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state nature publishing group art Safety Assessment and Dose Selection for First-in-Human Clinical Trials With Immunomodulatory Monoclonal Antibodies PY Muller1 and FR Brennan2 Modulating immune responses with monoclonal antibodies (mAbs) that target immune molecules has become a promising therapeutic strategy and is under investigation for the treatment of cancer and (auto)-immune diseases. A major hurdle to the development and early clinical investigation of many immunomodulatory mAbs is the inherent risk of adverse immune-mediated drug reactions in humans, such as cytokine storms, autoimmunity, and immunosuppression. Dose selection for first-in-human (FIH) clinical trials involving immunomodulatory mAbs, and mAbs in general, is based on specifically designed preclinical safety studies, primarily in nonhuman primates (NHPs), and on mechanistic ex vivo investigations. Dose selection in such trials is challenging for a number of reasons related to safety. In this context, safety-relevant differences between NHP and human immune systems, species selection/qualification and preclinical study design considerations, the receptor occupancy model and its calculation, the minimal anticipated biological effect level (MABEL) and its use in the selection of a safe starting dose in humans, microdosing and the impact of immunogenicity on safety assessment of mAbs, and safety-relevant formulation properties of therapeutic mAbs are critically reviewed. In addition, the current regulatory requirements are presented and discussed to demonstrate how the TeGenero TGN1412 case is leading to increased regulatory scrutiny regarding dose selection for FIH clinical trials. Immunomodulatory monoclonal antibodies (mAbs) are biopharmaceuticals that either enhance or suppress immune responses. With their ability to enhance the immune response against tumor cells, they have an unprecedented potential for use in the treatment of cancer.1,2 Using mAbs to stimulate the immune response against cancer cells employs an indirect mode of action, achieved primarily by either blocking inhibitory receptors such as cytotoxic T-lymphocyte-associated protein 4 or triggering/activating costimulatory receptors such as 4-1BB, CD40, or CD28. The underlying pharmacodynamic (PD) effects are mediated primarily by T cells or natural killer cells. Conversely, the immunosuppressive properties of immunomodulatory mAbs are explored for possible use in the treatment of autoimmune diseases and suppression of transplant rejection3 by suppression of immune-function cells, by prevention of their homing to lymphoid organs and inflammatory sites, or by induction of anergy or depletion of these cells. An alternative mode of action involves blocking of soluble targets, such as cytokines, by mAb-mediated sequestration. For a general overview of licensed mAbs, their isotypes, targets, indications for use, and adverse reactions, the recent compilation by Tabrizi and Roskos is recommended.4 In general, mAbs (including immunomodulatory mAbs) have proved to be safe and (in many cases) effective pharmaceuticals, whose toxicity is usually related to exaggerated pharmacology which can, in many cases, be predicted. However, the recent well-publicized adverse events observed with an immunomodulatory anti-CD28 superagonist mAb (TGN1412, intended to induce antitumor T-cell response in patients with B-cell chronic lymphocytic leukemia) in a first-in-human (FIH) clinical trial in the United Kingdom5 has highlighted how, on rare occasions, mAbs can be highly toxic. It is for this reason that preclinical safety assessment programs for mAbs, together with their dose selection for first clinical entry, will be scrutinized more than ever by the regulatory authorities in the years to come. Dose selection for FIH clinical trials (usually of double-blinded, placebo-controlled, single ascending dose design) needs to carefully balance safety of clinical trial subjects (particularly at first clinical dose administered) and efficient dose escalation to reach therapeutically active dose ranges. The latter requirement is an 1Novartis Institutes for BioMedical Research, Basel, Switzerland; 2Novartis Horsham Research Centre, Horsham, UK. Correspondence: PY Muller ([email protected]) Received 28 October 2008; accepted 2 December 2008; advance online publication 28 January 2009. doi:10.1038/clpt.2008.273 Clinical pharmacology & Therapeutics | VOLUME 85 NUMBER 3 | MARCH 2009 247 state art Table 1 Guidance documents supporting first-in-human trials with mAbs in general Guideline title Authority Year Ref. Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use FDA/CBER 1997 12 Guideline on Production and Quality Control of Monoclonal Antibodies EU 1994 13 S6: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals ICH 1997a 7 S8: Immunotoxicity Studies for Human Pharmaceuticals ICH 2005 11 S9: Nonclinical Evaluation for Anticancer Pharmaceuticals (draft, step 2) ICH 2008 18 Nonclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals FDA/CDER Tissue crossreactivity In vivo preclinical safety b Microdosing, eIND, and eCTA Position Paper on Non-Clinical Safety Studies to Support Clinical Trials With a Single Microdose EMEA/CHMP 2004 43 Exploratory IND studies FDA/CDER 2006 42 Guidance to the Conduct of Exploratory Trials in Belgium FAMHP 2008 41 Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers FDA/CDER 2005 16 Guideline on Strategies to Identify and Mitigate Risks for First-in-Human Clinical Trials With Investigational Medicinal Products EMEA/CHMP 2007 17 EMEA/CHMP 2007 46 First-in-human dosing and MABEL Immunogenicity (preclinical and clinical) Guideline on Immunogenicity Assessment of Biotechnology-Derived Therapeutic Proteins CBER, Center for Biologics Evaluation and Research; CDER, Center for Drug Evaluation and Research; CHMP, Committee for Medicinal Products for Human Use; EMEA, European Medicines Agency; EU, European Union; FAMHP, Federal Agency for Medicines and Health Products; FDA, US Food and Drug Administration; ICH, International Conference on Harmonisation; IND, investigational new drug; MABEL, minimal anticipated biological effect level. aIn revision. bDraft guidance, not yet officially released. ethical obligation for (open-label) trials involving patients rather than healthy volunteers, as is usually the case in oncology. This review aims to present a comprehensive overview of the scientific and regulatory considerations for safety assessment of immunomodulatory mAbs, particularly those targeting cell surface immune receptors, leading to dose selection for first clinical entry. It should assist clinical investigators and sponsors to critically and independently assess clinical protocols and investigators’ brochures for safety and dose selection of mAb therapeutics to be administered in FIH trials. A readily applicable representation for dose estimations aiming at low receptor occupancy (RO) following the minimal anticipated biological effect level (MABEL) approach is presented. TeGenero’s TGN1412 is used as an example, both to support this receptor saturation approach and to highlight how this case is leading to increased regulatory scrutiny and rapidly changing the regulatory framework (outlined in Table 1). Preclinical Safety Assessment For Immunomodulatory Mabs A major hurdle in the development and early clinical investigation of immunomodulatory mAbs with agonistic, immuneactivating mode of action is the relatively high inherent risk of adverse drug reactions in humans, such as systemic inflammatory reactions or manifestation of (generally reversible) autoimmunity.6 Risk prediction of such adverse reactions and dose selection for FIH clinical trials is based on preclinical safety 248 assessment in at least one pharmacologically relevant animal species.7 The selection of a relevant animal model (i.e., one in which the mAb binds to the target and elicits the same pharmacological effect as that expected in humans) and data interpretation of toxicity studies in such a model are paramount and need to be considered and justified on a case-by-case basis. It is acceptable to conduct studies in only one species of animal if only one relevant model can be identified, which is often the case for mAbs. Animal studies should be supported by ex vivo investigations using human and animal cells and tissues to determine the relative potency of the mAb in humans and the chosen animal model, to characterize the pharmacological activity in humans, and to examine specific aspects of mAb safety (discussed later).7 PD end points, if available, should be routinely assessed in toxicity studies to demonstrate pharmacological activity of the mAb in vivo in the test model. Generally, repeat dose toxicity studies using dose levels and exposures representing multiples of the starting dose and highest dose in humans, with a dosing duration of 4, or sometimes 13 weeks (depending on the duration of exposure in the FIH study) followed by an exposure-free recovery period of 4–8 weeks (the duration depending on the predicted duration of exposure and pharmacological activity; refer to “Pharmacokinetic Considerations”) are used for generating data to support human entry. The dosing duration in FIH studies for non-life-threatening indications is usually limited to the dosing duration covered in the animal studies.7 However, for life-threatening indications, such as in VOLUME 85 NUMBER 3 | MARCH 2009 | www.nature.com/cpt state oncology, treatment duration in FIH studies may considerably exceed preclinical coverage in animal studies. Most of these considerations are applicable to mAbs in general. The high species specificity of the sequence/structure of most immune receptors targeted by mAbs makes the selection of an appropriate animal model challenging. Given its genetic and pharmacological similarity to humans, the nonhuman primate (NHP) is the most commonly selected animal model for safety assessment of mAbs. For practicability/availability reasons the cynomolgus monkey (Macaca fascicularis, belonging to the family of Old World monkeys—Cercopithecidae) is the preferred NHP species. Occasionally marmosets (New World monkeys) and rhesus macaques are used. NHPs are normally the most relevant model for safety testing of mAbs, because the pharmacological activity in monkeys often resembles that in humans more strongly than that in lower species, such as dogs, rabbits, and rodents. In addition, the NHP immune system is generally more akin to that in humans than to that in lower species. Furthermore, human/humanized mAbs are likely to be less immunogenic following chronic dosing in NHPs than in lower species. In some rare cases, the only naturally occurring species in which certain very selective mAbs exhibit pharmacological activity is our closest evolutionary relative, the chimpanzee (Pan troglodytes, belonging to the family of great apes, Hominidae). However, toxicity studies involving terminal investigations are justified only in exceptional circumstances in this species, because of ethical considerations. Apart from the NHP, the relevance of a second species, e.g., rodent, needs to be assessed. In relatively infrequent instances when the mAb shows target binding and pharmacological activity in a rodent, most health authorities expect the rodent to be used as the second species for testing.7 In such cases, short-term toxicity studies (e.g., up to 4 weeks) are often performed in rodents. The immunogenicity of human/humanized mAbs often results in the generation of antidrug antibodies (ADAs) that facilitate mAb clearance and/or neutralize the pharmacological action, thereby restricting the duration of toxicity studies in rodents (refer to “Immunogenicity Considerations”). Toxicity studies in rodents and NHPs will not be informative and should therefore be ended when ADAs neutralize/reduce mAb exposure to levels that are not biologically meaningful or exert toxicities that are unlikely to be relevant to humans given the relative immunogenicity across species. However, if pharmacologically active exposure can be maintained in rodents, studies of longer duration would be expected. If no pharmacologically relevant animal model is available, the use of a surrogate mouse mAb (recognizing the mouse homolog of the human target)8 or testing of the human mAb in human target transgenic mice9 could be considered as options, depending on the specific pharmacological/toxicological end points intended to be assessed and whether they are likely to be predictive of the outcome in humans in these alternative toxicology models. What is common to both these approaches is the technical challenge of demonstrating the pharmacological relevance of the model in assessing safety in humans. Differences are potentially likely between rodents and humans with respect Clinical pharmacology & Therapeutics | VOLUME 85 NUMBER 3 | MARCH 2009 art to target ortholog/human transgene expression, turnover, and signal transduction, and this makes the extrapolation to humans of any animal-study findings very difficult. In addition, mAbs against different epitopes on the same target may have different toxicological profiles, thereby challenging the relevance of the surrogate approach, at least for some targets. In the case of a surrogate mAb, a second manufacturing process needs to be developed in order to produce a high-quality, well-characterized surrogate mAb for good laboratory practice toxicology studies, whereas testing of the human mAb in transgenic mice gives rise to problems of potential immunogenicity, resulting in restriction of the dosing duration. However, if the NHP is the only relevant naturally occurring animal model, neither surrogate mAb testing nor transgenic mice models expressing the human target are routinely required as a second species for study (authors’ experience). For mAbs with low general toxicity, the establishment of a maximum tolerated dose, as is done for new chemical entities, is generally not feasible in toxicity studies because doses can be so high (i.e., g/kg range) that potential effects might be confounded by protein overload. Instead, animal-study doses and/or exposures that provide an adequate number of multiples (typically ≥10×) relative to the anticipated clinical dose or exposure should be used as the (highest) doses with the aim of complete target saturation for the duration of the study. The frequency of dosing may be greater than the one intended to be used in the clinic, so as to compensate for potential differences in clearance and/or pharmacology. For mAbs with certain modes of action (refer to “The MABEL Approach to Determining Safe Starting Dose”), studying the intended clinical dose(s) as the (lowest) dose in the toxicity study might be warranted. Dose selection needs to be based on interspecies scaling, taking into account species-specific relative affinity/potency of a mAb, which is often lower in preclinical animal models than in humans. For immunomodulatory mAbs with agonistic/antagonistic mode of action, the doses in animal toxicity studies should be increased accordingly in relation to the intended clinical doses, in order to achieve complete activation/inhibition of the target. For immunomodulatory mAbs with agonistic mode of action, careful investigation of potential species-specific differences in effector function and/or signal transduction (refer to “The MABEL Approach to Determining Safe Starting Dose”) is highly recommended, and should be taken into account for dose selection in toxicity studies. For mAbs with an immunomodulatory mode of action, extended histopathology of lymphoid organs10 and immunophenotyping of lymphocytes is recommended as part of toxicity studies. Apart from the intended 1° immunopharmacological effects (PD end points), emphasis should be placed on detection of potential pharmacologically unintended and irreversible changes in immune parameters and/or overt immunotoxicity before first human entry. The need for screening of further immune-function-related end points (T-cell-dependent antibody responses, specific immune cell function assays) depends on the mode of action of the mAb and the signals observed. A tiered approach similar to the one commonly performed 249 state art for immunotoxicity assessment of new chemical entities at later development stages11 should be performed preclinically to support FIH studies involving immunomodulatory mAbs. Furthermore, immune-function biomarkers may be identified for use in detecting signs of potential immunotoxicity in clinical trials. Tissue crossreactivity (TCR) studies (in which binding of the mAb to human tissues and tissues from the chosen toxicology species is assessed) are performed both to identify the potential for mAb binding to tissues in humans and to confirm the relevance of the toxicology species (in which the mAb has a similar binding profile on human and animal tissues). TCR data also aid in the interpretation of any potential pathology findings in toxicity studies and in determining whether the toxicity findings in animals are relevant to human safety. Specific risks for immunotoxicity, reproductive toxicity, and adverse safety pharmacology effects may be flagged on the basis of TCR findings. Usually a full tissue list (32–35 fresh snap-frozen tissues) from three human and two animal donors is tested for TCR.12,13 Special absorption, distribution, metabolism, and elimination studies using radiolabeled mAbs are usually not informative for mAbs, because cleavage by proteases makes data interpretation difficult. These kinds of studies are therefore not required for mAbs (the TCR assesses potential distribution in humans). In vitro cardiac safety pharmacology assays (hERG channel activity) are not considered necessary for assessment of QT interval prolongation risk for mAbs as part of the preclinical safety strategy.14 mAbs therapeutics have very low potential to interact with intracellular (unlike low-molecular-weight compounds) or extracellular domains of the hERG channel and are therefore highly unlikely to inhibit hERG channel activity based on their targeted, specific binding properties. Furthermore, mAbs do not gain access to the cytosol of intact cells, because they reside in the endosomal compartment. Similarly, given the very low potential for direct DNA interaction, genotoxicity studies are usually not required for mAbs.7 Potential in vivo cardiovascular effects can be evaluated by standard electrocardiograms and blood pressure measurements in restrained animals within NHP toxicology studies or in conscious, freely moving NHPs fitted with noninvasive telemetry jackets. Along with cardiovascular parameters, potential cytokine release should be measured (preferably 2–6 h after the dose) wherever there is any concern relating to the mode of action of the mAb. A dedicated NHP cardiovascular safety pharmacology study by telemetry in conscious, freely moving animals to measure arterial blood pressure, heart rate, and ECGs may be warranted on the basis of specific study findings, if there is a mechanistic basis for a potential cardiovascular effect or if the mAb binds to tissues such as heart, lung, or brain in TCR studies (particularly for mAbs in the immunoglobulin G1 (IgG1) format due to Fc effector function).14 On the other hand, if the TCR studies show the absence of binding of mAb to major organ systems, separate safety pharmacology studies are not considered to be required. There are other important safety end points that are required to be assessed later in the process of clinical development and 250 prior to registration of mAbs, and these are not covered in detail in this review. These end points depend on the duration of treatment, the target population, and the mechanism of action; they include chronic toxicity (usually 26–39-week studies), reproductive toxicity (e.g., a combined embryo-fetal development and peri-/postnatal development study; there are usually no dedicated fertility studies, but fertility parameters can be assessed in chronic toxicology studies in sexually mature animals), and carcinogenicity (standard 2-year studies in rodents, rarely performed because of immunogenicity and lack of pharmacology in rodents). Potential adverse effects may also be predicted depending on the distribution and function of cells expressing the t arget receptor and on its species specificity, under both healthy and pathophysiological conditions. However, despite very similar target distribution, target expression (TE), and target sequence in NHPs and humans, downstream biological effects may differ considerably following target modulation, as evidenced by the adverse reactions reported during the FIH trial with TGN1412, an IgG4-based CD28 superagonist that activates T cells.5 Therefore, preclinical safety studies for immunomodulatory mAbs should be supplemented with science-based, mechanistic, ex vivo data using human cells and a range of mAb concentrations in order to assess the shape of the dose–response curve. Such experimental data may provide essential information for dose selection in FIH trials based on the MABEL, by incorporating all available in vitro and in vivo data.15 The MABEL approach is now routinely supplementing the classic concept of dose selection in FIH studies based solely on the “no observed adverse effect level” (NOAEL) in preclinical toxicity studies combined with appropriate interspecies scaling, i.e., the human equivalent dose (HED).16 For biopharmaceuticals >100 kDa that are administered intravenously, and for which distribution is largely confined to the vascular space, the HED should be calculated on the basis of body-weight (mg/ kg) scaling.16 For biopharmaceuticals <100 kDa, body surface area scaling (mg/m2) may be considered (e.g., for mAb fragments such as Fabs or nanobodies). Ultimately, the dose levels calculated by the NOAEL and MABEL approaches are weighted by appropriately justified safety factors (usually ≥10) based on potential risk/hazard, and then compared against each other in order to select the lower value as the actual starting dose in humans. The general aspects of risk mitigation relating to the organizational conduct of FIH trials with higher-risk medicinal products, including dosing sequence, rescue medication, and the qualifications of the investigators, are discussed in terms of regulatory issues in a recently released European Medicines Agency (EMEA) guideline.17 Special Considerations For Safety Assessment In Oncology Indications The early development of oncology drugs most often involves first-in-human clinical trials in cancer patients with latestage or advanced disease with limited therapeutic options. VOLUME 85 NUMBER 3 | MARCH 2009 | www.nature.com/cpt state Therefore, the study type and timing and the flexibility called for in designing nonclinical safety studies of anticancer pharmaceuticals can differ from those for other pharmaceuticals. A more flexible safety-assessment approach is endorsed in an only very recently released International Conference on Harmonisation Guideline, “Nonclinical Evaluation for Anticancer Pharmaceuticals” (S9, draft, step 2), which applies equally to low-molecular-weight drugs and biotechnologyderived pharmaceuticals such as mAbs.18 Phase I clinical trials in cancer patients may include dosing up to a maximum tolerated dose and dose-limiting toxicity. Therefore, determination of an NOAEL in preclinical toxicology studies is not considered essential to support human entry. Dose escalation in clinical trials in patients with cancer should not be limited by the highest dose tested in preclinical studies. However, when a steep dose–response curve is observed in toxicology studies, or when no preceding marker of toxicity is available, a slower escalation should be considered. For anticancer mAbs with agonistic mode of action, selection of the starting dose in first-in-human clinical trials should be considered using the MABEL approach (see “The MABEL Approach to Determining Safe Starting Dose”). Safety-Relevant Functional Differences Among Primate Immune Systems Despite the fact that NHPs and humans share a close evolutionary relationship, their immune systems have only limited similarity. This is because of the rapid evolution of the immune system in the direct ancestors of humans. There are a number of significant safety-relevant functional differences between the immune systems of NHPs and humans, and these need to be considered while assessing the safety of immunomodulatory mAbs. Compared with the T cells of our closest evolutionary relative, the chimpanzee (which, however, is not a direct human ancestor), human T cells give much stronger proliferative responses upon activation via the T-cell receptor. The underlying mechanism involves human-specific loss of T-cell Siglec expression during the later stages of evolution, subsequent to the last common ancestor of humans and great apes.19 CD33-related Siglecs, which are inhibitory signaling molecules expressed on most immune cells, downregulate cellular activation pathways via cytosolic immunoreceptor tyrosine-based inhibitory motifs.20 Among human immune cells, T lymphocytes are a striking exception, expressing few to none of these molecules, in contrast to those of chimpanzees and other primates such as cynomolgus macaques. Moreover, several common human T cell–mediated diseases, such as bronchial asthma, rheumatoid arthritis, and type 1 diabetes, have not been reported in chimpanzees or other great apes. These human-specific differences in T-cell activation need to be carefully considered during safety assessment of immunostimulatory mAbs against T-cell targets (see later text). Fc receptors are plasma membrane glycoproteins that bind to the Fc region of antibodies. Crosslinking of Fc receptors by Ab-opsonized antigen complexes initiates cellular immune Clinical pharmacology & Therapeutics | VOLUME 85 NUMBER 3 | MARCH 2009 art responses, including phagocytosis, Ab-dependent cell–mediated cytotoxicity (ADCC), respiratory burst, release of cytokines and inflammatory mediators, and antigen presentation.21 For certain anticancer mAbs, ADCC is intended as a direct mediator against tumor cells. Human FcγRIII, also known as CD16, is specific for IgG1 and IgG3. In humans, CD16a is expressed on monocyte subpopulations, macrophages and natural killer cells whereas the CD16b isoform is exclusively expressed on neutrophils and eosinophils that have been exposed to interferon-γ (IFN-γ). However, in NHPs only one CD16 gene, homologous to the human CD16a, is present. In baboon, rhesus, and cynomolgus monkeys, CD16a expression is restricted to natural killer cells and monocytes.21 These differences in cell-type expression should be carefully considered for safety evaluation of therapeutic mAbs, particularly if neutrophils and/or eosinophils are expected to be involved in any kind of human-specific downstream effects. Regarding IgG4 effector function, it has been shown that human IgG4, the underlying format of TGN1412, binds FcγRI relatively weakly but is capable of activating FcγRI-expressing cells.22 According to an internal report by TeGenero, the F(ab)2 fragment of TGN1412, in contrast to the full Ab, is not capable of inducing T-cell stimulation (see later). In view of the ongoing uncertainty regarding species-specific IgG effector functions, there is an urgent need for quantitative data on systematic crossspecies comparisons of the binding of human IgG subclasses to Fc receptors in NHPs and in humans. Most of the therapeutic mAbs that have received approval to date were developed in the IgG1 format, which is capable of mediating complement activation (binding to C1q). However, for immunomodulatory mAbs that bind to membrane-based targets, the IgG2 or IgG4 format (or silent IgG1 with reduced Fc effector function) is often used because of the absence of Fc effector function (to avoid cell depletion). In contrast to IgG1 and IgG3, IgG2 shows little complement activation and IgG4 shows none at all. Apart from complement-dependent cytotoxicity, which is an intended pharmacological effect of certain mAbs (e.g., direct killing of tumor cells), complement activation may lead to a number of safety-relevant consequences, including cytokine release, hematological effects, and/or potentially life-threatening hemodynamic disturbances.23 In spite of the fact that cynomolgus monkey serum indicates comparably high titers of complement components, hemolytic activity was shown to be very similar in the sera of cynomolgus monkeys and humans.24 This finding is supportive of comparable complement potency in cynomolgus monkeys and humans. However, mechanistic differences in terms of (cross-)speciesspecific IgG/Fc-mediated complement activation,25 particularly related to IgG aggregation status, cannot be excluded and deserve further investigation. Infusion Reactions: Hypersensitivity Reactions Vs. Cytokine Storms Acute infusion reactions upon administration of mAbs (black box warning for several approved mAbs) can be true hypersensitivity reactions, namely, IgE-mediated type I hypersensitivity 251 art reactions (anaphylactic reactions), or anaphylactoid reactions not mediated by IgE.26 True anaphylactic reactions usually do not occur upon initial infusion and require a certain sensitization. In contrast, the pathophysiology of anaphylactoid reactions appears to be secondary to the release of cytokines consequent to mAb binding to circulating antigen-expressing cells. However, anaphylactoid reactions are not equivalent to so-called cytokine storms (see later text). The clinical manifestations of anaphylactic and anaphylactoid reactions overlap, and both may lead to lifethreatening conditions involving the cardiovascular, respiratory, central nervous, gastrointestinal, and cutaneous systems. The predictive capacity of animal models with respect to these reactions is at best very limited, and the occurrence of infusion reactions in animals should not be extrapolated to humans. In humans, the management of anaphylactic and anaphylactoid reactions involves immediate administration of epinephrine, vasopressors, bronchodilators, corticosteroids, and/or antihistamines.26 If untreated, these reactions may be fatal. Therefore, in managing severe infusion-related reactions, it is crucial that risks are understood and early signs and symptoms are well recognized. Pleiotropic cytokines or mAb drugs that induce activation of a large population of (mainly) T cells or natural killer cells have the potential to initiate a cascade of systemic release of cytokines. This has been termed a “cytokine storm” and is characterized by the massive release of both proinflammatory (tumor necrosis factor–α, IFN-γ, interleukin-1 (IL-1), IL-6) and anti-inflammatory (IL-10) cytokines.27 A cytokine storm is a potentially fatal systemic immune reaction consisting of a positive feedback loop between cytokines and recruitment of immune effector cells. The immediate clinical manifestations are characterized by life-threatening systemic inflammatory reactions that may lead to rapid deterioration of cardiopulmonary, renal, and hepatic functions.5 Furthermore, tissue injury may occur as a result of cytokines activating vascular endothelial cells, and this in turn may result in vascular inflammation/damage and may promote intravascular coagulation and peripheral ischemia.27 Given the differences in T-cell activation between humans and NHPs (discussed in the previous section), T-cell-mediated cytokine storms are hard to predict on the basis of animal models (cf. the TeGenero case). In humans, the management of cytokine storms involves administration of corticosteroids and anti-IL-2 receptor antibodies combined with agents for stabilization of cardiopulmonary parameters if required.5 used for assessment of safe human starting doses and dose escalation of mAbs for which potential adverse reactions are thought to be predominantly mediated by on-target effects (exaggerated pharmacological response). In Figure 2, the antibody dose leading to an RO of 10% is calculated based on Eq. 3/4 in Figure 1 for a variety of TE levels ranging from 0.1 to 10 nmol/l. This plot can be readily used to determine dose levels leading to 10% RO (and/or RO <10% by linear reduction of the dose; see below). Eq. 1: K D = [ Ab free ] ⋅ [ Ta free ] ( Ab tot − Ab o Ta ) ⋅ ( Ta tot − Ab o Ta ) = [ Ab o Ta ] Ab o Ta Eq. 2: RO = Ab o Ta Ta tot Eq. 3 : Ab tot = Eq. 4 : Ab tot Abmolar dose Vinitial,plasma ⇒ Ab dose = Ab tot ⋅ MWAb ⋅V Vinitial,plasma K RO ⋅ Ta tot ⋅ RO − 1 − D Ta tot = RO − 1 Figure 1 Equations 1–4. Receptor occupancy (RO) for monoclonal antibody/target interaction and calculation of dose. Under equilibrium, the binding of an antibody (Ab) to its target (Ta), leading to the formation of an Ab–target complex (Ab ° Ta), is expressed by the mass–action law outlined in Eq. 1, whereby KD represents the dissociation constant. In Eq. 2, RO is expressed as the fraction of the Ab–target complex relative to total target expression, TE (Tatot). In Eq. 3, the dose (Abdose) of the Ab is expressed by total Ab concentration (Abtot) multiplied by its molecular weight (MWAb; assumed to be 150 kDa) and the initial plasma distribution volume after intravenous administration (Vinitial,plasma; assumed to be 0.036 l/kg). By combining Eqs. 1 and 2, the Abtot value can be expressed as shown in Eq. 4. 100.00 10.00 10 nmol/l TE Dose (µg/kg) state TGN1412 1.00 1 nmol/l TE 0.10 0.1 nmol/l TE The Ro Model For blood-based immune receptors with known quantitative TE, the RO can be calculated as a function of the mAb administered. Complete, i.e., 100%, RO is expected to lead to virtually maximum PD effect of a certain mAb, with the duration of saturation being dependent on the actual dose administered. The main parameters that influence doses as calculated by the RO model are outlined in Eqs. 1–4 in Figure 1. The concept behind this model is to predict and/or support a safe human starting dose that leads to a very low RO by the mAb administered (typically <10%), thereby giving rise to only very minor or virtually no PD effects mediated by the target. The RO model should therefore be 252 0.01 0.001 0.01 1 0.1 KD (nmol/l) 10 100 Figure 2 Graphical representation of monoclonal antibody dose leading to 10% receptor occupancy (RO). The antibody dose leading to an RO of 10% is calculated based on Eqs. 3 and 4 in Figure 1 as a function of the dissociation constant (KD) for total target expression ranging from 0.1 nmol/l (red line) through 1 nmol/l (blue line) to 10 nmol/l (green line) in 10 scaling steps each. Molecular weight is assumed to be 150 kDa; initial plasma distribution volume is assumed to be 0.036 l/kg. The TGN1412 dose leading to a predicted RO of 10% in humans is depicted. VOLUME 85 NUMBER 3 | MARCH 2009 | www.nature.com/cpt state Based on the characteristics of Eq. 3 in Figure 1, changes in molecular weight or initial plasma distribution volume after intravenous administration linearly translate into the respective changes in dose; e.g., a 2× lower molecular weight (75 kDa; e.g., antibody fragments) would result in a 2× lower dose, and a 2× higher initial plasma distribution volume after intravenous administration (0.72 l/kg) would result in a 2× higher dose. However, only at low RO levels (<10%) would changes in intended RO linearly translate into the respective changes in dose; e.g., a 10× lower RO (1%) would result in a 10× lower dose. Conversely, at higher RO levels, particularly toward the saturation range, there is considerable nonlinearity between RO and dose (based on the characteristics of Eq. 4 in Figure 1). This nonlinearity needs to be taken into account for finding dose-escalation strategies and should be calculated using the equation in Figure 3. For medium- and lower-affinity mAbs (i.e., dissociation constant (KD) >10–20 nmol/l), TE has only a very limited influence on the dose that leads to 10% RO (upper right area in the plot). Conversely, for high-affinity mAbs (i.e., KD <1 nmol/l), the dose that leads to 10% RO shows a proportional relationship with TE, particularly when the TE is relatively high (the area to the upper left in the plot with function lines parallel to the abscissa). This relationship needs to be carefully considered while determining dose selection of mAbs against targets with respect to which the expression is modulated by the underlying disease. In such cases, the use of simplified models for calculation of RO without consideration of TE should be discouraged. For high-affinity mAbs, the actual affinity (sometimes difficult to determine accurately) has virtually no influence on dose, particularly in the case of high TE (the area to the left in the plot with function lines parallel to the abscissa). Alternatively, RO can be calculated for any combination of KD, total target expression (i.e., TE) and total antibody concentration by combining Eqs. 1 and 2 in Figure 1 and subsequently solving the quadratic equation (resulting in the equation in Figure 3), whereby total antibody concentration is calculated as a function of the Ab dose administered (based on Eq. 3 in Figure 1). Such a calculation of RO should be routinely performed with respect to every dose-escalation step in FIH trials. Examples for calculation (with reference to the TGN1412 case) are shown in the respective section. The Mabel Approach To Determining Safe Starting Dose For certain risk profiles of therapeutic mAbs, the EMEA recommends calculation of safe human starting dose on the basis of the MABEL by incorporating all available in vitro and in vivo RO = KD + Ab tot + Ta tot − (− KD − Ab tot − Ta tot ) 2 − 4 ⋅ Ab tot ⋅ Ta tot 2 ⋅ Ta tot Figure 3 Equation 5. Receptor occupancy (RO) as a function of dissociation constant (KD), total target expression (Tatot), and total monoclonal antibody (mAb) concentration. By using this equation, RO can be calculated for any combination of KD, Tatot, and total antibody concentration (Abtot), where Abtot is calculated as a function of the mAb dose administered (based on Eq. 3 in Figure 1). Clinical pharmacology & Therapeutics | VOLUME 85 NUMBER 3 | MARCH 2009 art data.17,28 The calculation of starting dose should utilize all relevant information and follow a weight-of-evidence approach. Factors to be taken into account include the novelty of the agent and its mechanism of action, the degree of species specificity of the mAb and the target distribution, the steepness of the dose– response curves of biological effects in human and animal cells, dose–response data from in vivo animal studies wherein relevance to humans has been validated, the calculation of RO vs. concentration, and the calculated exposure of targets or target cells in humans in vivo. The species specificity of the mAb/target interaction should address and quantitatively compare affinity and potency, as well as signal transduction effects consequent to binding, such as duration of pharmacological action.29 Furthermore, the pharmacological impact of potential genetic polymorphisms of the target in preclinical safety models and in humans should be assessed. The EMEA recommends the MABEL approach in the investigation of medicinal products that are associated with increased risk with respect to (i) mode of action, (ii) nature of the target, and (iii) relevance of animal species and models. Similarly, with respect to immunomodulatory mAbs, the MABEL approach is encouraged in the investigation of medicinal products that fulfill one of the following criteria:15 1. The mechanism of action is: • Through acting on a master switch of the immune system (e.g., CD28 or cytotoxic T–lymphocyte-associated protein 4), • Through induction or modulation of pleiotropic cytokines; 2. The type of engineered scaffold is divalent, trifunctional antibodies, etc.; and 3. The target lacks appropriate animal models for safety testing—i.e., the mAb is not pharmacologically active on the target in animal models (e.g., the epitope or subepitope is not present in the animal model). For mAbs fulfilling criteria 1 and/or 2, it might be warranted to study the doses (scaling based on mg/kg) that will be admini stered to humans in relevant animal safety models first. The classic principle—that the highest dose in animal tests that does not lead to adverse effects (NOAEL) is the most relevant dose for determination of safety/safety margin—might not apply to immunomodulatory mAbs, for which lower doses could have a divergent effect (and even enhanced potency) as compared with higher doses (with the possibility of U- or bell-shaped dose– response curves). Furthermore, in such cases, the RO should be calculated in the animal model for the doses administered. Species-specific differences in target affinity as well as in effector functions mediated by Fc domains of mAbs may lead to differences in avidity, i.e., multivalent binding/crosslinking, capable of ultimately triggering variable downstream biological effects.30,31 Therefore, depending on the downstream effects mediated upon target binding, an intended RO <10%, e.g., 1%, may be warranted in FIH starting doses for certain mAbs, particularly for mAbs with agonistic modes of action. 253 state art Applying arbitrarily chosen safety margins (>>10) to the MABEL dose is no guarantee that safety is indeed sufficiently ensured. On the contrary, such an approach could quickly lead to unrealistically high safety factors and, in turn, to inappropriately low starting doses. It may be more advisable to obtain the necessary data that could reduce the uncertainty and that would enable a more accurate extrapolation from animals to humans than to keep increasing the safety factor. The major limitation of the MABEL approach is that it cannot avoid anaphylactic/anaphylactoid and/or other hypersensitivity reactions. Pharmacokinetic Considerations Clearance Further important factors to be considered are species-specific TE levels and/or rate of target turnover—potentially mediated by the underlying disease. In the case of a soluble target, the binding of the mAb to the target to form a complex will alter the clearance rate of most targets to one that conforms to the clearance rate of mAb-target complex, i.e., decelerating the clearance process. As a result, mAb-target complex will accumulate in the circulation. Conversely, for membrane-bound targets, the binding of the mAb to the target may alter the clearance rate of the mAb from a relatively slow process to one wherein the mAb is more rapidly cleared by internalization of the mAb-target complex (target-mediated disposition). However, the clearance of mAbs by cell surface targets represents a saturable process (“antigen sink”) (Figure 4). Therefore, clearance rate does decrease with increasing dose, leading to an overproportional increase in exposure and half-life, as is seen for trastuzumab32— particularly when achieving maximum RO. These relations need to be carefully considered during dose escalation and repeated dosing in early clinical trials. Knowledge of the expected (species-specific) behavior of such processes will take into consideration the distribution and clearance of the mAb in an Clearance mainly by targetmediated internalization Half-life Clearance mainly by RES (saturation of target-mediated clearance) Clearance mAb dose Figure 4 Clearance of monoclonal antibodies (mAbs) against cell surface targets. The total clearance represents the sum of (i) specific, targetmediated internalization that is nonlinear and saturable and (ii) nonspecific clearance that is linear and attributed to the reticuloendothelial system (RES). Plasma half-life has an inverse relationship with clearance, leading to relatively long initial half-life (at higher doses or higher plasma concentrations) and relatively short terminal half-life because of the prevailing target-mediated clearance (at lower doses or lower plasma concentrations). At higher doses or higher plasma concentrations, when the “antigen sink” is saturated, the clearance value approaches the nonspecific clearance by the RES. 254 appropriate pharmacokinetic (PK)/PD model. Such a model also needs to take into consideration species-specific FcRn-mediated IgG recycling and potentially reduced cross-species FcRn-IgG binding leading to increased clearance.33 In humans, FcRns are expressed on phagocytic cells of the reticuloendothelial system, thereby protecting against the rapid clearance of IgG1/2/4 but also mediating cross-placental and milk transfer. The downregulation of FcRs on cells of monocyte lineage by concomitant immunomodulatory drugs, such as methotrexate, may reduce the clearance of mAbs.34 Taking these factors into consideration, an estimate of the extent and duration of response for a given species, including humans, can be made. Furthermore, speciesspecific (terminal) clearance rates may need to be taken into account for selection of the duration of exposure-free off-dose periods, both in preclinical safety studies (“recovery period”) and in clinical trials (“washout”) with mAbs. In practice, four to five (terminal) half-lives followed by a 1-month true exposurefree period prove to be adequate. Mabel Approach Based On Ro Vs. Hed Based On Noael: The Tgn1412 Case For TGN1412, 10% RO would be reached in humans at a dose of 1.5 µg/kg (assuming a KD of 1.88 nmol/l and a total TE, total target expression, of 0.648 nmol/l, using Eqs. 3 and 4 in Figure 1; Figure 2), which is more than 60 times lower than the actually administered starting dose (0.1 mg/kg) leading to >90% RO (calculated based on the equation in Figure 3) and causing life-threatening cytokine release syndrome in healthy volunteers.5,35 Following this severe adverse drug reaction, a study was carried out on cynomolgus monkeys, which were dosed at 0.1, 0.5, 5.0, or 50 mg/kg using TGN1412 from the same batch that had been used clinically. All of these dose levels can be assumed to lead to complete receptor saturation in cynomolgus monkeys. In contrast to humans, cynomolgus macaques did not experience any adverse reactions at clinical dose level (0.1 mg/kg) as assessed by general health, blood pressure, heart rate, temperature, hematology, biochemistry, and liver function.28 In addition, the higher dose levels that had been used in the regulatory toxicity studies of TGN1412 were reconfirmed to be free of any adverse reactions. The NOAEL was considered to be at 50 mg/kg. Therefore, the HED corresponding to the NOAEL in cynomolgus monkeys (mg/kg scaling) is more than 30,000 times higher than the MABEL dose that is predicted to lead to 10% RO in humans. TGN1412 provides an impressive example of the divergence between the HED corresponding to the NOAEL in animal safety studies and the MABEL dose calculated using the RO model, and it demonstrates that the latter method is the most suitable for mAbs with such a mode of action. In rodents, CD28 superagonists induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. However, in humans, CD28 superagonists, including TGN1412, induce a delayed but extremely sustained calcium response in naive and memory CD4+ T cells, leading to pronounced cytokine release, an event not observed in cynomolgus T lymphocytes.36 VOLUME 85 NUMBER 3 | MARCH 2009 | www.nature.com/cpt state In vitro, cynomolgus lymphocytes did not proliferate when stimulated with immobilized TGN1412 unless IL-2 was added to the cultures. In contrast, in in vitro assays using human peripheral blood mononuclear cells, surface-immobilized TGN1412 stimulated the release of cytokines, including tumor necrosis factor–α, IFN-γ, IL-2, IL-6, and IL-8 and induced a profound proliferation of human CD4+ lymphocytes. This is in line with the finding of massive tumor necrosis factor–α and IFN-γ induction in humans5 as compared to the virtual absence of release of these cytokines in cynomolgus monkeys, even at 500× higher doses37 (Figure 5). However, borderline induction of IL-5 and IL-6 was observed in cynomolgus monkeys, which, in retrospect, needs to be considered as a safety-relevant signal indicating cytokine release. It was recently demonstrated that ex vivo cytokine production of peripheral blood mononuclear cells can be induced by TGN1412 in the presence of human endothelial cells.38 However, it is unclear whether endothelial cells of human and NHP origin can similarly crosslink anti-CD28 mAbs and, if so, whether such crosslinking plays a critical role in cytokine production in vivo and, further, which Fc receptor is involved.39 TGN1412 did not induce cytokine release or proliferative responses when presented in the aqueous phase or when crosslinked in aqueous phase. These findings clearly demonstrate that the absence of adverse findings in cynomolgus monkeys, even at exactly the same dose levels that are planned to be administered clinically, do not provide sufficient support for extrapolation to arrive at a safe 10,000 50 mg/kg TGN1412 in cynomolgus monkeys 0.1 mg/kg TGN1412 in humans 1,670 Fold induction 1,000 704 100 18 18 10 1 1 TNF-α 3 2 IFN-γ 1 IL-5 IL-6 IL-2 IL-4 Figure 5 Comparison of cytokine induction profiles of TGN1412 in nonhuman primates and in humans. Cytokine induction profiles are compared for TGN1412 doses of 50 mg/kg in cynomolgus monkeys (as part of the 4-week good laboratory practice toxicity study) and 0.1 mg/kg in humans. Maximum induction was detected 2–24 h postdose in cynomolgus monkeys (relative to vehicle control) and 4 h postdose in humans (relative to predose). Furthermore, in humans, massive induction of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12p70 was detected 4 h postdose (no quantitative data available). The most striking difference between cynomolgus monkeys and humans in terms of cytokine induction is the massive induction of tumor necrosis factor–α (TNF-α) and interferon-γ (IFN-γ) in humans as against the virtual absence of release of these cytokines in cynomolgus monkeys even at 500× higher TGN1412 dose. However, borderline induction of IL-5 and IL-6 was observed in cynomolgus monkeys. In retrospect, this needs to be considered a safetyrelevant signal indicating cytokine release. Clinical pharmacology & Therapeutics | VOLUME 85 NUMBER 3 | MARCH 2009 art starting dose in humans. Therefore, assessment of RO and relative pharmacology between animal models and humans should be routinely performed, both for preclinical safety studies and for FIH studies involving mAbs. Microdosing In Humans Microdosing is performed to assess pharmacokinetics, distribution, and/or imaging in FIH clinical trials without therapeutic intent. Such “phase 0” trials require only limited preclinical safety testing as outlined in the respective guidelines (Table 1). The concept is to administer a maximum dose of 100 μg of an investigational drug and less than 1/100th of the dose of the test substance that is calculated to yield a pharmacological effect in humans, as determined on the basis of data from animal studies.40 This approach refers mainly to small molecules and is outlined in guidance documents from the European Medicines Agency (EMEA), the FDA, and, recently, the Belgian Federal Agency for Medicinal and Health Products (FAMHP).41–43 However, in the FDA guideline, a maximum dose of 30 nmol is mentioned for protein products. For mAbs with a molecular weight of 150 kDa, this corresponds to 4.5 mg, or a dose of 64 μg/ kg (for a 70-kg subject). This dose is considerably beyond 10% RO, on the basis of KD and TE, for most mAbs, as outlined in Figure 2. Therefore, for microdosing studies with mAbs, 1/100th of the predicted pharmacologically active dose in humans is generally the more relevant safety parameter according to currently accepted safety standards, and it should be determined using the MABEL approach. Immunogenicity Considerations Most mAb therapeutics—even fully humanized mAbs or Fab fragments—elicit some level of antibody response (antidrug antibodies, ADAs) against the therapeutic product when administered to humans (and more so when administered to animals). Immunogenicity can lead to potentially serious side effects, alterations in PK parameters (mostly increased clearance), and loss of efficacy. It has been shown in mice that multimeric ADA immune complexes are rapidly cleared by the reticuloendothelial system—primarily in the liver.44 ADA responses against mAb therapeutics may be raised as early as after the first exposure; however, such responses are usually much stronger upon re-exposure. This property needs to be considered in the context of repeated dosing of mAbs in early clinical trials when the immunogenicity profile of an investigational mAb is poorly characterized. Shankar et al. suggest a risk-based strategy for the assessment of ADA responses against biological drugs.45 In accordance with the classification of Shankar et al., ADAs against immunomodulatory mAbs targeting cell surface receptors are to be considered as “low risk” (antagonistic mode of action) or “medium risk” (agonistic mode of action; possible overstimulation of endogenous mechanisms by therapeutic mAbs crosslinked by ADAs, “superagonist”). Other possible adverse reactions secondary to ADAs include injection-site reactions and immune-complex depositions. Furthermore, longer-term effects of therapeutic mAbs may be masked by the development of immunogenicity, possibly neutralizing the 255 state art harmacological/toxicological action of the mAb (neutralizing p antibodies)—both during safety assessment in NHPs and during clinical investigation in humans. In preclinical safety studies ADA determination is recommended at baseline, end of dosing, and end of (exposure-free) recovery. Analysis of potential ADAs in animal studies is important for the interpretation of PK parameters and is therefore essential in determining and confirming drug exposure and interpreting toxicity.45 However, the finding of immunogenicity of mAbs in animals, even in NHPs, has only a very limited capacity to predict the occurrence of ADA in humans. Conversely, the lack of ADA-related biological consequences in animal models cannot be interpreted as a predictor of safety in humans. For potential low- and medium-risk ADA responses, determinations should be carried out more frequently early in clinical development and less frequently at later stages, after the immunogenicity profile has been characterized.45 However, during routine prescription, it is of debatable value to monitor potentially neutralizing ADAs against immunomodulatory mAbs that target cell surface receptors, because the presence of neutralizing ADAs will probably not preclude the treatment of patients. The recently released EMEA draft guideline suggests a three-tiered analytical approach to monitoring ADAs levels during clinical trials: (i) screening assays, (ii) confirmation assays, and (iii) neutralization assays.46 It is of paramount importance to implement robust assays for immunogenicity assessment as early as possible during preclinical development of mAbs therapeutics. Assay design and assay performance parameters have been thoroughly reviewed by Mire-Sluis et al.47 The species-independent “bridging ELISA” format is often favored. In this context, the assessment of assay interference/competition of ADAs with PK measurements (ADAs binding to epitopes of mAb drug) and interference/ competition of mAb drug with ADA measurements (“drug-onboard”) is crucial.48 These interferences need to be thoroughly addressed during method implementation and method validation before FIH testing as well as before initiation of regulatory toxicology studies. Considerations Relating To Dosing Formulations Very low concentrations of mAbs in dosing formulations may be associated with a considerable proportion of nonlinear surface adhesion of proteins to plasticware such as tubes and infusion systems. Such adhesion may result in substantial reduction of the actually achieved concentrations of dosing formulations. This leads to considerable overestimation of the true concentration in dose formulations and, consequently, to overestimation of the administered dose. Therefore, surface adhesion needs to be assessed in all stages of the process of development, manufacturing, and analysis of dosing formulations—even during the preparation of calibrator solutions used in analysis of dosing formulations during preclinical safety studies as well as for release of the investigational drug product. In particular, this issue needs to be addressed in low-concentration dosing formulations administered in FIH testing. Furthermore, the compatibility of 256 the mAb and the formulation with primary packaging materials and administration systems should be investigated.17 The formulation properties of mAbs that affect immunogenicity, such as formulation and storage conditions, contaminants and impurities, exposure of neoepitopes on account of denaturation or fragmentation, and aggregation and adjuvant potential of inactive ingredients, need to be considered as well.45 This is particularly important given that manufacturing and formulation processes frequently change during early development of mAbs. Therefore, biological activity/potency as well as preclinical testing in general should be determined using defined reference material that is representative of the batch used clinically and validated/qualified assays. Conclusions And Recommendations This review summarizes safety assessment and dose selection for FIH clinical trials with immunomodulatory mAbs, based on the latest scientific and regulatory considerations. The MABEL approach, including the RO model, is discussed using TeGenero’s TGN1412 as an example, in order to both support this receptor saturation approach and highlight how this case is leading to increased regulatory scrutiny. Safety-relevant differences between human and NHP immune systems are highlighted, demonstrating that safety assessment of mAbs against T-cell targets is very challenging in NHPs and that NHP safety data need to be supplemented by data from ex vivo studies using human cells. The recent review by Melero et al. provides a comprehensive overview of potential T–cell targets for immunomodulatory mAbs for oncology indications.2 Based on the known physiological functions of these immune (co-)receptors, the risk of cytokine release in humans when these (co-)receptors are activated might be predicted to some extent. However, during safety assessment of TGN1412, this was not adequately performed. In the case of CD28, three key parameters are very similar, or even identical, in cynomolgus monkeys and in humans: the number of CD28 molecules expressed by T cells, 36 the affinity of TGN1412 to CD28, and the amino acid sequence of the extracellular CD28–binding domain.49 Furthermore, the Fcγ receptor binding of TGN1412 is expected to be comparable in cynomolgus monkeys and humans, given the high degree of Fcγ receptor sequence homology.39 However, none of these similarities turned out to be predictive for in vivo potency of TGN1412 on human T–cell activity relative to that seen in NHPs. Sequence comparison of CD28 between NHPs and humans revealed three amino acid exchanges in the corresponding transmembrane domains.36 These might influence the lateral interactions of CD28 with other signal-transducing molecules within the cell membrane and thereby alter the outcome of CD28 stimulation. The calcium signal observed in human T cells after CD28 activation was associated with the activation of multiple intracellular signaling pathways, culminating in the rapid de novo synthesis of high amounts of proinflammatory cytokines, particularly tumor necrosis factor–α and IFN-γ.36 This is in line with the VOLUME 85 NUMBER 3 | MARCH 2009 | www.nature.com/cpt state massive cytokine induction observed during the TGN1412 trial in humans5 but not in cynomolgus monkeys37 (Figure 5). The results reported by Waibler et al.36 clearly demonstrate that ex vivo models that assess cytokine release as an indicator of a cytokine storm in humans are valuable tools for assessing the relative potencies of immunostimulatory drugs in NHPs and in humans. The observation that calcium flux and cytokine secretion in human cells require crosslinking of TGN141236 raises the question of which Fcγ receptor–expressing cell types could have provided TGN1412 crosslinking in vivo and whether such crosslinking is also observed in NHPs. In case the pharmacological or toxicological mechanism of action of a certain mAb depends on receptor crosslinking, the possibility of a bell-shaped dose–response curve (for stoichiometric reasons) needs to be considered for both NHPs and humans. In this context, it needs to be emphasized that crosslinking of mAbs or Fab fragments bound to cell surface receptors can also be mediated by ADAs.45 This is of particular importance under conditions of repeated dosing in human subjects when ADA immune response becomes more likely than it is with single dosing. In vitro and ex vivo assays are required in order to justify and validate the species selection for preclinical safety testing. When no relevant, naturally occurring animal model can be identified for a certain mAb/target, a surrogate antibody or a transgenic mouse model expressing the human target might be considered. However, it remains questionable whether the use of a surrogate antibody or expression of the human target would be able to adequately mimic the intrinsically high ability of human T–cell activation19 and to identify a relevant NOAEL in any animal model. Therefore, the MABEL approach, together with RO-guided dose selection, needs to be followed in such cases so as to predict safe starting doses for first clinical entry. Only recently, an alternative ex vivo flow cytometric method was developed to experimentally determine the RO that can be taken to define the MABEL dose for TGN142.50 Despite the differences in sensitivity between NHPs and humans19 with respect to T-cell stimulation, risk prediction of cytokine storms in humans needs to account for any observation of even limited cytokine induction in NHPs, such as was observed with TGN1412.37 In retrospect, such a finding should be considered a safety-relevant signal. Similarly, changes in cytokine profiles, even in case very limited, should be critically reviewed during ongoing FIH trials, particularly from the viewpoint of justifying dose escalation. It is now recognized, based on a sound body of data, that findings in NHPs are not predictive for humans with respect to cytokine storms and that probably no other naturally occurring animal model is able to fully mimic those events.27 A way forward to prevent the risk of cytokine storms in humans might be to modify Fc regions or to use Fab fragments that would minimize effector-cell stimulation associated with cytokine release (if not required for efficacy). However this strategy has the drawback of being associated with increased plasma clearance profiles because of impaired or absent FcRnmediated mAb recycling. In conclusion, safety assessment for FIH clinical trials with immunomodulatory mAbs calls for a careful assessment of the Clinical pharmacology & Therapeutics | VOLUME 85 NUMBER 3 | MARCH 2009 art intrinsic safety-relevant properties of the investigational mAb itself and must also address human-specific target properties through in vitro, ex vivo, and in vivo investigations. 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