Development of Immunotoxicity Testing Strategies for Immunomodulatory Drugs

Toxicologic Pathology, 40: 288-293, 2012 Copyright # 2012 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623311430238 Development of Immunotoxicity Testing Strategies for Immunomodulatory Drugs THOMAS T. KAWABATA AND ELLEN W. EVANS Drug Safety R&D, Pfizer, Inc., Groton, Connecticut, USA ABSTRACT The ICH S8 immunotoxicity testing guideline for human pharmaceuticals was published in 2006 and was intended to provide guidance for assessing the immunotoxicity potential of low-molecular-weight drugs that are not intended to alter the immune system. For drugs intended to modulate the immune system, immunotoxicity testing strategies are generally developed on a case-by-case approach since the targets, intended patient population, and mechanisms of action of the test compound will determine the type of testing needed. Some of the general principles of ICH S8, however, may be applied to immunotoxicity testing strategies for immunomodulatory drugs. A weight-of-evidence approach using factors discussed in ICH S8 in concert with an assessment of the potential value of additional immunotoxicity testing should be considered. For most situations, immunotoxicity studies with immunomodulatory compounds evaluate off-target effects on the immune system and exaggerated pharmacology. The potential use of data from these studies and considerations such as translatability to humans are discussed. Keywords: immunotoxicity; immunotoxicology; immunomodulation; immunosuppression. INTRODUCTION The development of immunotoxicity testing strategies for drugs intended to modify the immune response can be a very challenging and controversial area. For the majority of situations, immunotoxicity strategies are best approached on a case-by-case basis rather than in a prescriptive fashion. Thus, in practice, the strategies may vary significantly between pharmaceutical companies, and regulatory agencies. The primary gray area of immunotoxicity testing strategies is in deciding if additional immunotoxicity testing is needed (assays beyond that normally done for standard toxicity studies [STS]). With some situations, only limited immunotoxicity testing may be needed since the effects on the immune system may be well characterized in nonclinical pharmacodynamic, efficacy, and general toxicity studies or because changes in immune response would be expected and predictable based on the nature of the intended target. On the other hand, there may be targets for which the biology is not fully understood and the types of pharmacodynamics and efficacy studies conducted do Address correspondence to: Thomas T. Kawabata, Drug Safety R&D, Pfizer, Inc., MS 8274-1206, Groton, CT 06340; e-mail: thomas.t.kawabata @pfizer.com. Conflict of interest/financial disclosure: The authors claim no conflict of interest and are employees of Pfizer, Inc. Abbreviations: BrdU, bromodeoxyuridine; EBV, Epstein Barr virus; FDA, Food and Drug Administration; HuLA, human lymphocyte activation; HSC, hematopoietic stem cells; ICH, International Conference on Harmonization; IMTX, immunotoxicity; JPMA, Japan Pharmaceutical Manufacturers Association; MHLW, Ministry of Health and Labor Welfare; NOD, nonobese diabetic; PBMC, peripheral blood mononuclear cells; PhARMA, Pharmaceutical Manufacturers of America; RAG, recombinant activating gene; RA, rheumatoid arthritis; TDAR, T-dependent antibody response; WoE, weight of evidence. not assess multiple arms of the immune response beyond those targeted for efficacy. The goal of this article is to discuss the history and current immunotoxicity testing guidelines and how they may be applied to developing an immunotoxicity testing strategy for immunomodulatory drugs. It will describe a general thought process and considerations for deciding if additional immunotoxicity studies should be done, how the additional immunotoxicity data will be used, and whether such studies will provide value to decisions made during the drug development process. The focus will be on immunomodulatory drugs that may suppress the immune response and possibly increase the risk for infections and malignancies. Finally, new approaches to address translatability of immunotoxicity from toxicity testing species to humans will be discussed. HISTORY OF IMMUNOTOXICITY TESTING GUIDELINES To better understand the current immunotoxicity testing guidelines for pharmaceuticals, a brief history of previous guidance documents is provided. The Committee for Proprietary Medicinal Products (2000) of the European Agency for the Evaluation of Medicinal Products (EMEA; currently referred to as EMA) updated the Note for Guidance on Repeat Dose Toxicity testing in 2000, including a section on immunotoxicity testing (section 6.4). This section recommends that all new medicinal products be screened for immunotoxic potential in at least one repeat-dose toxicity study. Initial screening for immunotoxicity consisted of standard toxicity testing, which includes hematology, lymphoid organ weights, histopathology of lymphoid tissues, and bone marrow cellularity. In addition to standard toxicity data, distribution of lymphocyte subsets (immunophenotyping) and natural killer cell activity were required as part of initial screening. According to the guidance 288

Vol. 40, No. 2, 2012 KAWABATA AND EVANS 289 document, if the latter two assays are not available, the primary T-dependent antibody response (TDAR) should be evaluated. These types of assays were primarily focused on detecting immunosuppression. In 2002, the Center for Drug Evaluation and Research of the U.S. Food and Drug Administration (FDA) published a guidance document titled Immunotoxicology Evaluation of Investigational New Drugs. This guidance focused on recommendations for testing approaches to characterize the effects of drugs on the immune system and when these studies should be conducted. In contrast to the EMA guidance, the FDA guideline included information on immunosuppression as well as other areas of immunotoxicology: immunogenicity, hypersensitivity, autoimmunity, and adverse immunostimulation. For immunosuppression, it was recommended that standard repeat-dose toxicity studies be used as the first tier of testing. If evidence of immunosuppression is found, follow-up studies may be appropriate. The two assays in particular that were recommended were the TDAR and immune cell phenotyping by flow cytometry or immunohistochemistry. With the publication of the EMA guidance document, the Ministry of Health and Labor Welfare (MHLW) of Japan and the Japan Pharmaceutical Manufacturers Association (JPMA) initiated the preparation of their own guidance document and discussions at meetings of the International Conference on Harmonization (ICH) in 2001 to develop a harmonized immunotoxicity testing guideline. Soon after the publication of the FDA immunotoxicity guidance document in 2002, the ICH endorsed the proposal from the MHLW and JPMA to prepare a harmonized guideline. An expert working group of representatives from the MHLW, JPMA, EMA, European Federation of Pharmaceutical Industries and Associations, FDA, and Pharmaceutical Research and Manufactures of America was formed to prepare the guidance. ICH S8 IMMUNOTOXICITY TESTING GUIDANCE After several years of discussions, the ICH S8 guidance on Immunotoxicity Studies for Human Pharmaceuticals was finalized and came into operation in May 2006 (ICH 2005). The scope of the S8 was focused on nonclinical testing for unintended immunosuppression and immunoenhancement. It excluded recommendations for biotechnology-derived pharmaceutical products (biologicals) since these were covered by ICH S6. Recommendations for hypersensitivity and autoimmunity testing were not included, and existing guidelines for these areas remained in force (primarily the 2002 FDA guidance). The key decision point for the guidance was the weightof-evidence (WoE) review of the factors to consider in the evaluation of potential immunotoxicity (Figure 1). These factors include results from standard toxicity studies, pharmacological properties of the test compound, the intended patient population, structural similarity of known immunosuppressive compounds, accumulation of the test compound in cells or organs of the immune system, and signs from clinical trials. If the WoE review indicates that additional immunotoxicity FIGURE 1. ICH S8 Decision Tree for Immunotoxicity (imtx) Testing. testing is warranted, then additional immunotoxicity tests should be conducted. The types of additional immunotoxicity tests selected should be based on the findings from the list of factors to consider (Fig. 1). If significant immunomodulation is observed with these additional tests, the sponsors need to determine if there is sufficient data for risk assessment and management of the potential immunotoxic effects. If not, additional studies may be needed. CURRENT THINKING ON IMMUNOTOXICITY TESTING OF IMMUNOMODULATORY DRUGS Since the scope of ICH S8 does not specifically include drugs intended to modify the immune response, the current practice for determining immunotoxicity testing strategies for immunomodulatory drugs is on a case-by-case basis. This practice makes the most sense since the type of testing should be based on the mechanism of action of the immunomodulatory drugs, the indication and use (acute vs. chronic), and intended patient population. It is not feasible or practical to develop guidelines that would apply to all situations because it would likely result in unnecessary testing. There is general agreement that the case-by-case approach is the most appropriate since it provides the flexibility needed to do the best science appropriate for the specific drug being developed. With the increasing number and diversity of immunomodulatory drugs in development along with inconsistencies in immunotoxicity testing practice between companies and requests for immunotoxicity testing by regulatory agencies, a workshop was held to discuss this topic in May 2007 (summarized in Piccotti et al. 2009). The primary goal of the meeting was to discuss testing strategies and identify key gaps in nonclinical and clinical immunotoxicity testing for anti-inflammatory and immunomodulatory drugs. Stakeholders from regulatory agencies,

290 IMMUNOTOXICITY TESTING STRATEGIES TOXICOLOGIC PATHOLOGY Immunomodulatory drug is being developed Conduct imtx assays in tox studies Concerns about translation Imtx data not used Consider including immuno-end points in clinical studies How can we make this assessment with non-clinical studies? Challenges regarding assay availability, costs and logistics Concerns about interpretation of data in regards to clinical impact (e.g. infections, malignancies) Primary reliance on infection and malignancy rate data Need assessment of potential infection and malignancy risk earlier in the development process FIGURE 2. Worst-Case Scenario of a Hypothetical Conundrum on Immunotoxicity Testing of Immunomodulatory Drugs academia, and industry were present and included nonclinical scientists and clinicians. There was consensus among participants that immunotoxicity testing for immunomodulatory drugs should continue to be conducted on a case-by-case basis where a WoE review of the different causes for concern drive the immunotoxicity testing strategy. Immunotoxicity testing may be conducted to evaluate off-target immunological effects and/or exaggerated pharmacology. The major gaps identified at this workshop were as follows: 1. Lack of appropriate testing methods for both nonclinical and clinical immunotoxicity testing. 2. Translation of immunotoxicity findings in animals to humans. 3. Determining when additional immunotoxicity testing would be helpful (triggers for testing). 4. Interpretation of changes in clinical immunological assays to actual risk for infections or malignancies. CHALLENGES IN DEVELOPING IMMUNOTOXICITY TESTING STRATEGIES The key nonclinical and clinical immunotoxicity testing gaps noted above have resulted in a conundrum that may play out as companies decide on the appropriate testing strategies for immunomodulatory drugs. The diagram shown in Figure 2 illustrates the worst-case scenario of this conundrum. The decisions described may not be applicable for all immunomodulatory drugs but for some components may play an important role in the decision process. The decisions regarding the immunotoxicity testing strategy will depend on a variety of factors including but not limited to the drug target and the availability of validated assays. As shown in Figure 2, it is possible to include immunotoxicity endpoints in repeat-dose toxicology studies (beyond endpoints included in STS) or to conduct stand-alone immunotoxicity studies (e.g., rat TDAR). However, there may be concerns about the translation of any findings to humans, so the data may have only minimal impact FIGURE 3. Thought Process in Developing an Immunotoxicity Testing Strategy on future development decisions. There also may be situations in which nonclinical immunotoxicity studies are not conducted since adverse immunomodulation may best be monitored in clinical trials. In addition, there may be significant challenges with incorporating the assays (e.g., technical challenges, lack of assay availability or expertise at testing sites, costs) in clinical studies. Concerns about interpreting the clinical laboratory data may also arise. For example, how much of a decrease in T-cell function will be associated with an increased incidence of infections and malignancies? This may then lead to the reliance of infection data and malignancy data observed in long-term clinical trials, but this occurs very late in the drug development process, so testing earlier in the development process may need to be reevaluated. This takes us back to the start of the conundrum. Therefore, to navigate around this issue, a better understanding is needed of the triggers or rationale for including nonclinical immunotoxicity studies in the thought process for toxicity testing of immunomodulatory compounds. In addition, methods to enhance our confidence in translating nonclinical data to humans are needed. PROPOSED THOUGHT PROCESS IN DEVELOPING AN IMMUNOTOXICITY TESTING STRATEGY It may be possible to apply the principles of the ICH S8 guidance document to immunomodulatory compounds by including additional considerations in the WoE review in deciding if additional immunotoxicity testing may be of value. For this thought process, one first needs to think about the key issue with immunomodulatory drugs that are immunosuppressive, that being the increased risk for infection and malignancies (Fig. 3). Second, one needs to evaluate how and if additional immunotoxicity data will help address the key issue. In addition, nonclinical pharmacodynamic and efficacy studies conducted during the discovery process need to be considered;

Vol. 40, No. 2, 2012 KAWABATA AND EVANS 291 TABLE 1. Potential Uses of Immunotoxicity Data Determined by Levels of Confidence in Translatability Level 1 confidence (hazard ID) Determine potential off-target effects Characterize the exaggerated pharmacological effects Identify potential unsuspected bad actors Identify potential biomarkers that can be used in future studies Level 2 confidence (risk assessment) All of the above Determine a safety margin or therapeutic index Competitor comparison Commercial viability Ranking compounds data from these studies may be adequate to characterize hazard or risk or may suggest a need for additional testing. In general, immunotoxicity studies for immunomodulatory drugs are primarily used to understand the potential immunological off-target and exaggerated pharmacologic effects. The findings from these studies may help to: (1) identify compounds that may unexpectedly have a significant or unacceptable impact on the immune response (so-called bad actors ), (2) rank compounds, (3) determine reversibility of these effects on the immune system, (4) determine a safety margin or therapeutic index for an effect on the immune system, (5) assist in the determination of commercial viability, and (6) identify potential biomarkers of immunomodulation (pharmacodynamics or immunotoxicity) for future nonclinical and clinical studies. The potential value of this type of information will depend on a number of factors for a particular compound such as therapeutic indication, patient population, length of drug therapy, and standard-of-care therapy. The value and utility of the data generated will also depend on the level of confidence regarding the translatability of the data to humans. This can best be understood by dividing levels of confidence and translatability into three levels. The first level simply involves translating the potential hazard from the animal to human (hazard identification). The second level involves translating alterations in immune function in animals to those in humans at equivalent exposure levels (risk assessment). The third and most difficult to determine is whether a specific alteration in an immunotoxicity endpoint correlates with an increased risk for infections or malignancies in humans. The potential uses of the data with the first and second level of confidence are shown in Table 1. Additional value from immunotoxicity testing data may be obtained with the second level of confidence as the risk assessment process can be applied, and thus a safety margin or therapeutic index may be determined. The level of confidence in data translation will depend on differences between the test species and humans that may be target (e.g., cytokine receptor) or compound specific (e.g., monoclonal antibody, low-molecular-weight chemical). This may include the distribution of the drug target among different tissues and cell types, sequence homology of the target, and the role of different cell types in generating immune responses (e.g., TDAR). However, in most situations, the confidence of translation between species and with humans is unknown and difficult to ascertain. Thus, as with toxicities in other organ systems, it is assumed that the toxicity observed in the toxicology species may identify target organs in humans and be used to determine a no adverse effect level unless data to the contrary are available. APPLICATION OF PROPOSED THOUGHT PROCESS TO A HYPOTHETICAL CASE STUDY To better understand the thought process described in the previous section, a hypothetical case study is discussed below. A monoclonal antibody (Mab) against receptor X on macrophages has been shown to inhibit the inflammatory responses in various mouse models through the inhibition of proinflammatory cytokine production. Although receptor X is primarily expressed on macrophages, low levels of receptor X have been reported on T and dendritic cells. The expression levels in the different cell types may differ between rats, monkeys, and humans. The project team plans to develop a Mab for receptor X (anti-x) for the treatment of rheumatoid arthritis (RA) and requests that an immunotoxicity testing strategy be developed. Evaluating potential off-target immunological effects and exaggerated pharmacology may be helpful in the early stages of development to determine if targeting receptor X will have a significant impact on multiple arms of immune responses (identifying bad actors ) and assist in the determination if anti-x will be commercially viable. For example, if anti-x dramatically inhibits adaptive immune responses with a low safety margin or therapeutic index, it may not be commercially competitive in comparison to the standard of care since most of the currently used RA therapies have a slight to moderate impact on adaptive immune responses. To assess the potential impact on adaptive immune responses, a holistic in vivo assay that assesses adaptive immune responses may be helpful. The in vivo TDAR is one of the most commonly used holistic approaches. This assay has been widely validated/qualified for sensitivity by using marketed immunosuppressive drugs with different mechanisms (e.g., cyclosporine A, dexamethasone, cyclophosphamide) and is commonly used in mouse, rat, and monkey immunotoxicity studies and occasionally in dogs. The level of confidence for translatability of the TDAR may vary with different targets or mechanisms of action, particularly if the target or biology in the nonclinical species varies from that of humans. This needs to be considered in determining if the TDAR should be conducted. Ex vivo or in vitro assays that specifically assess T-cell or dendritic cell function may also be helpful, particularly to determine the mechanism if there is an unexpected effect on the TDAR. These assays may also provide information that could help in determining the therapeutic index or safety margin when compared with efficacious exposure levels in mouse models of inflammation. Since inhibition of receptor X function at or near 100% may also theoretically inhibit macrophage host defense mechanisms, in vivo or in vitro exaggerated

292 IMMUNOTOXICITY TESTING STRATEGIES TOXICOLOGIC PATHOLOGY pharmacology studies that evaluate macrophage phagocytosis and killing of bacteria may be included. For example, if concentrations that inhibit receptor X mediated cytokine production are significantly lower than concentrations that inhibit macrophage killing of bacteria, there may be increased confidence of a lower risk for infections at efficacious doses (at least for infections in which macrophage killing plays an important role in host defense). In addition, these findings may refine or enhance the clinical monitoring strategy. For example, if there is decreased macrophage killing of fungi at clinically relevant concentrations in comparison to other pathogens, clinical studies in areas with high risk for histoplasmosis (e.g., Ohio River Valley) or other fungal infections with regional distributions may be monitored more intensely. APPROACHES TO ENHANCE CONFIDENCE IN TRANSLATABILITY To enhance the confidence of translatability of nonclinical immunotoxicity data, new approaches that use human cells may be helpful. The in vitro human lymphocyte activation (HuLA) assay (Collinge et al. 2010) may be used early in the development process since it requires low amounts of test compound and has a medium throughput. In contrast to other in vitro lymphocyte stimulation assays that use mitogens (e.g., conconavlin A, phytohemagglutin) or stimulating antibodies (e.g., anti-cd3, anti-cd40), this method evaluates influenzaspecific responses and thereby evaluates the effect of a test compound on multiple cells types and processes. The HuLA assay involves preparation of peripheral blood mononuclear cells (PBMCs) from healthy donors that had been recently vaccinated with the influenza vaccine. The PBMCs may be frozen and used when needed. PBMCs are incubated with test compound at different concentrations along with flu antigens (commercially available influenza vaccine). The flu antigens activate flu-specific T and B cells, and the proliferative response is measured by bromodeoxuridine (BrdU) incorporation (immunoassay for BrdU). The 50% inhibitory concentrations (IC50) of several immunosuppressive drugs have been determined in the HuLA assay (cyclosporine A, dexamethasone, mycophenolate, methotrexate). The IC50s were found to be similar to plasma concentrations at therapeutic doses. Additional endpoints may be included in the HuLA assay such as intracellular staining of cytokines by flow cytometry or the generation of flu-specific antibody-forming cells. This assay may be used early in the development process to rank compounds or identify bad actors. In addition, if the TDAR response is decreased in the rat, the HuLA assay may be used to determine if the findings in the rat may be translated to humans. Another approach that may be used in the future are humanized mouse models. These models have significantly improved over the past two decades and currently involve transplanting human hematopoietic stem cells into immunodeficient mouse strains (see history in the review by Brehm, Schultz, and Greiner 2010). One of the more recent models uses non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice with a mutated IL-2 receptor gamma chain (IL2ry null ) which express human HLA Class I heavy and light chains (NSG-HLA-A2-/HHD mice) (Shultz et al. 2010). When CD34þ HSC are transplanted into newborn mice, they develop functional CD4þ and CD8þ T cells in the bone marrow and spleen. Moreover, when infected with Epstein-Barr virus (EBV), they generate functional EBV-specific CD8þ cytotoxic T cells. Another model recently reported used NOD.Rag1KO. IL2RgcKO mice (NOD mice with recombinase-activating gene-1 and IL-2 receptor gamma chain knock out) that express HLA class II molecules (DRAG mice; Danner et al. 2011). Transplantation of adult DRAG mice with human stem cells resulted in a high rate of human T- and B-cell reconstitution. These cells were functional and when immunized with tetanus toxoid, the mice produced a robust tetanus-specific IgG response. Thus, with continual improvement, it may be practical to use these models to evaluate the effects of immunodulatory drugs on the human immune system in the future. CONCLUSIONS Although ICH S8 did not include guidance for drugs intended to suppress or enhance the immune response, some of the principles related to using a WoE approach can be applied. Further considerations in deciding if additional immunotoxicity studies are needed will depend on the value of determining immunological off-target effects and exaggerated pharmacology. The thought process in making this decision is focused on how the data will be used and the level of confidence in the translation of the data from animal studies to humans. In the future, this level of confidence may be enhanced by using in vivo and in vitro methods with human cells of the immune system. REFERENCES Brehm, M. A., Shultz, L. D., and Greiner, D. L. (2010). Humanized mouse models to study human diseases. Curr Opin Endocrinol Diabetes Obes 17, 120 25. Collinge, M., Cole, S. H., Schneider, P. A., Donovan, C. B., Kamperschroer, C., and Kawabata, T. T. (2010). Human lymphocyte activation assay: An in vitro method for predictive immunotoxicity testing. JImmunotoxicol7, 357 66. Committee for Proprietary Medical Products. (2000). Note for Guidance on Repeated Dose Toxicity. Available at: http://www.ema.europa.eu/docs/ en_gb/document_library/scientific_guideline/2009/09/wc500003102.pdf. Danner, R., Chaudhari, S. N., Rosenberger, J., Surls, J., Richie, T. L., Brumeanu, T. D., and Caseares, S. (2011). Expression of HLA class II molecules in humanized NOD.Rag.1KO.IL2RgckKO mice is critical for development and function of human T and B cells. PLoS ONE 6, e19826. Food and Drug Administration, Center for Drug Evaluation and Research, U.S. Department of Health and Human Services. (2002). Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs. Available at: http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryin formation/guidances/ucm079239.pdf. International Conference in Harmonisation. (2005). Harmonised Tripartite Guideline: Immunotoxicity Studies for Human Pharmaceuticals S8. Available at: http://www.ich.org/lob/media/media1706.pdf. Piccotti, J. R., Lebrec, H. N., Evans, E., Herzyk, D. J., Hastings, K. L., Burns-Naas,L.A.,Gourley,I.S.,Wierda,D.,andKawabata,T.T.

Vol. 40, No. 2, 2012 KAWABATA AND EVANS 293 (2009) Summary of a workshop on nonclinical and clinical immunotoxicity assessment of immunomodulatory drugs. J Immunotoxicol 6, 1 10. Shultz, L. D., Saito, Y., Najima, Y., Tanaka, S., Ochi, T., Tomizawa, M., Doi, T., Sone, A., Suzuki, N., Fujiwara, H., Yasukawa, M., and Ishikawa, F. (2010). Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/ SCID/IL2rg null humanized mice. Proc Natl Acad Sci U S A 107, 13022 27. For reprints and permissions queries, please visit SAGE s Web site at http://www.sagepub.com/journalspermissions.nav.