preclinical development handbook || safety assessment of biotechnology-derived therapeutics

695

19 SAFETY ASSESSMENT OF BIOTECHNOLOGY - DERIVED THERAPEUTICS

Mary Ellen Cosenza Amgen Inc., Thousand Oaks, California

Preclinical Development Handbook: Toxicology, edited by Shayne Cox GadCopyright © 2008 John Wiley & Sons, Inc.

Contents

19.1 Introduction 19.2 Species Specifi city and Selection

19.2.1 Species Selection 19.2.2 Nonhuman Primates 19.2.3 Surrogates and Other Alternatives

19.3 Immunogenicity 19.3.1 Types of Antibodies to Biotechnology - Derived Pharmaceuticals 19.3.2 Study Design Issues

19.4 Special Studies 19.4.1 Genotoxicity 19.4.2 Reproductive Toxicology 19.4.3 Carcinogenicity 19.4.4 Safety Pharmacology 19.4.5 Local Tolerance

19.5 Future Trends References

19.1 INTRODUCTION

Over the course of the last 20 years, the biotechnology fi eld has grown and is now a large part of pharmaceutical therapeutics development. According to a 2006 report from the Pharmaceutical Research and Manufacturers of America (PhRMA)

696 SAFETY ASSESSMENT OF BIOTECHNOLOGY-DERIVED THERAPEUTICS

[1] , approximately 125 biotechnology - derived pharmaceuticals have been approved and are on the market. In addition, over 418 biotechnology - derived pharmaceuticals are in development for over 100 different diseases, and many of these potential therapeutics are being developed for grievous illnesses such as cancer, autoimmune diseases, and AIDS/HIV. Therapeutics derived from biotechnology (often referred to simply as “ biologics ” ) fall into several categories, including growth factors, hor-mones, enzymes, receptors, monoclonal antibodies, cytokines, vaccines, and gene and cell therapies (Table 19.1 ).

Many of the early biotechnology - derived therapeutics were replacement mole-cules, that is, recombinant - derived proteins largely unaltered from the endogenous counterparts. These included recombinant forms of human insulin, human growth hormone, interferon, erythropoietin, clotting factors, and colony stimulating factors (G - CSF and GM – CSF). Enzymes such as Activase (TPA) and Pulmozyme (DNAase) were developed in the late 1980s to early 1990s. Over time, modifi ed versions of these proteins have been developed (e.g., pegylated proteins) as well as humanized and fully human monoclonal antibodies. More complicated molecules have been developed as well, such as fusion molecules (e.g., Enbrel, a peptide fused to a human IgG Fc) and conjugated antibodies (e.g., Mylotarg, a recombinant humanized anti-body conjugated to a cytotoxic antibiotic) (Table 19.2 ). Early monoclonal antibodies were murine derived and chimerized; with the advent of more advanced technolo-gies, monoclonal antibodies recently approved or currently in development are more humanized or fully human. These molecules bind to a specifi c antigen or epitope on a cell receptor. In addition, biotechnology - derived molecules are being designed to address more complicated diseases. New molecules are being developed to subtly alter immune function and to target specifi c ligand and receptor interac-tions. The background disease states are also more complicated and many molecules will be used in combinations with traditional pharmaceuticals or other biologics.

The regulation of these products differs by region and by type of product (e.g., replacement proteins versus DNA vaccines). Several years ago, a guidance from the

TABLE 19.1 Categories of Biotechnology - Derived Pharmaceuticals

Proteins Hormones Blood products Cytokines Growth factors Ligands and receptors Modifi ed human proteins Antibodies Murine Chimeric Humanized Fully human Fragments Gene therapies Cellular therapies Engineered tissue products Vaccines

International Congress on Harmonisation was developed to address the preclinical development and safety issues of most of these types of products (ICH S6 Preclinical Safety Evaluation of Biotechnology - Derived Pharmaceuticals ). ICH S6 defi ned bio-technology - derived pharmaceuticals as “ products derived from characterized cells through the use of a variety of expression systems including bacteria, yeast, insect, plant, and mammalian cells. ” The scope of this guidance included proteins, peptides, derivatives of these, or products of which they are components. It also states that these principles may apply to “ recombinant DNA protein vaccines, chemically syn-thesized peptides, plasma derived products, endogenous proteins extracted from human tissues, and oligonucleotide drugs, ” but does not cover “ antibiotics, allergenic extracts, heparin, vitamins, cellular blood components, conventional bacterial or viral vaccines, DNA vaccines, or cellular and gene therapies. ”

TABLE 19.2 Examples of Marketed Biotechnology Products a

Year of Approval Approved Biologics Trade Name

1982 Insulin Humulin ® 1985 Growth hormone Protropin ® 1986 Interferon alfa Roferon ® , Intron A ® 1986 Muromonab - CD3 Orthoclone OKT ® 3 1987 Alteplase (TPA) Activase ® 1989 Epoetin alfa Epogen ® 1990 Interferon gamma Actimmune ® 1991 Filgrastim (G - CSF)

Sargramostim (GM - CSF) Neupogen ® Leukine ®

1992 Aldesleukin (IL - 2) Proleukin ® 1993 Interferon beta

Dornase alfa (DNase) Betaseron ® Pulmozyme ®

1994 Imiglucerase ( β - glucocerebrosidase) Abciximab

Cerezyme ® ReoPro ®

1997 Rituximab Daclizumab Oprelvekin (IL - 11)

Rituxan ® Zenapax ® Neumega ®

1998 Trastuzumab Infl iximab Basiliximab Palivizumab

Herceptin ® Remicade ® Simulect ® Synagis ®

2001 Pegfi lgrastim (pegylated G - CSF) Darbepoetin alfa Alemtuzumab

Neulasta ® Aranesp ® Campath ®

2003 Alefacept Tositumomab Efalizumab

Amevive ® Bexxar ® Raptiva ®

2004 Bevacizumab Natalizumab Palifermin (KGF)

Avastin ® Tysabri ® Kepivance ®

a DNase, deoxyribonuclease I; G - CSF, granulocyte colony - stimulating factor; GM - CSF, granulocyte - macrophage colony - stimulating factor; IL - 2, interleukin 2; IL - 11, interleukin 11; KGF, keratinocyte growth factor; TPA, tissue plasminogen activator. Source : www.fda.gov .

INTRODUCTION 697


Regulatory agencies throughout the world defi ne some of these products differ-ently, which may affect how they are regulated. The main theme of the ICH S6 guidance is to create a case - by - case, science - driven approach to biotechnology pre-clinical product development. Each molecule should be fully examined for both its physical and pharmacologic properties. Its pharmacologic attributes need to be understood before embarking on a preclinical safety assessment development plan. The goal of any preclinical development plan is the same: to provide information for designing and conducting clinical trials. The biggest challenge is to use all of the information available and to conduct the best preclinical, in vitro and in vivo , experi-ments to best predict what will happen in humans. A rational, science - based approach is the best way to develop biotechnology - derived products [2] .

Many traditional small - molecule studies are not very appropriate for biologics [3] : some for “ technical ” reasons (the protein may be too large to enter an ion channel) and other studies for scientifi c reasons (monoclonal antibodies would not be expected to have direct genotoxic effects on DNA or chromosomes). Carcino-genicity studies are also not generally conducted for biotechnology - derived thera-peutics unless there is a “ cause for concern. ” In the early days of biotechnology development, there was greater concern for the technical issues of conducting these types of studies. These concerns included immunogenicity of human proteins in rodents and the technical challenge of parenteral administration in rats and mice over a 2 year time period. Over time some of these concerns have been diminished as immunogenicity can sometimes be addressed via dosing changes.

Early concerns for biotechnology products focused on “ quality ” issues [4] or process - related impurities. The concerns at that time were for carry - over of DNA or other cellular proteins, endotoxins, chemical contaminants, and viruses. Of course, these concerns still exist, but methods for purifi cation and assays for evaluation of clearance have alleviated the need for the safety assessment scientist to focus on contaminants; current safety assessments focus on the pharmacologic activity of the molecules. This chapter therefore does not address the standard assays and methods (e.g., viral clearance) used to address purity issues. There are several guidances that address these issues, especially the FDA ’ s Guidance for Industry on content and format of investigational new drug (IND) applications for Phase I studies of drugs, including well - characterized therapeutic, biotechnology - derived products [5] and the ICH ’ s Q6B Specifi cations: Test Procedures and Accep-tance Criteria for Biotechnological/Biological Products [6] . Other product - related impurities that do need to be considered by the safety assessment scientist include genetic variants, aggregate forms, chemical linkers, and differences in glycosylation patterns.

The assessment of safety in the preclinical development of biotechnology - derived therapeutics differs from that of traditional small molecules primarily in the species specifi city and the potential for immunogenicity of the former. In addition, clinical indications, length of exposure, route of administration, and dosing intervals can be very different between biologics and traditional small - molecule pharmaceuticals. Determining safety margins can be challenging in a situation where “ toxicity ” is mainly due to exaggerated pharmacology. It is also not unusual to have trouble establishing “ no effect ” (NOEL) doses with these highly selective and potent mol-ecules. The development plans for traditional pharmaceuticals are more routine and driven by well - established guidelines, while the development plans for biologics are

SPECIES SPECIFICITY AND SELECTION 699

more driven by the pharmacology of the molecule under investigation (Tables 19.3 and 19.4 ).

This chapter focuses on the standard approaches to safety assessment and toxicity evaluation of biotechnology - derived therapeutics and current issues in the fi eld.

19.2 SPECIES SPECIFICITY AND SELECTION

Perhaps the greatest challenge in the preclinical development of biotechnology - derived therapeutics is species specifi city. As therapeutics have become more spe-cifi c over the years, this has become an even greater challenge. Unlike the pharmaceutical development of traditional small molecules, one cannot assume that a biotechnology - derived therapeutic will be active in a standard species used for

TABLE 19.3 Differences Between Small - Molecule and Biotechnology - Derived Pharmaceuticals

Parameter Small - Molecule Pharmaceuticals

Biotechnology - Derived Pharmaceuticals

Size < 500 daltons > 1 kD (macromolecules) Immunogenicity Nonimmunogenic Potential for immunogenicity Metabolism Metabolized Degraded Frequency of dosing Daily Variable Toxicity Often structurally based Exaggerated pharmacology Half - life Short Long Route of administration Oral, some IV Parenteral Species specifi city Active in several species Species specifi c Synthesis Organic chemistry

(synthesized) Genetic engineering (derived

from living material) Structure Well defi ned Often not fully characterized

TABLE 19.4 Typical Toxicology Studies for Small - Molecule and Protein Therapeutics

Small - Molecule Therapeutics Protein Therapeutics

Screening studies Range - fi nding studies Range - fi nding studies One month studies Acute GLP studies Three/six month studies One month studies Safety pharmacology Safety pharmacology Reproductive studies Mutagenicity studies Tissue cross - reactivity studies Three month studies Irritation/tolerance Reproductive studies Others as needed Six month – - rat One year – - dog/monkey Industrial toxicology Diet RF studies Carcinogenicity studies Total cost : 5.0 – 6.5 million Total cost : 2.5 – 3.0 million Linear time : 4.5 – 5 years Linear time : 2 – 2.5 years


toxicity testing. Rats and dogs are often selected for the assessment of safety of small molecules without regard to pharmacologic activity, the focus being on off - target toxicity due to structural effects. Species selection for small - molecule toxicity studies is often based on ADME considerations, particularly metabolite formation (i.e., which species has a metabolite profi le most like humans). Only in recent years, with companies developing both traditional pharmaceuticals and biologics, has more emphasis been placed on testing for pharmacologic activity in species for standard toxicity assessments. This has always been an issue for biologics, for which lack of pharmacologic activity in a species leads to no effects in that species. In addition, the use of standard studies in standard species for biologics had little purpose and was of questionable use (except for potential effects of containments). The early work performed on interferons is a classic example; studies performed in nonrelevant species gave misleading information [7 – 9] . Several studies were con-ducted in rodents with recombinant human interferons. No evidence of toxicity was noted in many of these studies; these results later proved to be not predictive of what was to occur in humans. Activity in monkeys was more predictive, but there was a relative difference in the level of activity between primates and humans, indicating again how complex these assessments can be. Further work was per-formed on many of the interferons using homologous (surrogate) molecules.

19.2.1 Species Selection

There are several ways to determine whether a preclinical species is appropriate for safety assessment. If the therapeutic target is a replacement protein, the sequencing of the protein across species is a good place to start. Sequencing of related targets (e.g., ligands and receptors) can also be compared. If the therapeutic target is a recep-tor, receptor or ligand distribution between species can be investigated even before developing or testing the molecule. Often, in vitro studies are done fi rst to look at cross - reactivity. Receptor binding can be a start, but binding itself is not suffi cient. There are human or humanized molecules that demonstrate binding in primate cells but have no bioactivity. Receptor affi nity and then occupancy or binding should be compared across species. Often there are log - fold differences between humans and nonhuman primates or lower species, especially in the immune system [10] .

Assays for biologic activity should be available to test potency and specifi city. Assays used for characterization in Good Laboratory Practice (GLP) test species should be validated. Assays can be cell culture - based or biochemical in nature. They may measure physiologic responses or enzymatic reaction rates. In vitro assays can be used to compare the potency and specifi city across species and to predict what will happen in humans by correlating the responses in vitro with in vivo pharmaco-dynamic studies. There are instances when in vitro assay data does not correlate with in vivo data; this is sometimes the case when a protein is pegylated (i.e., is conjugated to polyethylene glycol (PEG) and activity is related to pharmacokinetics [11] . In these cases, the in vitro activity of the molecule is actually reduced due to steric hindrance from the large (PEG) moiety added to the protein. The in vitro results would lead one to believe that the potency of the linked molecule is less than the unlinked molecule. In actuality, because of the extended in vivo pharmacokinetic half - life of the linked molecule, the in vivo potency is greater than the original molecule.

SPECIES SPECIFICITY AND SELECTION 701

Tissue cross - reactivity studies have traditionally been performed on monoclonal antibodies as described in the FDA guidance Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use [12] . These studies are usually conducted with tissues from three unrelated human donors. Comparison of the binding in tissues from different species can be helpful in determining the most relevant animal species for in vivo toxicology studies. These studies can help determine if an antibody binds to tissues in different species, and whether the dis-tribution (which tissues show signifi cant binding) is similar between human tissues and preclinical species. Unintended binding can also be evaluated and may give clues of what to look for in future in vivo studies (e.g., which target organs). These types of studies have several limitations, and better information can often be derived from studies examining mRNA across tissues and species. As stated in the FDA document [12] , newer technologies should be employed as they become available and validated.

At one time, lack of binding in these studies led to the consideration of not per-forming any in vivo studies with the clinical molecule. If the target is not found in any appropriate animal species, then surrogates or other approaches may need to be considered. Lack of binding may also refl ect a technical issue, and other methods to determine if the test species are relevant should be employed as well. Many targets in recent years are soluble receptors and traditional immunohistochemistry studies do not detect these receptors well (low receptor density, or the receptor is not easily accessible) [13] . Differences in target expression and binding in different species may be compensated for by altering doses, but these differences need to be identifi ed before starting the safety assessment studies.

In vivo studies also have applicability in the selection of test species. If there are disease models in the potential safety assessment species and activity can be deter-mined with the molecule intended for humans, safety assessment plans become more straightforward. The doses with activity can help determine margins of safety from the toxicology studies. Comparisons of in vitro data from human cells/tissues with data from the pharmacologically active species can greatly aid in the dose selection for early human studies. Animal models of disease often have provided useful information for safety assessment. When there are no disease models, phar-macologic activity can be measured using biomarkers or surrogate markers in normal animals. If this is not feasible, then receptor occupancy in vitro may be compared to in vitro activity. Other strategies may be needed if the molecule does not cross - react with an appropriate species. This is where exaggerated pharmacology and target liability (i.e., toxicity inherent to the receptor, ligand, enzyme, etc. being targeted) may need to be evaluated with a surrogate molecule, knock - out animals, or knock - in models (in which the human receptor/target is spliced into the genome of the test species) [13] .

19.2.2 Nonhuman Primates

Many biologics are only active in primates; this is especially common with monoclonal antibodies. There are many challenges for programs that are limited to testing in nonhuman primates. These include the availability of nonhuman primates and the availability of laboratories and historical databases. This is particularly true for reproduction toxicology (see Section 19.4.2 ). Although more safety pharmacol-


ogy (see Section 19.4.4 ) parameters are now validated in non - human primates, there are some that are not testable in this model. This is particularly true for develop-mental CNS/behavioral measurements that are well validated in rodents. Other challenges are in the immunotoxicology arena, where intended or unintended effects on the immune system are measured using fl ow cytometry, cytokine measurements, and functional assays. These have been validated in rodents over the years, and only a few laboratories have been able to validate these assays in primates. The historical databases for T - cell - dependent antibody response (TDAR) and keyhole limpet hemocyanin (KLH) assays in nonhuman primates are still small.

Marmosets were gaining popularity a few years ago due to their small size, hence reducing the amount of compound needed for chronic studies. This size advantage can be outweighed by other factors, such as the small amount of blood available in these animals. In addition, marmosets are sensitive to stress [14] . Few laboratories have much experience with these animals and there is only a small historical data-base available.

Some molecules are even more specifi c and are only active in humans and chim-panzees ( Pan troglodytes ), severely limiting the types of testing that can be con-ducted preclinically. This situation will lead the safety assessment scientist to consider the use of surrogate molecules and/or the use of transgenic or knock - out animals. The use of chimpanzees for safety assessment is often impractical [15] because of the limitations of the testing laboratories. There are very few such laboratories avail-able, and they are not resourced suffi ciently to run multiple complicated studies concurrently. These laboratories also are not equipped to run toxicity studies at the same standards for GLPs as traditional toxicology laboratories. Chimpanzees are a protected and endangered species and cannot be bred. Few tests and bleedings can be conducted as each of these requires sedation of the animals. They are also very expensive studies to conduct, they are nonterminal (i.e., animals are not euthanized at the end of the study), and only a small number of animals can be separately housed at any time, making long - term monitoring diffi cult. Because chimpanzees often weigh more than 45 kg, they require large amounts of test material [16] . Many of the chimps available in the United States have been infected with HIV, which can complicate test results especially for compounds that have effects on the immune system. Special studies such as reproductive toxicology and carcinogenicity cannot be conducted in chimpanzees for numerous ethical, technical, and logistical reasons.

19.2.3 Surrogates and Other Alternatives

Surrogates or homologous proteins have their own issues. They can be diffi cult to develop in the desired species and by defi nition will be different from the molecules being developed for humans. They may have different affi nity for receptors, differ-ent epitopes, different half - lives, and so on, all of which may change the dynamics and alter the pharmacologic and toxicologic effects of the molecules. They can delay the development of other programs because they will use the same manufac-turing facilities. Surrogates provide little or no information on the fi nal human product contaminants and degradation products. Overall, the decision whether to investigate a surrogate or homologous protein is the balance between the time, cost, and delay to other programs versus getting target liability information. In general,

any of these molecules need to undergo the same rigorous evaluation as the clinical agent [17] .

Other methods of getting target liability information include literature reviews and knock - out animals. Surrogate antibodies can help elucidate the mechanism of action [18] . Other tactics include the use of homologous ligands or the use of trans-genics expressing human receptors. This allows the use of human - specifi c molecules but may not address off - mechanism toxicities. Knock - out models also have been used to mimic the suppression of endogenous cytokines or proteins. Disease models have also been used for safety assessment [19] , especially when compound avail-ability is a concern.

All of these strategies have their challenges, advantages, and disadvantages. In evaluating all of these data, it is important to bear in mind the goals of preclinical safety of biologics: to determine the pharmacologic and toxicologic attributes and activities of the molecule to be tested in humans, to determine the target organs, and to assess the potential similarities and differences. This information will be used to predict appropriate doses (safe starting doses as well as maximal doses) for clini-cal testing and to determine what parameters may need to be measured in early clinical trials. In some cases, one species may be suffi cient if there is only one species that has activity or if the biologic activity or the molecule and its target are well understood.

19.3 IMMUNOGENICITY

It is generally well accepted that immunogenicity is not well predicted across species. It has also been well documented that many biologic compounds intended for human use are immunogenic in animals [20] . ICH S6 states that “ most biotechnol-ogy - derived pharmaceuticals intended for humans are immunogenic in animals. ” Traditional antigenicity studies or guinea pig anaphylaxis studies are not useful for predicting immunogenicity in humans and are now generally recognized (ICH S6) as not being appropriate studies for biologics. When these studies were conducted years ago, at the request of regulators, they were generally positive and led to adverse effects in animals. Since these studies have no predictive value, they are no longer considered appropriate and may be an unnecessary use of animals.

19.3.1 Types of Antibodies to Biotechnology - Derived Pharmaceuticals

All preclinical (and clinical) studies with biologics should include measurements of total incidence of antibodies and whether these antibodies are neutralizing. Assays to determine antibodies have become more sophisticated over the years, and the newest technologies allow detection at lower levels than were achievable with tra-ditional enzyme - linked immunoassays (ELISAs) [21] . The technology exists today to measure and characterize the production of antibodies and to evaluate their effects on pharmacokinetics and pharmacodynamics. Clinically relevant antibodies are those that clear, sustain, neutralize, or cross - react with an endogenous protein. It is important to screen for the presence and development of antibodies to the test molecule throughout development. Since the consequence of these antibodies may range from no clinically signifi cant effects to serious safety effects, assays need to

IMMUNOGENICITY 703


be developed to determine if the antibodies that appear are able to block the bio-logic activity of the test compound. This may occur either by direct binding to an epitope with activity or by blocking the active site through steric hindrance after binding to a site in close proximity [21] .

A “ clearing antibody ” is one that results in more rapid clearance of test molecule: that is, the binding of the antibody to the test molecule causes the test molecule to be removed more quickly from the circulation. This type of antibody can be detected by comparing the pharmacokinetic profi les of the test molecule in animals with and without antibodies, or by comparing the pharmacokinetic profi le of the fi rst dose (at which time no antibodies are expected) with a subsequent dose (see Fig. 19.1 ). A clearing antibody may or may not have a direct neutralizing effect, but the con-sequence of the rapid clearance may eradicate any pharmacologic activity of the test compound. This type of antibody can make the toxicology studies invalid; if there is not adequate exposure to the test molecule, then the lack of toxicity does not give assurance of lack of effect from the pharmacologic activity of the molecule itself.

A “ sustaining antibody ” is one that delays the clearance of the test molecule: that is, the binding of the antibody to the test molecule keeps the test molecule in the circulation longer. This type of antibody also can be determined by measuring the serum or blood concentrations in animals with and without antibodies (see Fig. 19.2 ). The binding of a sustaining antibody to the test compound may or may not be neutralizing. When there are sustaining antibodies, the recovery period of the toxicology studies may need to be extended to see true reversibility of effects. These types of antibodies can lead to even greater exaggeration of pharmacology, and dose levels may need to be altered to account for unanticipated accumulation.

Both clearing and sustaining antibodies may also be “ neutralizing antibodies ” : that is, the binding of the antibody to the test molecule changes the ability of the test molecule to produce its pharmacologic activity. The most worrisome type of antibodies are those that cross - react with an endogenous protein (see Fig. 19.3 ). In this case, the individual ’ s own endogenous proteins can no longer function pharmacologically because the antibody response is binding and neutralizing its

FIGURE 19.1 Clearing antibody.

1

10

100

1000

10000

1470Time (days)

Seru

m C

on

cen

trati

on

(n

g/m

L)

1st Dose Ab Negative

3rd DoseAb Positive

21

activity as well. These types of antibodies can have devastating effects in the clinic [22 – 24] .

There are ways to alter the incidence and robustness of antibodies in preclinical studies. Altering the dosing regimen and route is the most obvious manner. The subcutaneous and intramuscular routes are the most immunogenic with the intra-venous route being the least immunogenic [25] . This may be due to aggregation at the injection sites. The frequency and duration of dosing also can affect immunoge-nicity. The longer the dosing period, the greater the probability of an immunogenic response and the greater the response may be. The dosing frequency may also have an effect, but there is a balance between more immunogenicity due to greater expo-sure versus a frequency that mimics a pulse effect [23] .

One of the most controversial topics in this area is the ability or lack of ability of animal immunogenicity results to predict immunogenicity in humans. Generally, it has been accepted that an antibody response in animals cannot directly predict such a response in humans. But recent studies have shown that animal studies may

FIGURE 19.2 Sustaining antibody.

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5 6

Time (h)

Se

rum

Co

nc

en

tra

tio

n (

mg

/mL

)

Antibody Positive(Day 28)

Antibody Negative(Day 28)

FIGURE 19.3 Neutralizing antibody.

0

200

400

600

800

1000

-10 10 30 50 70 90 110 130 150 170 190Study Day

Bio

log

ical

Mark

er

IMMUNOGENICITY 705


be able to predict relative immunogenicity of different compounds (e.g., modifi ed versions of a protein) and the potential subsequent effects of antibody production [20, 26] . There is also evidence that glycosylated proteins are less immunogenic than nonglycosylated proteins [22, 27] . Other effects that need to be assessed include those due to antibody – drug complexes. These complexes can deposit in tissues and organs [28] and cause glomerulonephritis [29] . Infusion reactions and anaphylactic responses also need to be monitored [24] .

In summary, immunogenicity is a substantial complication for preclinical safety assessment studies. It can invalidate the model species, but antibody production alone should not stop the conduct of these studies. The antibodies need to be mea-sured with reproducible assays [30] . Longer recovery periods are often needed for these studies to wash out the drug from the test animals. This can greatly increase the ability of the assays to detect antibodies to the drug. If there are active drug levels in the blood samples, they can interfere with the ability to measure true anti-body levels [21] . The effects on pharmacokinetics and pharmacodynamics need to be measured and evaluated. The potential consequences of the antibodies on endog-enous molecules need to be evaluated as well. The secondary effects, such as anti-body deposition, should be measured. The lack of ability to predict human immunogenicity and the relative potential to predict this should be considered on a case - by - case basis.

19.3.2 Study Design Issues

Unlike most traditional small molecules, biologics are not given once or twice a day, orally, in a pill or capsule. They are almost all dosed via a parenteral route, are not given (or “ taken ” ) daily, and some are given in hospital settings or in clinician offi ces (e.g., monoclonal antibodies for the treatment of solid tumors such as Avastin ® and Erbitux ® ). Several are now self - administered on a weekly or even less frequent basis (e.g., TNF inhibitors such as Enbrel ® and Remicade ® ). The dose regimen for the safety assessment studies should refl ect the dosing regimen plan for the human studies. The dosing interval in animals may be different from that planned for humans, depending on the half - life of the molecule in animals versus humans. Also, the development of antibodies in animals may alter the pharmacokinetics, and modifying the dosing interval may reduce the incidence of antibodies.

Other design issues include dose level selection. Often when the dose - limiting toxicity is related to the pharmacology (often referred to as “ exaggerated pharma-cology ” ) it is diffi cult to establish a large margin of safety. The difference between the intended level of effect and an effect that leads to toxicity, even if it is the same effect, may be very small.

Other factors that need to be considered in dose selection are those that fall under the category of pharmacokinetics and drug metabolism. These factors will not be considered in any great depth in this chapter, but a few points to consider are listed here:

• Route of dosing (IV, SQ, infusion) • Half - life of the molecule (e.g., IgG isotypes) • PK/PD modeling (very useful for growth factors such as Epogen ® and

Neupogen ® )

• Dosing regimen in the clinic (weekly, monthly, once per cycle) • Length of intended clinical use (acute vs. chronic)

One study type generally not deemed relevant for biologics is metabolism studies. Mass balance studies have not proved useful when performed with proteins. Radio-labeled studies have limited usefulness as these labeled molecules undergo rapid metabolism and the label is often unstable. Protein biologics undergo proteolytic degradation into smaller peptides and then into individual amino acids. Distribution is viewed more from the perspective of target distribution, except for gene and cell therapies or vectors.

Toxicokinetics must be assessed in these studies, as with traditional pharmaceu-ticals. These data are necessary to prove exposure (which may differ with route) and to monitor the potential effects of antibodies on the exposure levels over time. One of the greatest differences between small molecules and biologics is the potential for immunogenicity, which can greatly affect pharmacokinetics, as previously discussed.

Recovery periods are very important in studies with biologics for several reasons. As already discussed, it is important to have a wash - out period of the drug to prop-erly test for antibody levels. This works best with samples that are clear of the test compound itself (assay interference).

19.4 SPECIAL STUDIES

19.4.1 Genotoxicity

It is generally accepted now that these studies are not applicable for biotechnology - derived products, unless there is a “ chemical ” linker or toxic conjugate to the mol-ecule. This is clearly stated in ICH S6 as well as the FDA document for monoclonal antibodies [12] .

19.4.2 Reproductive Toxicology

When a biologic cross - reacts with traditional reproductive toxicology species, then these studies should be conducted as appropriate for the intended clinical population. The main consideration for these studies, if conducted in rodents and rabbits, is the dosing regimen and immunogenicity. The regimen should be carefully chosen to ensure pharmacokinetic coverage during the pivotal parts of gestation. The frequency of dosing in these studies might need to be closer to ensure that there is adequate exposure. Also, this may address the potential immunogenic-ity as discussed earlier. The exposure period for the Segment II studies in rodents and rabbits is usually short enough to avoid a strong immunogenic response, but samples should be taken to measure drug levels and antibodies in the dose range - fi nding studies.

The more complicated situation is with those molecules that only work in nonhu-man primates. Here a decision must be made as to whether to conduct these studies in primates or to create a rodent surrogate. The decision should be based on good science as well as the practical and technical issues. There is no right or wrong answer

SPECIAL STUDIES 707


to this question, but there are several factors that need to be considered. There are differences between non - human primate subspecies to consider as well. For example, rhesus monkeys are seasonal breeders and cynomolgus monkeys are not. Cynomol-gus monkeys also have a similar duration of menstrual cycle and placental morphol-ogy and physiology similar to humans. Human IgGs are known to cross the placental barrier, and their response to teratogens has been shown to be similar to humans [31] . On the other hand, compared with rodents, nonhuman primates have a small number of offspring (usually just one offspring per mother), they are very expensive, and their gestation period is 150 days long. They have a low conception rate and a high spontaneous abortion rate. In addition, there are only a small number of labo-ratories that have the capabilities of conducting primate reproductive studies. The historical database is not that large.

As for specifi c studies, male fertility is easier to test in cynomolgus monkeys [32] , and these assays (e.g., hormones, sperm count) can often be incorporated into stan-dard toxicology studies [33] . Mating studies are much more diffi cult, but fertility parameters can be measured in females as well (e.g., menstrual cycle, FSH, LH). Segment II studies are usually dosed during organogenesis (days 20 to 50), and the mothers are C - sectioned on day 100 [34] . Placental transfer can also be measured with continuous dosing.

19.4.3 Carcinogenicity

Unless there is a specifi c “ cause for concern, ” these studies are generally considered inappropriate for biologics (ICH S6). This issue is also addressed in ICH S1A [35] , which states that these studies are not generally needed for molecules that are developed as replacement therapy (e.g., insulin and certain hormones). This guid-ance also recognizes that neutralizing antibodies may “ invalidate ” the results. As with the S6 document, ICH S1A suggests that these studies be considered depending on the clinical indication, patient population, and treatment duration. Further reasons to consider these studies might include modifi cations made to the molecule or indications of potential effects based on subchronic studies (e.g., unexpected hyperplasia).

For molecules that are only pharmacologically active in non - human primates, there are additional technical issues. Carcinogenicity studies would not be feasible in non - human primates due to the long life span, nor are they practical due to the large number of animals that would be needed for statistics. The alternative would be to develop a surrogate that would be active in rodents. This would carry the same issues as already discussed. In addition, the longer time span needed for testing would only increase the chance for neutralizing antibody production. Alternative approaches, such as in vitro proliferation studies or receptor binding studies, may be informative but still not defi nitive. Another complication is with molecules that are immunosup-pressive, as these might increase the incidence of any background tumors.

19.4.4 Safety Pharmacology

Often, parameters assessed in safety pharmacology studies can be incorporated into standard toxicology studies, including single - dose studies. For molecules that cross - react in traditional species (rodents and dogs), study designs can be more fl exible,

while study designs for molecules that are active only in nonhuman primates have more challenges. Non - human primates can be telemeterized for cardiovascular assessments if there are concerns and even many CNS evaluations can be performed in non - human primates as well now.

19.4.5 Local Tolerance

Local tolerance assessments can usually be conducted as part of subchronic or chronic toxicity studies, by taking sections from injection sites. They are often used to test formulation changes as well.

19.5 FUTURE TRENDS

In the future, biotechnology products are going to become more complex. We will see more conjugated monoclonal antibodies, oligonucleotides, fusion molecules, and gene or plural: cell therapies. The need to address the basic questions with these products will remain the same, but the challenges will be greater. There are new tools becoming available to the toxicologist to address these concerns. Global devel-opment strategies for development of biotechnology products will be more science driven, more problem focused, and with additional regulatory challenges. The overall goal should still be to evaluate preclinically the potential effects of these molecules in humans in the diseases of interest. The safety assessment scientist should develop a plan with a strong scientifi c rationale, using all of the necessary tools and good justifi cation for the animal model chosen.

REFERENCES

1. Pharma Org . www.pharma.org . 2. Cavagnaro JA . Preclinical safety evaluation of biotechnology - derived pharmaceuticals .

Nat Rev 2002 ; 1 : 469 . 3. Griffi ths SA , Lumley CE . Non - clinical safety studies for biotechnologically - derived phar-

maceuticals: conclusions from an International Workshop . Hum Exp Toxicol 1998 ; 17 : 63 . 4. Dayan AD . Safety evaluation of biological and biotechnology - derived medicines . Toxicol-

ogy 1995 ; 105 : 59 . 5. FDA IND Guidelines . www.fda.gov . 6. ICH Q6B Specifi cations: Test Procedures and Acceptance Criteria for Biotechnological/

Biological Products 1999 . 7. Green JD , Terrell TG . Utilization of homologous proteins to evaluate the safety of recom-

binant human proteins — case study: recombinant human interferon gamma (rhIFN - γ ) . Toxicol Lett 1992 ; 64/65 : 321 .

8. Serabian MA , Pilaro AM . Safety assessment of biotechnology - derived pharmaceuticals: ICH and beyond . Toxicol Pathol 1999 ; 27 : 2 .

9. Ryan AM , Terrell TG . Biotechnology and its products . In Haschek W , Ed. Handbook of Toxicologic Pathology , 2nd ed. London : Academic Press ; 2002 , p 479 .

10. Hart TK , et al. Preclinical effi cacy and safety of pascolizumab (SB 240683): a humanized anti - interleukin - 4 antibody with therapeutic potential in asthma . Clin Exp Immunol 2002 ; 130 : 93 .

REFERENCES 709


11. Elliott S , Lorenzini T , Asher S , et al. Enhancement of therapeutic protein in vivo activities through glycoengineering . Nat Biotechnol March 2003 ; 21 : 414 – 421 .

12. Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use . Washington, DC: FDA, CBER; 1997.

13. Bolon B , et al. Genetic engineering and molecular technology . In Krinke G , Ed. The Laboratory Rat . London : Academic Press ; 2000 , p 603 .

14. Zuhlke U , et al. The common marmoset ( Callithrix jacchus ) as a model in biotechnology . In Korte R , Vogel F , Weinbauer GF , Eds. Primate Models in Pharmaceutical Drug Devel-opment . Munster, Germany : Waxmann ; 2002 , p 119 .

15. Klingbeil C , Hsu D . Pharmacology and safety assessment of humanized monoclonal antibodies for therapeutic use . Toxicol Pathol 1999 ; 27 : 1 .

16. Tony Ndifor Presentation at CRL Biotechnology Symposium 2003 .

17. Clarke J , et al. Evaluation of a surrogate antibody for preclinical safety testing of an anti - CD11a monoclonal antibody . Regul Toxicol Pharmacol 2004 ; 40 : 219 .

18. Treacy G . Using an analogous monoclonal antibody to evaluate the reproductive and chronic toxicity potential for a humanized anti - TNFa monoclonal antibody . Hum Exp Toxicol 2000 ; 19 : 226 .

19. Sato K , et al. Effi cacy of intracoronary versus intravenous FGF - 2 in a pig model of chronic myocardial ischemia . Ann Thorac Surg 2000 ; 70 : 2113 – 2118 .

20. Bugelski PJ , Treacy G . Predictive power of preclinical studies in animals for the immu-nogenicity of recombinant therapeutic proteins in humans . Curr Opin Mol Ther 2004 ; 6 : 10 .

21. Gupta S , et al. Recommendations for the design, optimization, and qualifi cation of cell - based assays used for the detection of neutralizing antibody responses elicited to biologi-cal therapeutics . J Immunol Methods 2006 ; 321 : 1 – 18 .

22. Koren E , Zuckerman LA , Mire - Sluis AR . Immune responses to therapeutic proteins in humans — clinical signifi cance, assessment and prediction . Curr Pharm Biotechnol 2002 ; 3 : 349 .

23. Schellekens H . Immunogenicity of therapeutic proteins: clinical implications and future prospects . Clin Ther 2002 ; 24 : 1720 .

24. Rosenburg AS . Immunogenicity of biological therapeutics: a hierarchy of concerns . In Brown F , Mire - Sluis A , Eds. Immunogenicity of Therapeutic Biological Products . Basel : Karger AG ; 2003 , p 15 .

25. Working PK . Potential effects of antibody induction by protein drugs . In Ferraiolo BL , Mohler MA , Gloff CA , Eds. Protein Pharmacokinetics and Metabolism . New York : Plenum Press ; 1992 , p 73 .

26. Wierda D , Smith HW , Zwickl CM . Immunogenicity of biopharmaceuticals in laboratory animals . Toxicology 2001 ; 158 : 71 .

27. Gribben JG , et al. Development of antibodies to unprotected glycosylation sites on recombinant human GM - CSF . Lancet 1990 ; 335 : 434 .

28. Henck JW , et al. Reproductive toxicity testing of therapeutic biotechnology agents . Tera-tology 1996 ; 53 : 185 .

29. Terrell TG , Green JD . Comparative pathology of recombinant murine interferon - γ in mice and recombinant human interferon - γ in cynomolgus monkeys . In Richter GW , Solez K , Ryffel B , Eds. International Review of Experimental Pathology: Cytokine - Induced Pathology Part A: Interleukins and Hematopoietic Growth Factors . San Diego : Academic Press ; 1993 , p 73 .

30. Mire - Sluis AR . Progress in the use of biological assays during the development of bio-technology products . Pharm Res 2001 ; 18 : 1239 .

31. Weinbauer GF . The nonhuman primate as a model in developmental and reproductive toxicology . In Korte R , Weinbauer GF , Eds. Primate Models in Pharmaceutical Drug Development . Munster, Germany : Waxmann ; 2002 , p 49 .

32. Vogel F . How to design male fertility investigations in the cynomolgus monkey . In Korte R , Weinbauer GF , Eds. Towards New Horizons in Primate Toxicology . Munster, Germany : Waxmann ; 2000 , p 43 .

33. Weinbauer GF , Cooper TG . Assessment of male fertility impairment in the macaque model . In Korte R , Weinbauer GF , Eds. Towards New Horizons in Primate Toxicology . Munster, Germany : Waxmann ; 2000 , p 13 .

34. Weinbauer GF . The nonhuman primate as a model in developmental and reproductive toxicology . In Korte R , Weinbauer GF , Eds. Primate Models in Pharmaceutical Drug Development . Munster, Germany : Waxmann ; 2002 , p 49 .

35. ICH S1A: The Need for Long - Term Rodent Carcinogenicity Studies of Pharmaceuticals 1995 .

REFERENCES 711

preclinical development handbook || safety assessment of biotechnology-derived therapeutics

Documents