the aptamers. These single-stranded oligonucleotide molecules are selected not primarily for base-pairing with other nucleotides, but rather for their ability to interact in a highly selective manner with proteins. These aptamers are identified using a serial binding–enrichment– expansion paradigm called SELEX (systematic evolutions of ligands by exponential enrichment). Oligonucleotides identified through this process form stable structures that specifically bind the target protein, even though the protein and the aptamer are structurally different macromolecules. Like other nucleic acid-based therapeutics, aptamers are modified to limit digestion by endogenous nucleases. As with candidate protein therapeutics, demonstration of specific cross-reactivity of the aptamer to the target is needed to demonstrate pharmacologic relevance of the animal species proposed for non-clinical testing. The non-target-specific toxicity profile of aptamers is similar to that of other oligonucleotide therapies, with accumulation in mononuclear phagocytes being the most consistent finding. Additional modifications such as PEGylation can be made to optimize the PK profile. As with many other biomolecules, aptamers are typically administered via parenteral routes. Pegaptanib (MacugenÔ ) is an approved PEGylated aptamer specific for

vascular endothelial growth factor (VEGF) that is given via intraocular injection to treat macular degeneration. Overall, the oligonucleotide-based therapies have demonstrated strong potential for therapeutic benefit, but delivery, targeting, and exposure remain hurdles for successful application of the technology. Modifications of the backbone, development of alternative liposomal and synthetic nanodelivery systems, and in vivo expression systems using viral vectors for delivery are all being explored. The non-clinical assessment for these products typically requires a fairly extensive assessment of ADME, including mass balance, metabolism, and tissue distribution studies. In addition, the toxicity profile may be both target- and sequence-independent. Therefore, at present the development considerations for oligonucleotide therapeutics fall closer to the small molecule spectrum than to the development pathway typical of the protein therapeutics. CELL-BASED THERAPIES

Therapies based on the administration of intact, viable cells are an emerging therapeutic area in both human and veterinary medicine. The typical application is in the oncology setting to increase and/or accelerate a tumor-specific immune response. For instance, a common approach is to isolate a host’s leukocytes followed by selective ex vivo culture and activation prior to returning them cells to the host. An alternative strategy is to create a tumor-based whole-cell “vaccine” using concentrated tumor cells expressing one or more antigens. In these settings, each “product” is unique to the patient/host, so safety assessment has focused on the potential impact of processes conducted ex vivo on the isolated cells. Particular steps considered for evaluation include the isolation procedure and cell/antigen formulations, to ensure that no added and controllable toxicity is introduced by manipulating the cells ex vivo. Similar approaches have been considered for manipulated host/patient cells intended to restore missing hematopoietic cell lineages (e.g., hematopoietic stem cells from fetal umbilical cord blood to correct immunodeficiencies or treat autoimmune diseases or cancers) as well as to support structural repair of tissue defects (e.g., mesenchymal stem cells to repair osteoarthritic

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erosions in joint cartilage). In this regard, the bulk of current research is on the isolation and manipulation of various stem cell populations. The therapeutic use of stem cells remains controversial, chiefly due to the reliance on embryonic stem cells for early experiments. However, evolving technology for isolating autologous somatic stem cells from adult tissues may succeed over time in dampening the controversy. Isolation and introduction of more specific cell types, including precursors for glial cells, neurons, retinal cells, and pancreatic islets, are also being explored as cures for neurodegenerative diseases and diabetes. Finally, the potential reconstitution of full tissues, such as construction of an artificial organ like liver or lung, is also an area of active research. When whole-cell therapies are used, exposure to the test article may be determined through a combination of dose-ranging and biodistribution studies in lieu of standard PK endpoints. Although guidelines are available, toxicity testing does not follow a standard paradigm for whole-cell therapies. For these products or approaches, the safety and pharmacology may both be have to be determined using disease models. Such testing is abbreviated when the test article is comprised of host-origin cells. However, more extensive testing for adverse effects will typically be necessary when the product includes non-host components. Examples of such exogenous material may include the cells themselves (e.g., porcine islet cell xenografts) as well as the scaffolding needed to support or confine them (e.g., porous membranes to contain subcutaneous xenografts, connective tissue matrices for three-dimensional organ reconstruction). In these latter instances, the safety assessment profile will likely require testing of the isolated components as well as the combined product.

3. SAFETY EVALUATION STRATEGIES FOR BIOPHARMACEUTICALS 3.1. Regulatory Resources The FDA Center for Biologics Evaluation and Research (CBER) web page (http://www.fda.

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gov/BiologicsBloodVaccines/ResourcesforYou/ default.htm) offers the following definition for biopharmaceuticals (“biologics”): Biological products include a wide range of products such as vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins. Biologics can be composed of sugars, proteins, or nucleic acids or complex combinations of these substances, or may be living entities such as cells and tissues. Biologics are isolated from a variety of natural sources – human, animal, or microorganism – and may be produced by biotechnology methods and other cutting-edge technologies. Gene-based and cellular biologics, for example, often are at the forefront of biomedical research, and may be used to treat a variety of medical conditions for which no other treatments are available.

Initially, all biologics were regulated through the CBER. However, between June 2003 and October 2004, all recombinant protein therapeutics, with the exception of those designated as vaccines or blood products, instead were transitioned to the FDA Center for Drug Evaluation and Research (CDER). At present, the majority of new protein therapeutics intended for human use, including therapeutic mAbs, along with small molecule therapeutics are regulated in the US according to their target indication rather than their composition (biomolecule or chemical). This approach allows for a more efficient regulatory review by ensuring that clinical studies in a given therapeutic area are reviewed according to consistent criteria. However, the non-clinical programs may be quite different for a small molecule drug versus a biologic agent, even when they have the same target and are expected to produce similar outcomes. For example, small molecule kinase inhibitors such as erlotinib and gefitinib and mAbs like cetuximab and panitumumab all target the human epidermal growth factor receptor (EGFR). All of them are used to treat cancers such as non-small cell lung cancer (NSCLC), colorectal cancer, and squamous cell carcinoma of the head and neck where EGFR is a contributing factor to tumor growth. Nevertheless, while the indications for each are overlapping, they are not identical. The clinical toxicity profiles of EGFR antagonists overlap to some degree (i.e., each lists dermatologic toxicity occurring in > 20% of patients). However, these agents are also distinct for each; based on current label information, infusion reactions are slightly more common with cetuximab than

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panitumumab, and only the small molecule inhibitors list hepatotoxicity as a potential adverse effect. These variations coupled with the inherent differences between biomolecules and small molecules require divergent designs for the product registration pathways of these agents. During non-clinical safety assessment, the small molecules followed a conventional two-species toxicity testing paradigm. In contrast, the mAb products did not cross-react with rodent EGFR, so in vivo non-clinical studies for these proteins were limited to a single pharmacologically relevant species (cynomolgus monkey). This seemingly simple but profound difference highlights why no “standard” development pathway can be defined for registering biomolecules. In recognition of the different non-clinical testing challenges for biologics versus small molecule therapeutics, specific guidelines have been written by the FDA, Organisation for Economic Co-operation and Development (OECD), and other health authorities for the development of biologics. Furthermore, the scope of other guidelines intended more for traditional small molecule therapeutics often have been updated to contain remarks regarding their potential applicability to biopharmaceutical agents. Many of these guidelines have now been harmonized among the United States, European Union, Japan, and United Kingdom under the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The current ICH Safety (“S”) and Multidisciplinary (“M”) Guidelines of relevance to biologics development are listed in Table 25.2, with reference to the year each guideline went into effect. Of note, the ICH S6 guideline for Biotechnological Products has been recently revised, and several new guidelines are currently under development, including one for Gene Therapy (also listed in Table 25.2).

3.2. General Toxicity Studies for Biologics Single-Dose Studies Single-dose toxicity studies are commonly performed in the development of small molecular weight drugs for two reasons. First, small maximum tolerated dose (MTD) studies that

precede short-term repeat-dose studies are meant to identify doses that will be tolerated when used during longer-term repeat-dose studies. Second, single-dose studies with small molecules are intended to identify and characterize the acute toxicity profile that might occur if a subject taking the drug was exposed to unexpectedly high plasma concentrations over a short period, such as might occur with overdose or idiosyncratic metabolic characteristics of an individual patient or group of patients. For biotherapeutics, the former of these two reasons is relevant and typically drives the conduct of single-dose studies for molecules or targets where toxicity is expected and a tolerable exposure level needs to be defined. However, the second justification is of limited or no utility for biomolecules. Exposures among individuals given a biological agent are generally very consistent due to the lack of metabolism by inducible enzymes. Furthermore, mis-dosing is also less common with parenteral administration. Finally, the pharmacologically mediated toxicities associated with biotherapeutics are generally not related to high acute exposures (maximum concentration [Cmax]) but more often are related to sustained interdiction in the pharmacologic pathway over time (cumulative area under the curve [AUC]). For these reasons the primary place for single-dose toxicity studies for biotherapeutics, if indeed it is felt necessary to use them, is in early phases of development when the goal is understanding what dose levels to use in repeat-dose toxicity studies meant to support clinical development. In our experience, single-dose studies are most commonly employed to evaluate the single-dose PK profile over time. In general, single-administration dose-range finding is only considered for biomolecules if the anticipated physiological response will occur in a short time (e.g., glucose normalization following an insulin injection). Dose-range finding for biologics often requires long-term studies with extended exposure to produce and detect a response, and thus is typically explored using a repeat-dose design with multiple dose groups (see next section). Repeat-Dose Studies With the exception of routes of administration and the frequency of dosing, repeat-dose toxicity

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TABLE 25.2

ICH Guidelinesa

Guideline

Topic

In effect (year)

ICH S1 (A-C)

Carcinogenicity Studies

1995 (A), 1997 (B), 2008 (C)

ICH S2 (R1)

Genotoxicity Studies

2008

ICH S3 (A, B)

Toxicokinetics and Pharmacokinetics

1994

ICH S4

Duration of Chronic Toxicity Testing

1998

ICH S5 (R2)

Reproductive Toxicology

2000

ICH S6b (R1)

Biotechnological Products

2011

ICH S7a

Safety Pharmacology

2000

ICH S7b

(Prolongation of QT testing)

2005

ICH S8

Immunotoxicology Studies

2005

ICH S9

Nonclinical Evaluation for Anticancer Pharmaceuticals

2009

ICH M3

Nonclinical Safety Studies

2009

Gene Therapy

Draft

General Principles to Address the Risk of Inadvertant Germline Integration of Gene Therapy Vectors

2006

Oncolytic Viruses

2009

General Principles to Address Virus and Vector Shedding

2009

ICH M6c ICH Considerations (Gene Therapy Working Group)

c

a

ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) Guidelines have been harmonized across the regulatory authorities in Europe, Japan, and the United States. This table includes a listing of ICH guidelines that are specific for or include general references relevant to the non-clinical testing of biopharmaceutical products. b ICH S6 is the only guideline that is specific for products of biotechnology. ICH “S” (safety)-type guidelines can be found at http://www. ich.org/products/guidelines/safety/article/safety-guidelines.html, while ICH “M” (multidisciplinary)-type guidelines can be found at http:// www.ich.org/products/guidelines/multidisciplinary/article/multidisciplinary-guidelines.html (last accessed December 2011). c The Gene Therapy Guideline is currently in draft form, and is supported by the previously published considerations documents prepared through the Gene Therapy Working Group.

studies for biotherapeutics are generally conducted in a manner very similar to corresponding studies for small molecule drugs. As with all such studies involving novel therapeutic agents, the design, duration, and timing of studies needed to support clinical development for specific indications and in specific regulatory jurisdictions should take into consideration the relevant governing regulatory guidelines. In the event that such documents are lacking, an ad hoc approach to assembling the non-clinical package is required. In this case, sponsors should consult regulatory agencies well in

advance regarding potential design parameters for toxicity testing. Twenty-eight day (1-month) toxicity studies are generally sufficient to support most first-inhuman (FIH) clinical studies, with the duration of longer-term repeat-dose studies determined by the particular clinical development program under consideration. If pharmacologically relevant, both rodent and non-rodent studies should be conducted prior to establishing clinical experience. Many biopharmaceuticals are highly specific and may not cross-react with the rodent target, in which case such studies are not

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informative to the program and should not be conducted. In the extreme instance of specificity excluding non-human primates (NHP) as well, a single non-clinical safety study, often only in a rodent, may be conducted to assess the potential for off-target toxicity, but these studies are considered of very limited value to inform clinical safety. This approach might apply to viral antigen targets where no relevant infection model or homolog exists. There is more variability in the applicability, design, and conduct of longer-term studies, including the use of a single or multiple species. In some cases, these long-term animal studies may also be informed by results from the earliest human clinical studies. In an effort to reduce NHP use in non-clinical safety studies, and assuming that the needs of the clinical development program allow this modification, it may be desirable to eliminate the 13-week (3-month) repeat-dose study in a non-rodent species for biologics development even though such studies are nearly always a component of small molecule development programs. In such cases, a 26-week (6-month) repeat-dose toxicity study in NHP would be used to support longer term clinical exposures and to fulfill requirements for a biological licensing application (BLA). Likewise, if the FIH study requires a longer dosing period, a longer initial study of 2–3 months may be considered instead of 1-month study, again reducing the total number of NHP studies ultimately required to support licensing. Because they are biologic molecules and will be enzymatically destroyed if given orally, biotherapeutics are typically administered by parenteral routes (e.g., subcutaneous, intravenous, intrathecal, or intravitreal injection). Accordingly, thoughtful collection and careful microscopic examination of the administration sites are vital components of the overall safety assessment of biotherapeutics administered by these routes. If the route of administration is intravenous or subcutaneous, injection sites will commonly be rotated throughout the course of a repeat-dose study; thus, collection and examination of more than one of these sites will afford an evaluation of both acute and chronic effects of the biomolecule under study. In general, repeated intradermal injections of biologics intended for therapeutic purposes are not used due to the potential for activating antigen-presenting cells

in this location, resulting in a potent immune response against the therapeutic. Unlike most small molecule drugs, dosing frequency in a repeat-dose toxicity study with a biopharmaceutical is rarely daily, due to the relatively long elimination half-life. Instead, a common schedule for mAbs is to dose on a weekly basis, as this generally corresponds to a single half-life for most antibodies in nonhuman primates and is appropriate for maintaining a relatively consistent steady-state blood concentration. Other biologics may permit dose intervals of 2 weeks, a month, or even several months. The dose interval is set using the PK profile established during the single-dose study, although additional confirmatory PK data are often acquired during the repeat-dose studies. Safety Pharmacology Studies There are clear guidelines for the evaluation of cardiovascular, respiratory, and neurologic safety pharmacology (“core battery”) as part of the non-clinical assessment of a novel therapeutic. The potential impact of biopharmaceuticals on these organ systems is relevant to the overall non-clinical safety assessment of biomolecules, but a standard “small molecule”-like safety pharmacology testing approach is rarely appropriate for biologics. Although inclusive of biologics in scope, the ICH guidelines for safety pharmacology were largely written from a small molecule development program perspective. Foundational assumptions in these guidelines are that the following practices are all relevant to product development: toxicity testing in both a rodent and a non-rodent species is needed; the test agent has a short half-life (enabling a rapid “washout period”); each animal may be used as its own control; and comparison to vehicle controls and, where necessary, small molecule positive controls is useful for interpretation of results. In addition, the small molecule setting, without the likelihood of test article-induced immunogenicity, allows for non-naı¨ve animals to be serially reused for non-terminal in vivo studies. For most biopharmaceuticals, however, recommended practices formulated under these small molecule-oriented assumptions are difficult if not impossible to apply. As an example, let us consider the suitability of cardiovascular safety pharmacology guidelines

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as they might apply to development of a biologic. The general concern for small molecules in this regard is their potential for altering ion channel (hERG) conductance, and thus the electoral rhythm, of cardiac muscle cells. However, large proteins have not been shown to have a direct effect on ion channel conductance to date. In addition, typical formulations for protein therapeutics are not compatible with patch clamp testing, as the presence of polyethoxylated sorbitan emulsifiers needed to minimize protein aggregation will impact cell membrane-based ionic gradients. Therefore, the in vitro hERG screen, used to identify risk of Torsade de Pointes with small molecules, is generally not directly applicable to proteins. To date, an integrated approach to safety pharmacology assessment for protein therapeutics has been the norm applied to most biopharmaceuticals, with directed mechanism-ofaction (MOA) studies for a specific issue being performed only following the identification of non-clinical or clinical signals suggesting such an assay will provide relevant data. Thus, the pathology findings from general toxicology studies for biologics, or even the tissue-binding profile (e.g., “cross-reactivity” of mAbs with non-target tissues), may be used to influence the types of investigation that may be required. When conducted, the spectrum of tests used for biologics is comparable to that employed for small molecules, although the study design typically must be adapted to acknowledge the protein rather than the chemical nature of the test article. Developmental and Reproductive Toxicity (DART) Studies Assessment of potential male and female reproductive toxicity should be guided by the expected pharmacology, the target patient population, and any relevant signals in the toxicology program. If rodents and rabbits are pharmacologically relevant, reproductive toxicity testing of biologics can be done in these species. In particular, the period of embryofetal development is short enough that the gradual onset of a neutralizing antibody response may not preclude adequate coverage (i.e., sufficient accumulation of the candidate molecule at the target site) and eventual interpretation. However, many biopharmaceuticals are

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pharmacologically active only in non-human primates (NHP). A reproductive toxicity assessment in NHP is primarily an exercise in hazard identification rather than a robust and translatable risk assessment. Even so, the evaluation may have several components, including evaluation of male and female fertility parameters, embryofetal development, and pre- and postnatal growth and development (see Embryo and Fetus, Chapter 62). The first look at potential reproductive toxicity typically occurs during general toxicology studies of at least 3 months’ duration that include at least some mature male and female animals. In this setting, the histologic assessment of reproductive tissues should identify any consistent findings likely to affect fertility. In males, histology of the testis is considered to be the most sensitive indicator of an effect on fertility, and typically should incorporate qualitative spermatogenic staging (see Male Reproductive System, Chapter 59). In females, even if all tissues are normal, the tissues available for histological analysis should include all the different phases of the menstrual cycle (see Female Reproductive System, Chapter 60). Mature female macaques do not typically cycle together even with prolonged co-housing, so if all females in treated groups are in the same phase of the cycle, it may reflect a treatment-related effect on cycle progression. If these morphological assessments of adult tissues do not identify any effect, additional fertility studies may not be needed. However, serial hormone assessments in males or females, semen evaluation in males, and evaluation of menstrual-cycle length in females may be conducted for additional information on an asneeded basis. Breeding studies are not typically considered appropriate when assembling the biopharmaceutical safety in NHP due to the high variability in natural conception rates and the relatively high early gestation losses in these species. Pregnancy studies for biologics in NHP should be conducted for cause (i.e., an apparent effect of treatment on gestational outcome), and not as a matter of course. Other information that may influence the need for an in vivo pregnancy study in NHP includes the patient population (age demographics; disease type, severity, and prognosis; concomitant medication) and knowledge of the targeted biology

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and pharmacology, including information derived from animal models. In other words, if a genetic knockout of a target is known to be lethal to a developing mouse embryo, then saturating inhibition of that target in a pregnant human patient is assumed to carry an inherent risk to the developing fetus. In this situation, a pregnancy study in NHP may not be warranted as the existence of the risk is already established, while the primate study will not be able to quantify the degree of risk given the small group sizes. However, there are numerous instances where pregnancy studies are needed to inform appropriate labeling and use of biopharmaceuticals intended for use by women of childbearing age. Recently, efforts have been invested in combining the embryofetal and pre- and postnatal development studies where possible to reduce animal use. In this setting, an interim report including data available through postpartum day 7 can be submitted (if needed) prior to registration to support the clinical program. The interim report would contain data such as pregnancy losses, stillbirths, neonatal losses, and body weights of dams and offspring, as well as information regarding the external physical examination, neurobehavioral assessment, and radiography of the neonates. These assessments replace the more traditional strategy of dosing through closure of the hard palate at gestation day (GD) 50 and cesarean section on GD100. The new approach is influenced by the well-documented active transfer of mAbs across the placenta primarily in the third trimester, or GD100–150, in the cynomolgus monkey. However, even very low concentrations of biopharmaceuticals may be pharmacologically active in a developing fetus. The combination design described above also supports evaluation of non-teratogenic effects on the developing fetus when a biopharmaceutical is administered to the dam during pregnancy. Post-partum development at least through weaning is typically also included, with evaluation up to 12 months post-partum in some cases. The offspring are often necropsied at the end of the post-partum evaluation period, at which time any soft-tissue developmental abnormalities can be identified. In addition, for some immunomodulatory agents, functional assessment of immune system development

(see Immune System, Chapter 49) may be incorporated into the infant/juvenile assessments. Assessment of Carcinogenic Potential As directed by national and international regulatory guidelines, the need to assess the carcinogenic potential of a therapeutic biomolecule should be determined by the likely conditions of clinical use, a situation that is similar to that used for small molecular weight drugs. In essence, for any therapeutic where there is the reasonable potential for intermittent or continuous drug exposure up to 6 months in duration, there should be an assessment of carcinogenic potential. For small molecular weight drugs, this has traditionally been done through the use of lifetime (2-year) carcinogenicity bioassays in two rodent species, rat and mouse, although the recent trend has been to replace the 2-year assay in wild-type mice with a 6-month assay in a mouse line genetically engineered to exhibit enhanced susceptibility to carcinogens (see Carcinogenicity Assessment, Chapter 27). However, this long-term bioassay approach is generally not feasible for protein therapeutics, particularly for mAbs and related molecules targeted to human antigens, due to the lack of appropriate animal models. There are multiple reasons behind the conclusion that the traditional rat and mouse carcinogenicity testing models are generally not appropriate for use with biologics. First, protein therapeutics are catabolized to their constituent amino acids and not metabolized to DNAreactive or interactive molecules. Thus, adverse effects of biologics, including carcinogenic potential, are due to intended or unintended epigenetic (non-genotoxic) pharmacologic effects. Second, the highly species-specific nature of interactions between therapeutic biomolecules and their targets typically precludes even highly homologous proteins from different species from interfacing in a manner sufficient to trigger the pharmacologic effects that are being assessed for carcinogenic potential. Thus, CDRs from mAbs designed to bind to human protein targets may bind to the rodent homolog target proteins only very weakly or not at all, preventing an adequate examination of the effects of prolonged pathway inhibition on carcinogenic potential. Finally, because the common humanized and human mAbs and other therapeutic proteins contain

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amino acid sequences that are perceived as “foreign” by rodent immune systems, immune responses may be mounted against those proteins which will block their pharmacologic effects and/ or increase their elimination through FcRn-mediated clearance. This activity prevents the sustained, lifetime exposure in rodents required to assess carcinogenicity in those models. An additional consideration is that prolonged immune stimulation by repeated dosing with the biologic could even incite a false positive carcinogenic response if one or more lineages of hyperactive leukocytes (typically B lymphocytes) were to undergo spontaneous neoplastic transformation. These reasons should not be construed as an absolute negation of the rodent 2-year bioassays for assessing the carcinogenic potential of biotherapeutics, but instead as a note of caution that the instances in which this platform is likely to generate relevant and translatable results for a biologic product will represent a relatively low proportion of the total number of candidate biotherapeutics. Other potential means of performing risk assessments for carcinogenic potential of protein therapeutics have been devised. The list is fairly impressive: 1. Review of the literature to glean data regarding the potential role of the impacted pharmacologic pathway in carcinogenesis. 2. In vitro and/or in vivo examination of the effects of the biotherapeutic on cell proliferation. 3. In vivo examination of the effects of the biotherapeutic on tumor xenograft growth enhancement. 4. Use of genetically modified animals as indirect probes of carcinogenic potential, using one or more of the following approaches: a) Thorough phenotyping of animals lacking the target against which the protein therapeutic is directed (knockouts) to provide data regarding the lifetime effects of target pathway blockade. However, knockout animals may bring to bear physiologic compensatory mechanisms such that full absence of the targeted function may not occur, even in knockout animals confirmed to lack the targeted molecule.

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b) Humanized mice, containing the fully human form of the target (i.e., identical in amino acid sequence to the human protein) may be used as an animal model to examine the effects of a human-directed biotherapeutic that is not active against the native murine form of the drug target. However, use of such a model requires extensive, thorough, and resource-intensive biologic characterization of the upstream and downstream molecular features of the affected pathway to understand the similarities and dissimilarities between rodent and human systems. In particular, expression of a human target protein in a mouse does not ensure that the human protein will interact effectively with the remainder of the murine signal transduction pathway. 5. In vivo rodent assessment conducted with a rodent-specific “homolog” molecule – in other words, production of a parallel biologic engineered to recognize the endogenous rodent form of the target against which the human biotherapeutic is directed – may be used for toxicologic assessments, including those concerning carcinogenic potential. This approach theoretically overcomes the abovementioned obstacles with respect to the lack of species-specific drug–target interactions and the likelihood that chronic exposure to foreign biomolecules will elicit an immunogenic response. However, as noted above for other genetically altered mouse models, extensive characterization is necessary to understand the relevance and utility of models using rodent homolog molecules to their human counterparts. 6. Consideration of findings from chronic toxicology studies. While absence of precancerous lesions from a chronic nonhuman primate toxicity study does not ensure the absence of a potential carcinogenic effect of the biotherapeutic in other species, the presence of tumors in non-human primates would be taken to suggest that it is possible that the biologic agent had the potential to induce a carcinogenic response. 7. Examination of the status of oncogenic viruses. Biotherapeutics with intended immunomodulatory effects may alter mechanisms of viral control, particularly with

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regard to oncogenic and latent gammaherpesviruses. Numerous methodologies are in common use to quantify levels of virus and viral gene products in tissues and blood in non-human primates. Elevations in those endpoints may indicate a propensity toward virally-mediated tumor formation. However, it is reasonable to assume that all potent immunomodulatory agents carry this risk.

comprising the general toxicology program, especially for non-human primates, and may be used as a matter of course to evaluate any potential impact of a biologic agent on reproductive maturity. If the relative sexual maturity of the animals on the study is documented according to histologic attributes, developmentally sensitive toxicities may become apparent without the need for additional dedicated studies.

In summary, while traditional rodent lifetime (2-year) bioassays are generally not appropriate to assess the carcinogenic potential of biotherapeutics, numerous other means are available. One or possibly more of these methods typically should be employed to form an overall assessment for carcinogenic risk for biologics intended for chronic therapeutic indications.

Tissue Cross-Reactivity Studies Tissue cross-reactivity studies are intended for antibodies and antibody-like molecules. These studies utilize immunohistochemistry as one means of evaluating the possible spectrum of binding activity for candidate therapeutics that include a CDR. When initially requested by regulatory authorities, these studies were intended to identify potential unexpected tissue localization (“off-target” binding) of biomolecules in human tissues to inform monitoring during subsequent clinical development. By labeling, in parallel, tissues from the animal species selected for in vivo toxicity studies, cross-reactivity data also became part of the support for the pharmacologic relevance of those test species. For example, if a candidate therapeutic specifically stained a few tissues or compartments from humans, but also reacted with the same compartments in a non-clinical species, then there would be increased confidence that hazards identified in the in vivo safety program with that animal species might be relevant to predicting outcomes in human studies as well. However, these studies are only one component of the overall safety assessment package for antibody therapeutics, and results should be interpreted in the context of other available data. In addition, the application of the antibody to tissue sections often identifies positive staining that is not uniformly relevant. For example, cytoplasmic labeling by antibodies directed against an extracellular target is unlikely to reflect potential in vivo binding, as the cytoplasmic compartment is not typically exposed in vivo. In designing a strategy for performing an immunohistochemical cross-reactivity study for a mAb, it is important first to identify appropriate positive and negative controls with which to test the method. Ideally, a positive control tissue will have the expected distribution of the

Studies to Support Pediatric Use As with the rest of the toxicology program, testing strategies to support use of biopharmaceuticals in pediatric populations should follow the known pharmacology of each product, and be mapped closely to specific attributes of the target patient population. Important drivers for expanding clinical use of any therapeutic agent in a younger patient population include potential differences in PK between juvenile and adult patients, and a thorough knowledge of potential effects on general growth and development, neurobehavioral maturation and learning, immune system development, and future reproductive potential. For large molecule biopharmaceuticals, the role of enzyme induction or metabolism as a mechanism for affecting exposure levels is much less than that of some small molecules. Most protein therapeutics are eliminated by relatively simple metabolism by the liver into small peptides or amino acids which are either recycled into new proteins or cleared by the kidney. A thorough understanding of the test article’s pharmacology during development is important, including consideration of possible developmental effects on humans based on the expected mechanism of action (with or without confirmatory animal data). As a routine practice, it can be helpful to capture data supporting the relative maturity of animals used in the general toxicity studies. A spectrum of sexually immature, peri-pubertal or mature animals may be allocated to studies

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target antigen as this will most closely mimic the presumed reactivity in other tissues. Other positive control “tissue” options, of decreasing relevance to the tissue reactivity being investigated in non-control tissues, include (1) cell pellets made with established cell lines known to express the target antigen, (2) cell pellets prepared after transfection of cultured cells with an expression vector encoding the target antigen, and (3) recombinant or isolated target antigen crosslinked to beads or directly to the slide. For each of these methods, a relevant and similar tissue, cell pellet, or protein that is not expected to include the target antigen should be selected as a negative control tissue. These controls are all effective for confirming that the test antibody is intact and capable of binding the target antigen with some specificity, although differences in antigen concentration in some of these controls make it difficult to optimize staining for tissue expression levels. Not all mAbs are good immunohistochemical reagents. Therefore, a robust method cannot always be developed for performing such studies. Conduct of tissue cross-reactivity studies is not recommended in the absence of confirmation of the method with relevant controls.

3.3. Toxicologic Pathology Findings with Biopharmaceuticals Discussion of Exaggerated Pharmacology vs Toxicity More so than perhaps with small molecular weight drugs, interpretation of toxicology and pathology data from safety assessment studies for biomolecules often requires very thorough knowledge of the pharmacologic mechanism of action for the test article under study. A fundamental underlying principle regarding nonclinical safety assessment of protein therapeutics is the concept of “exaggerated pharmacology.” In other words, both non-adverse (i.e., therapeutic) or adverse (so-called “toxic”) effects of biomolecule exposure can be predicted based on the pharmacologic mechanism of action of the molecule under study. This expectation that adverse findings from biotherapeutic toxicity studies will only be excessive “on-target” outcomes of supra-therapeutic exposure, as opposed to “off-target” toxic

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damage in other tissues, is based on several unique features of biomolecules. First, mAbs, fusion proteins, and other protein therapeutics will be catabolized to their constituent amino acids, and thus will not be metabolized to other reactive molecules that may interact with molecular and structural components of the target cells and/or other non-targeted cells. Second, biotherapeutics are exquisitely specific with respect to the target molecules with which they will bind. While there are examples in nature of receptor–ligand promiscuity, until proven otherwise the working assumption is that a biomolecule will interact only with the molecule it was designed to recognize and bind. Finally, due to their chemical structure, protein therapeutics are not endowed with properties that would elicit non-specific “chemical toxicity” similar to those of small molecules, such as phospholipidosis, lipid peroxidation, or mitochondrial toxicity. A caveat to this last point is that some proteins are conjugated to chemical polymers in the hope of extending their half-life in circulation, and some of these polymers (e.g., PEG) can accumulate over time in non-target cells. Examples of exaggerated pharmacology observed in the course of biotherapeutic nonclinical safety assessment are readily obtained by a brief review of the published literature. Bevacizumab (AvastinÒ ) has been linked to physeal dysplasia in cynomolgus macaques secondary to reduced capillary invasion of epiphysis due to VEGF receptor (VEGFR) blockade. Alefacept (AmeviveÒ ) induces widespread lymphoproliferation in cynomolgus macaques due to recrudescence of gamma-herpesviral lymphocryptovirus resulting from anti-CD2-mediated T lymphocyte depletion. Natalizumab (TysabriÒ ) decreases red cell mass in fetuses of pregnant cynomolgus monkeys because the anti-VLA4 antibody crosses the placenta and disrupts sites and patterns of fetal hematopoiesis. Similarly, the occurrence of certain pathogen-mediated diseases in association with specific marketed biotherapeutics that target signaling pathways in the immune system is assumed to be due to an excess of those pharmacologic actions expected to be elicited by the therapeutic. Examples of this kind include progressive multifocal leukoencephalopathy associated with administration of rituximab (RituxanÒ ), natalizumab, or efalizumab as well as enhanced granulomatous

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responses to some intracellular pathogens following treatment with the TNF inhibitors etanercept (EnbrelÒ ), infliximab (RemicadeÒ ), or adalimumab (HumiraÒ ). Several explanations have been defined to account for those instances where findings from biotherapeutic non-clinical safety assessment studies do not seem to fit with the expected consequences of interdiction in the targeted pathway. One possibility is that cross-reactivity occurs between the biotherapeutic and one or more molecules in a related or unrelated biological pathway. An alternative is that unexpected findings are, in fact, due to engagement of the intended target, but that current understanding of the anatomical distribution of the target and/or functional roles of the biological pathway within which the target is found is too incomplete to have permitted a reliable prediction of the full range of expected “on-target” effects. Deconvolution of unexpected findings in these instances may require additional hypothesisdriven investigative experiments that lie outside the scope of traditional regulatory safety assessment studies. During the early phases of developing a biotherapeutic, there may be variable and evolving levels of understanding of the relationship between test article-exposure, pharmacology, pharmacodynamic (PD) markers, and, by extension, toxicity. For many new pharmaceuticals, including biotherapeutics, reliable PD markers may not have been identified in the early stages of drug development when the initial safety assessment studies to enable first-in-human (FIH) clinical trials were being planned or performed. Also in these early stages, attempts are often made to correlate effects observed in animal models (usually mice) with effects visible in toxicity models (usually macaques) with anticipated effects in clinical subjects (healthy or diseased humans), each of which is a fundamentally different experimental population. If a reliable PD marker is available, it would ideally have the properties of being highly related to the pharmacologic site of action, be sensitive to dose–response pharmacologic effects, possess a wide dynamic range, and be easily measurable in peripheral blood samples. The point in emphasizing PD markers here is to reiterate how useful they can be when trying to interpret exaggerated and adverse

pharmacologic effects during the early stages of drug development when one often may be operating in the dark with respect to drug exposure levels that constitute the range bracketing minimal to maximal pharmacologic effects. For example, the lack of an apparent dose response with regard to the occurrence of an adverse pharmacologic effect may be explained by results of PD marker measurements that support saturation of pharmacologic effects at the lowest doses. Conversely, if expected adverse pharmacologic effects are not noted, PD marker measurements may indicate that drug exposure was not sufficient to have elicited expected pharmacologic effects. One such example would be a case where anti-drug antibodies do not result in accelerated elimination of the biotherapeutic (i.e., adequate exposure levels are maintained) but instead act to neutralize the biomolecule and prevent its interaction with its target. In such instances the PD marker reveals the lack of the predicted pharmacologic effect, indicating that adequate levels of test article in the absolute sense did not translate into adequate levels of active test article. In general, development programs for biomolecules require a greater up-front investment in basic biological understanding (i.e., more focus on pharmacologic mechanism-of-action) than may be necessary for traditional small chemical entities which tend to have both on-target and off-target activities. This perspective is backed by the amount of knowledge required to effectively translate pharmacologic data from mice to understand toxicity findings in macaques so that predictions can be made regarding safe use conditions in humans for a novel biomolecular entity. One of the most important factors in this regard is knowing the degree to which relevant biological pathways are conserved across the species. Is the relevant signal transduction pathway more or less the same in all three species? If not, what are the differences, and how might they affect efficacy and toxicity outcomes? Another critical factor is to know the comparative pharmacologic activity of the homologous therapeutic molecules used in the three species. In other words, when setting expectations about effective doses in humans, are the pharmacologic effects of a murinespecific antibody used in mouse studies similar qualitatively and quantitatively to effects elicited by the human or humanized antibody in

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monkeys or humans? While greater clarity is evolving with practice, in our experience the development pathways for biotherapeutics are unlikely to readily fall into line with the fairly regimented numbers and tests that are used routinely when developing a small chemical entity.

4. SUMMARY AND CONCLUSIONS Overall, the biomolecule products of recombinant DNA technology have redefined therapeutic possibilities, and have notably changed the practice of human and veterinary medicine. In the process, these accelerating technological advances have also challenged the conventional non-clinical testing paradigms established for synthetic small molecule therapeutics. In particular, the pharmacologic relevance of the test systems plays a key role in the justification of non-clinical studies and interpretation of data when developing biotherapeutics. Drug metabolism and excretion is generally more predictable for biopharmaceuticals. Most adverse effects related to biomolecules are aspects of “exaggerated pharmacology” (too much of a good thing), while non-specific or off-target effects may be limited or even absent. With accumulated experience, we have also learned more about the effects of immune responses to xenoproteins, and the translatability of non-clinical safety assessment to the clinical setting, thereby permitting even more refined designs that should help shorten the timeline for future biomolecule development efforts. Although it can be argued that the safety assessment of biopharmaceuticals has included more challenges than were initially expected, there have also been repeated confirmations of their early promise as agents capable of inducing highly targeted therapeutic effects with little if any baggage. The scientific community continues to gather information that can help guide the interpretation of results and design of appropriate studies and development programs for new generations of biopharmaceuticals. An integrated assessment of the pathology, pharmacology, and general physiologic effects of the biopharmaceutical, in both the non-clinical and clinical settings, should be

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considered in the overall risk assessment. Based on long familiarity with the “whole animal” approach to data analysis and interpretation, as well as the need for case-by-case modifications of a development program to address unexpected issues, the toxicologic pathologist is the ideally situated person to undertake such integrations. Discerning product development teams will recognize this expertise, and incorporate one or more toxicologic pathologists as central members of the team that is responsible for assembling the final summary for individual study reports and the pharmacology and toxicology sections of Biological Licensing Applications.

SUGGESTED READING General Books and Reviews Baumann, A., 2009. Nonclinical development of biopharmaceuticals. Drug. Discov. Today 14 (23–24), 1112–1122. Buckley, L.A., Benson, K., Davis-Bruno, K., Dempster, M., Finch, G.L., Harlow, P., Haggerty, H.G., Hart, T., Kinter, L., Leighton, J.K., McNulty, J., Roskos, L., Saber, H., Stauber, A., Tabrizi, M., 2008. Nonclinical aspects of biopharmaceutical development: discussion of case studies at a PhRMA–FDA workshop. Int. J. Toxicol. 27 (4), 303–312. Cavagnero, J.A., 2008. Preclinical Safety Evaluation of Biopharmaceuticals. John Wiley & Sons, Inc., Hoboken, NJ. Golan, D.E., 2012. Principles of Pharmacology, third ed. Lippincott Williams & Wilkins, Philadelphia, PA. Green, J.D., Tsang, L., Cavagnaro, J.A., 2003. “Generic” or “follow-on” biologics: scientific considerations and safety issues. Expert Opin. Biol. Ther. 3 (7), 1019–1022. Leader, B., Baca, Q.J., Golan, D.E., 2008. Protein therapeutics: a summary and pharmacological classification. Nat. Rev. Drug. Discov. 7 (1), 21–39. Rader, R.A., 2008. (Re) defining biopharmaceutical. Nat. Biotechnol. 26 (7), 743–751. Reilly, R.M., 2010. Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer. John Wiley & Sons, Inc., Hoboken, NJ. Strand, V., Kimberly, R., Isaacs, J.D., 2007. Biologic therapies in rheumatology: lessons learned, future directions. Nat. Rev. Drug. Discov. 6 (1), 75–92. van Tongeren, S., Fagerland, J.A., Conner, M.W., Diegel, K., Donnelly, K., Grubor, B., Lopez-Martinez, A., Bolliger, A.P., Sharma, A., Tannehill-Gregg, S., Turner, P.V., Wancket, L.M., 2011. The role of the toxicologic pathologist in the biopharmaceutical industry. Int. J. Toxicol. 30 (5), 568–582. Zuniga, L., Calvo, B., 2010. Biosimilars: pharmacovigilance and risk management. Pharmacoepidemiol. Drug. Saf. 19 (7), 661–669.

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II. SAFETY ASSESSMENT INCLUDING CURRENT AND EMERGING ISSUES IN TOXICOLOGIC PATHOLOGY