INTRODUCTION

Biologic pharmaceuticals are substances derived from living organisms, including humans, animals, plants and microorganisms, or produced by biotechnology methods (i.e., recombinant DNA technology). Proteinaceous biologics produced by biotechnology methods, which are the focus of this publication, include monoclonal antibodies, fusion proteins, cytokines and enzymes. Indeed, the majority of the United States Food and Drug Administration (FDA)-approved biologics fall into one of those four classes. Although extracted proteins, gene and cell therapies, vaccines and blood-derived products are also classified as biologics, they will not be addressed in this article.

Biologics have been approved for multiple indications, including cancer, infectious diseases, and cardiovascular and immune-mediated conditions, such as rheumatoid arthritis (RA) and multiple sclerosis (MS). Recognized mechanisms of action for monoclonal antibodies, which can also apply to fusion proteins, include the following: (1) blocking an activity by binding to soluble mediators, such as tumor necrosis factor-alpha (TNFα) or to cellular receptors for such mediators; (2) cell killing through complement-dependent cytotoxicity (CDC) and/or antibody dependent cell-mediated cytotoxicity (ADCC); and, (3) inducing an activity, such as apoptosis, through signaling (Glennie and Johnson, 2000). Recombinant cytokines and enzymes act through the same mechanisms as their native human counterparts.

Because biologics tend to be highly target specific and undergo proteolytic degradation to small peptides and constituent amino acids, adverse effects are generally related to the pharmacology of the compounds and are referred to as exaggerated pharmacology. Whether biologics are targeting the immune system or other systems, their safety needs to be evaluated (1) in appropriately designed pharmacology and toxicology studies in laboratory animals to support the safe use of products in humans during clinical trials and post-approval and (2) in clinical trials in the intended patient population.

In order to initiate clinical trials with biologics or other products in the United States (US), biopharmaceutical and pharmaceutical companies, referred to as sponsors, must submit an Investigational New Drug Application (IND) to the US FDA. The majority of the monoclonal antibodies, fusion proteins, cytokines and enzymes were originally regulated within FDA's Center for Biologics Evaluation and Research (CBER) but now fall under the jurisdiction of the FDA's Center for Drug Evaluation and Research (CDER). In the initial IND submission, sponsors inform FDA/CDER that they are seeking to proceed with the clinical development of a product. The original IND contains the following: Introductory Statement; General Investigational Plan; Investigator's Brochure; Protocol; Chemistry, Manufacturing and Control (CMC) Data; Pharmacology and Toxicology Data; Previous Human Experience; and Additional Information. Any subsequent submissions to an IND are referred to as IND amendments and include clinical protocol amendments, information amendments (CMC, microbiology, pharmacology and toxicology, and clinical), IND safety reports and annual reports (Novak and Whisenand, 2004). In order to obtain approval for marketing a new product, sponsors must submit a Biological License Application (BLA) to the FDA. BLAs contain an introduction and summary statement, manufacturing and control data, pharmacology and toxicology data and clinical data (Mathieu et al., 2004). Upon approval of a BLA, sponsors can begin marketing their product. Guidance documents are available to assist sponsors with multiple aspects of the IND and BLA processes. FDA/CDER has its own guidance documents on numerous topics. Additional guidance documents have been authored by the International Conference on Harmonisation (ICH), an international organization comprised of scientists from regulatory agencies and regulated industry in the United States, Japan and Europe.

The development of products that are specifically intended to affect the immune system (immunomodulatory) and can be used to treat medical conditions ranging from life-threatening cancers to less serious inflammatory conditions is an area of active pursuit. This article will provide an overview of immunomodulatory biologics, define approaches to identify potential adverse effects of these products on the immune system (hazard identification) and define the steps taken to assess and manage risks in patients.

OVERVIEW OF IMMUNOMODULATORY BIOLOGICS

The term “immunomodulatory biologic” (ImB) generally refers to products intended to elicit effects on the immune system. Examples of approved ImBs are shown in Table 1. Monoclonal antibodies and fusion proteins exert their therapeutic effects by binding to targets on immune cells and to soluble cytokines. For example, natalizumab (Tysabri®) binds to the α₄ subunit of integrins expressed on all leukocytes except for neutrophils and prevents binding to adhesion molecules expressed on vascular endothelium, which inhibits leukocyte migration into inflamed tissues (US FDA, 2006a). Tysbari is approved for the treatment of MS in a selected patient population. Recombinant human cytokines, such as interleukin (IL)-2, possess the biologic activities of the native proteins. For example, recombinant human IL-2 (PROLEUKIN, aldesleukin) produces multiple immunological effects, including activation of cellular immunity, profound lymphocytosis and production of cytokines (Chiron, 2000). PROLEUKIN is approved for the treatment of adults with metastatic renal cell carcinoma and metastatic melanoma.

When considering the properties and safety evaluation of ImBs, it is important to clearly define immunopharmacology and immunotoxicity. Immunopharmacology refers to the intended effect of a product, while immunotoxicity refers to undesirable/unintended extensions of immunopharmacology. For example, the intended effect of anti-TNFα products such as infliximab (Remicade) is to diminish the effects of this cytokine in tissues affected by inflammatory conditions such RA, Crohn's Disease and ankylosing spondylitis (US FDA, 2006b). However, because of the normal role of TNFα in the immune system's response to infection, the use of these products is associated with an increased risk of serious infections, which is considered an immunotoxicity of these products. In addition to increased infections and other types of immunosuppression-related effects, ImBs have been associated with autoimmunity and adverse immunostimulation. These effects have been linked to approved ImBs and to products being evaluated in clinical trials.

Autoimmune events, including thyroiditis and vitiligo, have been associated with treatment of cancer patients with interferon-α (Gogas et al., 2006; Koon and Atkins, 2006). Similarly, patients with malignant melanoma receiving treatment with an investigational monoclonal antibody, which is directed against cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), an immunoregulatory molecule expressed on activated T-lymphocytes and resting CD4⁺CD25⁺ T-lymphocytes, in conjunction with peptide vaccination, have exhibited autoimmune hypophysitis (Blansfield et al., 2005), colitis, dermatitis, uveitis, or enterocolitis (Attia et al., 2005). In March 2006, a striking example of adverse immunostimulation occurred in a clinical trial conducted in the United Kingdom (Suntharalingam et al., 2006). In that trial, normal healthy volunteers received TGN1412, a monoclonal antibody that stimulates and expands T-lymphocytes independently of ligation of the T-cell receptor by binding to CD28.

Within 90 minutes after receiving a single intravenous (IV) dose of TGN1412, the volunteers experienced a systemic inflammatory response, which resulted in a critical illness characterized by rapid induction of pro-inflammatory cytokines. The resulting cytokine storm was characterized by multiple symptoms including headache, myalgia, rigors hypotension, tachycardia, fever, multi-organ failure in two patients, desquamation and difficulty concentrating. The six volunteers receiving TGN1412, all of whom required treatment in an intensive care unit, ultimately survived. Serious adverse reactions such as those observed with TGN1412 are extremely rare.

IDENTIFYING EFFECTS ON THE IMMUNE SYSTEM

The hazard identification for ImBs as well as other types of therapeutics is a multi-step safety evaluation process, which can be accomplished using two frequently overlapping approaches: (1) background research/literature search and (2) experimental. The critical first step is to obtain as much information as possible on the biological system(s) to be targeted by the product. Sources of information include, but are not limited to, basic scientific literature, package inserts for approved products, redacted FDA reviews of approved BLAs, and publicly available information relating to products being evaluated in clinical trials. Information on approved products can be obtained from publications in peer-reviewed journals, scientific meetings, and the FDA website.

The FDA's redacted reviews of data that companies submit to support marketing approval can be found at the Drugs@FDA link on the FDA/CDER website. An evaluation of basic science can lead to an increased understanding of the biology of the system being targeted. Such information can be used to define potential concerns associated with targeting specific systems. Another goal of a thorough background/literature search is to gain as much information as possible about the differences and similarities between specific systems in humans and laboratory animals.

Experimental approaches to safety evaluation are comprised of nonclinical studies (also known as preclinical studies) that are conducted in in vitro systems and in laboratory animals to support the initiation and continuation of clinical trials through to approval and marketing. Nonclinical studies relevant to ImBs include nonclinical pharmacology, pharmacokinetics, general toxicology, and immunotoxicology. The specific types and designs of these studies can be influenced by concerns identified in the literature and/or from other sources. Basic properties of biologics, including ImBs, need to be considered in order to conduct scientifically meaningful nonclinical studies. First, biologics are highly targeted to a specific receptor or epitope (in the case of monoclonal antibodies). Second, they undergo proteolytic degradation to small peptides and constituent amino acids, as opposed to metabolism via cytochrome P450 and/or other enzyme systems to form active or reactive metabolites. Third, their relatively large size can limit their tissue distribution.

Due to the reasons cited here, effects associated with biologics, including ImBs, tend to be related to their intended pharmacology with “off target” effects being of limited concern. Therefore, nonclinical studies with biologics should be conducted in animal models with a molecular target and associated pharmacological mechanisms similar to humans. Such models are referred to as relevant models or species. Toxicology studies in non-relevant species can be misleading and are discouraged by the ICH guidance document addressing the nonclinical safety evaluation of biologics, which is entitled “Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals” (ICH, 1997). Therefore, accurately identifying a pharmacologically relevant animal model is a key to conducting scientifically meaningful nonclinical studies with biologics, including ImBs. The types of experiments needed to successfully identify a relevant animal model depend upon the type of product and mechanism(s) of action. Possible methodologies include, but are not limited to, immunohistochemistry, flow cytometry, and functional assays. The ultimate goal is to assess the relative pharmacodynamics of the product in animal models compared to humans.

As stated previously, toxicology studies conducted in non-relevant animal models can be misleading. For example, the humanized anti-human monoclonal antibody Hu1D10 binds to an HLA-DR variant expressed on normal B-lymphocytes and on B-cell lymphomas (Klingbeil and Hsu, 1999). The goal of toxicology studies conducted with Hu1D10 would be to evaluate the effect of the product's binding to B-lymphocytes in animals. Flow cytometry data have shown that the expression level of the antigen on B-lymphocytes varies among individuals. Based on flow cytometry data, monkeys can be divided into three categories with respect to antigen expression: strong positive, weak positive, and negative. When strongly positive and negative rhesus monkeys were treated with Hu1D10, B-lymphocyte depletion occurred in antigen-positive animals only, showing that B-lymphocyte depletion is antigen-specific.

Because biologics are generally specific for human targets, it is not surprising that the only relevant animal model for nonclinical studies is frequently a non-human primate (NHP), with the cynomolgus monkey (Macaca fasicularis) being the most commonly used. In certain cases, the biological activity of ImBs (and other types of biologics) is confined to humans, or to humans and chimpanzees. Although it is possible to conduct nonclinical studies in chimpanzees, the studies are limited in scope due to the protected status of the animals. For example, because chimpanzees cannot be sacrificed at the end of toxicology studies, it is not possible to obtain histopathology, which is frequently the most sensitive endpoint for assessing toxicity. It is possible, however, to obtain biopsies of some tissues from chimpanzees. In order to obtain sufficient toxicology data for molecules with such limited activity, alternate approaches, including analogous/surrogate molecules recognizing the target in animal models, or transgenic animals possessing the human target may need to be used.

For example, efalizumab (Raptiva) binds to CD11a in humans and chimpanzees only. In order to fully assess the safety of efalizumab, the product's sponsor developed and used an antibody that recognized CD11a in mice (Clarke et al., 2004). Such approaches require significant effort to characterize the analogous/surrogate product and its pharmacology in animal models.

General toxicology studies are conducted in NHPs and/or other species as appropriate to support the safety of products used in humans. The goal of general toxicology studies and other safety evaluation studies is to: (1) establish an initial safe starting dose and dose escalation scheme in humans; (2) estimate an acceptable risk:benefit ratio in humans; (3) identify potential target organs of toxicity and/or activity; (4) identify parameters for clinical monitoring; (5) delineate patient inclusion/exclusion criteria; and, (6) support eventual label claims (Serabian and Pilaro, 1999). General toxicology studies include both routine and specialized endpoints. Routine endpoints are included in toxicology studies for ImBs and other types of products, whereas specialized endpoints focus on the concern(s) associated with each specific compound. As will be discussed below, under certain circumstances, specialized endpoints can be included in general toxicology studies.

Routine endpoints for general toxicology studies include body weight, food consumption, clinical observations, ophthalmological evaluations, clinical pathology (clinical chemistry, hematology, coagulation and urinalysis), gross necropsy, organ weights and histopathology (Wilson et al., 2001). Those endpoints that are most relevant to assessing effects of compounds on the immune system include clinical pathology (hematology and clinical chemistry), gross pathology, organ weights, and histopathology. Standard hematological evaluations provide quantitative data on the broad classes of white blood cells (neutrophils, eosinophils, basophils, monocytes and lymphocytes) but not on subsets, such as T- and B-lymphocytes. Routine clinical chemistry tests assess globulins, which are a heterogeneous class of proteins that include immunoglulins, specific transport proteins, and mediators of inflammation (Hall, 2001).

Although it is possible to assess individual globulin components using protein electrophoresis, the practice is not recommended as standard procedure for toxicology studies (Haley et al., 2005). Necropsy should include gross evaluation, weighing of lymphoid tissue, and standard histopathology. Histopathological evaluation of lymphoid tissue has been the topic of numerous publications in recent years (Kuper et al., 2000; Germolec et al., 2004; Haley et al., 2005), with the goal being to obtain the most meaningful data possible from histopathological evaluation of lymphoid tissue.

Specialized endpoints that can be utilized include immunophenotyping (flow cytometry [FC] and immunohistochemistry [IHC]) and functional assays. Immunophenotyping allows for the identification of subsets of cells based on the expression of one or more distinctive antigens (Lappin and Black, 2003). It allows for different subtypes of cells with similar morphology (e.g., T- and B-lymphocytes) to be examined and quantified. Flow cytometry can be used to evaluate cells in peripheral blood and in single cell suspensions of lymphoid and other tissues. Immunohistochemistry allows for more detailed evaluation of cells within a tissue section.

Both FC and IHC can be readily incorporated into general toxicology studies. For example, Vugmeyster and co-workers (2005) used FC and IHC to characterize B-lymphocyte depletion and recovery in cynomolgus monkeys following treatment with an anti-CD20 monoclonal antibody being evaluated for treatment of B-lymphocyte-mediated diseases, including RA. FC and IHC are routinely included in toxicology studies conducted in NHPs for immunophenotyping.

One challenge that has faced sponsors developing ImBs is that, like many biologics, the activity of ImBs tends to be limited to humans and non-human primates. In comparison to rodents, however, assays intended to evaluate immune function in non-human primates are less developed and characterized. However, such assays are available, and in recent years progress has been made in developing them for use in NHPs.

Examples of functional assays include T-lymphocyte-dependent antibody response (TDAR), delayed-type hypersensitivity (DTH), and natural killer cell (NK) assays (De Gagne et al., 2006; Rechetnik et al., 2006; Satterwhite et al., 2006). The TDAR is perhaps the best characterized of the functional assays used in non-human primates. It measures primary and secondary antibody responses and is considered to be the best stand-alone functional assay. The ability of the contract research organizations (CROs) that perform many of the nonclinical studies needed to support drug development to conduct the TDAR is improving. Functional assays, such as the TDAR, can be included in general toxicology studies or in separate immunotoxicology studies. To date, the development of specialized endpoints to address autoimmunity and inappropriate immunostimulation lags behind that for immunosuppression. Methods are available, however, for measuring cytokine levels.

As discussed above, there are a variety of endpoints that can be used to assess the effects of ImBs on the immune system. Knowing which endpoints are considered appropriate from a regulatory perspective is a concern for those involved in the nonclinical safety evaluation of products intended for use in humans. In 2002, the US FDA/CDER published a guidance document entitled “Immunotoxicology Evaluation of Investigational New Drugs” (US FDA, 2002). However, it is specifically stated in the Introduction that it “is intended for drug products and does not apply to biological products.” It should be noted, however, that the document provides approaches to evaluate immunotoxicity that can be useful in assessing the effects of ImBs and serves as a general reference for these approaches. In 2006, the ICH published “ICH S8, Immunotoxicity Studies for Human Pharmaceuticals” (ICH, 2006). However, similar to the FDA/CDER document, the document “does not apply to biotechnology-derived pharmaceutical products covered by ICH S6 and other biologicals.” ICH S6 addresses the manner in which biologics can affect the immune system and states that “routine tiered approaches or standard testing batteries, however, are not recommended for biotechnology-derived pharmaceuticals.”

Unfortunately, ICH S6 does not provide any specific or detailed guidance for assessing the effects of ImBs or other biologics on the immune system. The lack of such information in ICH S6 is likely a reflection of the state of the technology available for assessing immune function in non-human primates in 1996 when the document was published. Additionally, the number of ImBs in clinical trials and being approved for chronic indications has increased since ICH S6 was written (Green and Black, 2000). Until more detailed guidance documents for assessing the effects of ImBs on the immune system become available, the appropriate use of routine endpoints and incorporation of specialized endpoints based on concerns associated with the pharmacology of the product can address safety concerns.

ASSESSING AND MANAGING RISK IN PATIENTS

Once data from nonclinical studies and other sources have been obtained, it should be possible to assess the potential risks that products pose to humans and to develop a strategy for managing those risks. FDA conducts an independent review of data obtained from nonclinical studies and other sources, such as published literature and previous experience with similar products. Unacceptable risk can result in a clinical hold, non-approval, or removal of the product from the market place. Risk can be managed in patients through clinical trial design, risk communication, and specialized programs. Risk management through clinical trial design should include, but not be limited to, establishing the safe starting dose, defining the appropriate patient population, and establishing appropriate monitoring.

In July 2005, FDA/CDER published a guidance document entitled “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” (US FDA, 2005). The document outlines a process (algorithm) for determining the Maximum Recommended Starting Dose (MRSD) for first-in-human clinical trials of new molecular entities in adult healthy volunteers. The document applies to both traditional pharmaceuticals and biologics, including ImBs. It does not, however, apply to endogenous hormones and proteins (e.g., recombinant clotting factors) used at physiologic concentrations or to prophylactic vaccines. Although the primary intent of the document is to address starting doses in normal healthy volunteers, many principles and some approaches may be applicable to trials conducted in patients. Included in the document are considerations that present an increased cause for concern. Those considerations are shown in Table 2.

In addition to establishing the appropriate starting dose, risk management through clinical design includes defining the appropriate patient population and endpoints for monitoring based upon nonclinical data, clinical data for similar products, and the biology of the product being evaluated. Risk communication is essential for all products and, in certain cases, specialized programs are needed to manage risks. Risk communication begins prior to initiation of first-in-human clinical trials and continues after a product has been approved for marketing.

According to the Code of Federal Regulations (CFR) 21 CFR 312, each IND should contain an Investigator's Brochure (IB) to be prepared by the sponsor (CFR 2006). The IB is intended to provide the investigators responsible for conducting the clinical trial with a summary of the available information for the product. The IB should include, but not be limited to, the following: a brief description of the product and the formulation, a summary of the pharmacological and toxicological effects of the product in animals and, if available in humans, a summary of safety and efficacy data from prior clinical trials, a description of possible risks and side effects anticipated in humans and precautions or special monitoring to be included in clinical trials. The IB undergoes FDA review. Inaccurate or misleading statements in an IB can result in a clinical hold (Mathieu, 2004). Sponsors communicate potential risks in lay language directly to volunteers/patients through the use of an Informed Consent document. Risk communication continues after approval of the product. By law, adequate directions for use must be provided for biologics and other medicinal products (Mathieu et al., 2004). The content of the product package insert, which is defined in 21 CFR 201, includes information on a number of topics that are applicable to risk management in the post-marketing scenario (CFR, 2006). For example, the contraindication section describes situations under which a product should not be used because the risks clearly outweigh any potential benefit. Specialized prescribing programs can be employed to minimize risks presented by serious adverse events. In summary, properly assessing the risks that products, including ImBs, pose to human subjects/patients and developing strategies for managing and communicating risks are needed to ensure safe development and use of ImBs, as well as other products. Numerous examples of risk assessment, management and communication are available among approved ImB products.

CASE EXAMPLES

Information derived from FDA reviews, product package inserts, and other publicly available sources relating to approved products provide examples of hazard identification and risk management scenarios for ImBs. Available examples include Amevive® and Tysabri, both of which will be discussed next.

Amevive (alefacept) is a fusion protein consisting of the CD2-binding portion of LFA-3 (CD58) linked to the F_c portion of IgG₁. By binding to CD2, it inhibits the LFA-3·CD2 interaction and interferes with lymphocyte activation. Additionally, Amevive reduces subsets of CD2 T-lymphocytes, presumably due to the F_c portion of the molecule engaging effector cells. Amevive is indicated for adult patients with moderate to severe plaque psoriasis who are candidates for systemic or photo-therapy. The non-clinical toxicology program for Amevive included a 52-wk toxicology study conducted in cynomolgus monkeys (US FDA, 2003). In that study, the product was administered IV at doses of 0, 1 or 20 mg/kg/wk. All treated animals were positive for lymphocryptovirus (LCV, an endemic primate herpes virus). Although latent LCV infection is generally asymptomatic, it can lead to B-lymphocyte hyperplasia and/or lymphoma in immunosuppressed animals. One animal in the 20 mg/kg group developed a B-cell lymphoma that was detected after 28 wk of dosing. Additional animals in both treatment groups developed B-cell hyperplasia of the spleen and lymph nodes. No evidence of B-cell hyperplasia or lymphoma was observed one year post-treatment.

Treatment of patients with immunosuppressive products, such as Amevive raises concern about increased risk of malignancy in humans. The occurrence of B-cell lymphoma in a monkey receiving alefacept raised the concern for malignancy associated with Amevive. Additionally, an increased incidence of malignancy was noted in patients enrolled in clinical trials conducted with Amevive (1.3% in treated patients vs. 0.5% in the control group) (US FDA, 2003). Communication of the risk of increased malignancy was accomplished by including the findings from the toxicology study and the clinical trial in the warnings section of the package insert. The risk to patients was managed by including the following statement in the package: (1) Amevive should not be administered to patients with a history of systemic malignancy; (2) caution should be used when considering the use of Amevive in patients at high risk for malignancy; and, (3) Amevive treatment should be discontinued if a patient develops a malignancy.

Tysabri (natalizumab) is an IgG₄ monoclonal antibody to the α 4β 1 and α 4β 7 integrins expressed on the cell surface of all leukocytes (except for neutrophils) and inhibits binding to adhesion molecules. Interference with the interaction between integrins and adhesion molecules prevents transmigration of leukocytes into inflamed parenchymal tissue (US FDA, 2006a). The nonclinical safety evaluation of Tysabri included 28-d and 6-mo general toxicology studies conducted in cynomolgus monkeys. In those studies, animals exhibited an increase in circulating leukocytes, primarily lymphocytes, which was attributed to the pharmacological activity of the product (US FDA, 2006c). Additionally, a 28-d study was conducted in rhesus monkeys to evaluate the toxicity of Tysabri in combination with Avonex (interferon-β_1α), which is approved for the treatment of MS. That study did not reveal any interaction between the two products.

In November 2004, Tysabri was approved for the treatment of patients with relapsing forms of MS to reduce the frequency of clinical exacerbations. In February 2005, at the sponsor's initiative, the marketing of Tysabri was suspended and clinical trials with the product halted due to the diagnosis of progressive multi-focal leukoencephalopathy (PML) in two MS patients receiving Tysabri in combination with Avonex. Subsequently, PML was diagnosed in a third patient who had received treatment with Tysabri for Crohn's disease. PML is a rare opportunistic demyelinating infection that is caused by JC virus (JCV) and occurs in immunocompromised patients (Greenlee, 2006). In June 2006, after an extensive review of data from ∼ 3000 patients failed to reveal any additional cases of PML, FDA approved an application to resume marketing of Tysabri.

The following steps were taken to communicate and manage the risk of PML: (1) Tysabri was approved as monotherapy for the treatment of patients with relapsing forms of MS; (2) Tysabri was generally recommended for patients who have had inadequate response to, or have been unable to tolerate, alternative therapies; (3) a black box warning, (wording, enclosed by a black border, at the beginning of the approved package insert to alert prescribers and patients about serious adverse events) regarding the risk of PML was included in the approved package insert for Tysabri; and, (4) Tysabri was made available only through a prescribing program known as TOUCH, which limits those who are able to prescribe, distribute or infuse Tysabri and the patients who can receive the product (Greenlee, 2006; US FDA, 2006d).

SUMMARY AND CONCLUSIONS

ImBs are produced using biotechnology methods and are intended to augment or diminish immune system function. Although these products are intended to modulate the immune system, they can still cause immunotoxicity (exaggerated immunosuppression, immune stimulation, and autoimmunity). In order to support the use of ImBs during development and post-approval, toxicology and other nonclinical studies are conducted in relevant animal models (i.e., animals possessing a receptor or epitope for the product). Effects of ImBs on the immune system can be assessed using endpoints that are routinely included in nonclinical toxicology studies (e.g., hematology, organ weight, and histopathology) as well as specialized endpoints intended to further characterize cell type (immunophenotyping) and function (TDAR). Data obtained from nonclinical studies, in combination with information from other sources, can be used to assess the risks that ImBs pose to humans. Risks to humans can be addressed through clinical trial design and communication.