Symposium International Journal of Toxicology 1-11 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1091581815584918 ijt.sagepub.com

Cytokines: The Good, the Bad, and the Deadly Thulasi Ramani1, Carol S. Auletta1, Daniel Weinstock2, Barbara Mounho-Zamora3, Patricia C. Ryan4, Theodora W. Salcedo5, and Gregory Bannish1

Abstract Over the past 30 years, the world of pharmaceutical toxicology has seen an explosion in the area of cytokines. An overview of the many aspects of cytokine safety evaluation currently in progress and evolving strategies for evaluating these important entities was presented at this symposium. Cytokines play a broad role to help the immune system respond to diseases, and drugs which modulate their effect have led to some amazing therapies. Cytokines may be ‘‘good’’ when stimulating the immune system to fight a foreign pathogen or attack tumors. Other ‘‘good’’ cytokine effects include reduction of an immune response, for example interferon b reduction of neuron inflammation in patients with multiple sclerosis. They may be ‘‘bad’’ when their expression causes inflammatory diseases, such as the role of tumor necrosis factor a in rheumatoid arthritis or asthma and Crohn’s disease. Therapeutic modulation of cytokine expression can help the ‘‘good’’ cytokines to generate or quench the immune system and block the ‘‘bad’’ cytokines to prevent damaging inflammatory events. However, care must be exercised, as some antibody therapeutics can cause ‘‘ugly’’ cytokine release which can be deadly. Well-designed toxicology studies should incorporate careful assessment of cytokine modulation that will allow effective therapies to treat unmet needs. This symposium discussed lessons learned in cytokine toxicology using case studies and suggested future directions. Keywords cytokines, biotherapeutics, immunomodulators, cytokine release syndrome, biomarkers

Introduction Cytokine Explosion: Rocking the World of Toxicology (Thulasi Ramani and Carol S. Auletta, Huntingdon Life Sciences) This symposium began with a thorough overview of cytokine biology and a discussion of the role of cytokines in the regulation of the immune system. Strategies for developing cytokine-targeted therapies were presented and included a discussion of regulatory guidelines and preclinical development strategies. Nonclinical development was discussed further in a presentation on safety evaluation of cytokine therapeutics with illustrations of translation of preclinical data to the clinic. Subsequent presentations summarized special considerations required in designing and interpreting nonclinical safety studies for biologics that either induce cytokines or block a key cytokine pathway as part of their mechanism of action and a thorough discussion of cytokine release syndrome with suggestions for predicting and managing this adverse immunemodulated reaction. A separate presentation reviewed the importance of including biomarkers for evaluation of immunotoxicity in development strategies. Illustrative case studies were included throughout.

Therapeutic Cytokine-Blocking Antibodies (Daniel Weinstock, Janssen Research and Development) Introduction to anticytokines therapy Understanding the structure, function, and biology of cytokines is essential for developing a rational strategy to intervene in cytokine-mediated disease processes. Knowledge of regulatory guidance and a case-by-case application to those principles is required for successful development of anticytokine therapeutics. Cytokine comes from the Greek root words ‘‘cyto’’ for cell and ‘‘kinos’’ for movement. Cytokines are small proteins

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Huntingdon Life Sciences, Somerset, NJ, USA Janssen R&D, LLC, Spring House, PA, USA 3 ToxStrategies, Inc, Bend, Oregon, USA 4 MedImmune, Gaithesburg, MD, USA 5 Bristol Myers Squibb Company, New Brunswick, NJ, USA 2

Corresponding Author: Thulasi Ramani, Huntingdon Life Sciences, 100 Mettlers Road, Somerset, NJ 08873, USA. Email: [email protected]

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released by cells that act via receptors to regulate the growth, maturation, and responsiveness of particular cell populations. Typically, cytokines are approximately 5 to 30 kDa peptides, proteins, or glycoproteins. They have a very short half-life and act locally at picomolar concentrations. Cytokines are produced by a wide variety of immune and nonimmune cells and are known by many different names including lymphokines, monokines, chemokines, interleukins (ILs), interferons (IFNs), colony stimulating factors, and growth factors. All these are soluble factors that bind to cell surface receptors, triggering differential gene cell expression by the targeted cells. They form complex interreactive networks with potential autocrine, paracrine, and endocrine functions. Cytokines are most well known for the key role that they play in regulation of the immune system, and therefore, cytokine pathways have been utilized as targets for successful therapeutic intervention via marketed products. Examples of cytokines and receptors targeted by approved large therapeutics include tumor necrosis factor (TNF), IL-1, interleukin 2 receptor (IL-2R), IL-6R, IL-12, IL-23, and receptor activator of nuclear factor-kB ligand. Indications include immunemediated diseases such as inflammatory bowel disease, rheumatoid arthritis, asthma, and psoriasis. Cytokines may be classified by structure or function. There are 4 structural families: 4 alpha helix bundle family (includes IL2 subfamily, IFN subfamily, and IL-10 subfamily), IL-1 family, IL-17 family, and cysteine-knot cytokines (transforming growth factor b [TGF-b] superfamily). Functionally, there are type 1 and type 2 immunomodulatory cytokines. Type 1 favors regulation of cellular immune responses (IFN-g, TNF-a, etc), while type 2 cytokines favor regulation of antibody production (TGF-b, IL-4, IL-10, IL-13, etc). Cytokine receptors are classified into 5 major families: immunoglobulin superfamily, hematopoietic receptor family (class I), IFN receptor family (class II), TNF receptor family, and chemokine receptor family. Although the cytokine ligands for the receptors are quite variable, class I and II receptors signal through the Janus kinase family of tyrosine kinases. The TGF-b receptors signal through serine/threonine protein kinases. Chemokine receptors couple to G-proteins for signal transduction. Production of multiple cytokines by different cell types and variable expression of receptors by multiple cell types results in complex functional networks. Cytokines can be pleiotropic with different effects on different cell types. Cytokines can act synergistically. There are redundancies within cytokine networks. Cytokines can have autocrine, paracrine, or endocrine effects but must be differentiated from hormones. Cytokine release can result in cascading expression of subsequent cytokines and receptors in other cells or set up feedback loops. As an example, IL-4 drives differentiation of naive T-helper (Th) cells to Th2 cells. Additional IL-4 is produced by Th2 cells driving proliferation of activated T and B cells while also causing immunoglobulin E (IgE) class switching in B cells. IL-4 upregulates expression of MHC class II and decreases production of Th1 cells, IFN-g, and dendritic cell-derived IL-12. IL-4 drives differentiation of macrophages into M2 while inhibiting classical

activation to M1 phenotype. IL-5 also plays a role in B-cell proliferation and IgE class switching, which may be inhibited IFN-g. The receptor for IL-4 exists in several different forms. Type 1 binds IL-4 alone while type 2 binds both IL-4 and IL-13. Therefore, successful therapeutic intervention requires detailed knowledge of the network function of the targeted cytokine. Strategies for therapeutic intervention can target the cytokine or the receptor. Inhibition of function can be achieved by neutralization of the soluble cytokine and/or removal from circulation using monoclonal antibodies (mAbs) or alternate protein scaffolds or by scavenging via a soluble receptor or receptor fusion protein. Receptor blockade may be achieved by inhibition of binding sites or by inhibition of signaling cascade. Multiple different types of novel engineered biotherapeutic proteins are being developed as therapeutic agents. Engineered mAbs and immunoglobulin fractions have altered pharmacology versus the native immunoglobulin, such as altered half-life and altered binding of Fc receptors. Novel protein scaffolds derived from intrinsic human covalent protein binding structures demonstrate unique pharmacologic properties depending upon size, charge, and moiety. Thorough understanding of the structure, function, and pharmacology of the therapeutic agent is essential for design and implementation of a successful development strategy. Regulatory guidance for large-molecule immunomodulatory therapy is publically available in the form of ICH guideline S6 (R1)—Preclinical Safety Evaluation of Biotechnologyderived Pharmaceuticals.1 ‘‘The primary goals of preclinical safety evaluation are (1) to identify an initial safe dose and subsequent dose escalation schemes in humans, (2) to identify potential target organs for toxicity and for the study of whether such toxicity is reversible, and (3) to identify safety parameters for clinical monitoring.’’ ‘‘Preclinical safety testing should consider (1) selection of the relevant animal species; (2) age; (3) physiological state; (4) the manner of delivery, including dose, route of administration, and treatment regimen; and (5) stability of the test material under the conditions of use.’’ Preclinical development strategies vary tremendously depending on the disease indication, target, and therapeutic molecule. Programs for chronic inflammatory diseases versus oncology are quite different. In general, a preclinical development program for a large-molecule immunomodulator should consider appropriate toxicology species (usually nonhuman primate [NHP]), routes of administration (intravenous [IV]/subcutaneous [SC]), dosing frequency (1-2/wk), pharmacokinetic/ pharmacodynamic (PK/PD) assays, immunogenicity, safety pharmacology, carcinogenicity, developmental and reproductive toxicity, and peri- and postnatal development studies. Studies typically conducted prior to initiation of FIH dosing include a non-GLP 1-month NHP toleration study with PK and antidrug antibody (ADA) assessments, a GLP 1- or 3-month NHP study, Good Laboratory Practice (GLP) tissue cross-reactivity, cytokine release assay (CRA), blood, and serum compatibility assays. Adverse and nonadverse findings are often observed in preclinical and clinical studies with immunomodulaory therapeutics. Understanding the pathogenesis of these responses and their

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Table 1. Approved Cytokine and Cytokine Blocking/Neutralizing Therapeutics. Biotherapeutic

Target, mechanism of action

Indication

Adalimumab (Humira)

Neutralization of TNF-a activity

Rheumatoid arthritis, psoriasis, Crohn disease, ulcerative colitis, ankylosing spondylitis, and juvenile idiopathic arthritis Psoriasis Rheumatoid arthritis Hepatitis C and hepatitis B

Ustekinumab (Stelara) Neutralization of IL-12 and IL-23 activities Tocilizumab (Actemra) Inhibition of IL-6R signaling Peginterferon alfa-2a (Pegasys) Recombinant a-2a-interferon; induces innate antiviral immune response Interferon alfa-2b (Intron A) Recombinant a-2b interferon; induces innate antiviral immune response Aldesleukin (Proleukin)

Basiliximab (Simulect) Daclizumab (Zenapax)

Malignant melanoma, follicular lymphoma, AIDS-related Kaposi sarcoma, hepatitis C, and hepatitis B Metastatic renal cell carcinoma

Recombinant interleukin-2; inhibition of tumor growth via activation of cellular immunity (exact mechanism of action unknown) IL-2Ra; mAb that binds and blocks IL-2 receptor a chain Prophylaxis of acute organ rejection in renal (CD25 antigen) on the surface of activated T cells transplant patients IL-2R; mAb that binds a subunit of IL-2 receptor (CD25); Prophylaxis of acute organ rejection in renal inhibits IL-2–IL-2R interactions transplant patients

Abbreviations: mAb, monoclonal antibody; IL, interleukin; IL-2R, interleukin 2 receptor; TNF-a, tumor necrosis factor a.

relevance to humans is necessary. Regulatory guidance is provided in ‘‘Guidance for Industry, S8 Immunotoxicity Studies for Human Pharmaceuticals.’’2 Immune end points in standard toxicity studies are important considerations and additional standalone studies may be required. Standard immune end points include lymphoid organ weight, appearance and histology, hematology and clinical chemistry as well as surveillance for opportunistic infection and increased incidence of neoplasia. Some additional end points that may be included as part of standard toxicity studies are serum cytokine levels, immunophenotyping of peripheral blood cells and T-cell-dependent antigen response as well as any unique biomarkers of toxicity and/or efficacy. Additional stand-alone studies may include host resistance animal models and a variety of ex vivo and in vivo cell-based assays. In summary, cytokines and their receptors are validated targets for multiple therapeutic areas. Detailed knowledge of the systems biology of the targeted cytokine and receptor is essential. Knowledge of the pharmacology, effector function, and idiosyncrasies of the large-molecule scaffold of the therapeutic agent is required. Monitoring of immune end points specific for the target is a necessity for an effective development program.

Safety Evaluation of Cytokine Therapeutics: Translation of the Preclinical Data to the Clinic (Barbara Mounho-Zamora, ToxStrategies, Inc) Cytokines are a family of proteins that are an important component of the immune system acting as mediators between cells to regulate the human immune response.3 Cytokines modulate the balance between humoral- and cell-based immune responses as well as regulate the maturation, growth, and responsiveness of certain cell populations. Cytokines are grouped into different categories based on function and/or source, and include:

  

mediators of innate immunity (TNF-a, IL-1, IL-10, IL-12, INF-a, and INF-g), mediators of adaptive immunity (IL-2, IL-4, IL-5, TGF-b, IL-10, and INF-g), and mediators of hematopoiesis (granulocyte–macrophage colony-stimulating factor [GM-CSF]).

Cytokines can act on several different types of cells and regulate different immune functions. Different cytokines can also have similar function; for example, both IL-1 and TNF-a act as inflammatory mediators. Imbalances in cytokine production and/or cytokine receptor activation can result in various pathological disorders such as rheumatoid arthritis or systemic lupus erythematosus, which are associated with a dysregulation of various cytokines, such as TNF-a, IL-1, and IL-6. It has been long recognized that cytokines and cytokine receptors can be excellent targets for medicinal products.4 However, it is critical to consider the complex biology of cytokines when selecting a cytokine as a therapeutic target, particularly the pharmacological action of the therapeutic (eg, blocking a cytokine, anticytokine therapy). There are numerous approved cytokine biotherapeutics for the treatment of various conditions (Table 1). The pharmacological action of cytokine biotherapeutics involves modulation (activation or inhibition) of immune system pathways. Modulation of the immune system by cytokine biotherapeutics can result in multiple effects, which can be desirable or undesirable depending on the disease being treated. Thus, it is important to characterize the potential adverse effects of cytokine biotherapeutics in toxicology studies (in vitro and/or in vivo). Several points should be considered for the nonclinical safety assessment of cytokine biotherapeutics, such as the availability of relevant models (in vitro and animal models) and the variability of the assays. In addition, since the biology

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Table 2. Repeat-Dose Toxicology Study of Ustekinumab in Cynomolgus Monkeys.

Group 1 2 3

Dose, mg/kg

No. males/ females (terminated week 13)

No. males/ females (terminated week 26)

No. males/ females (recovery)

0 (vehicle) 22.5 45.0

3/3 3/3 3/3

3/3 3/3 3/3

2/2 2/2 2/2

of the immune system can vary across species, it is important to consider how the data generated from toxicology studies translate to the human response. Furthermore, there are certain aspects of toxicology studies that may limit their ability to fully predict the potential adverse of effects of the cytokine therapeutic in patients. Toxicology studies, for example, are generally conducted in normal, healthy animals where the expression of the cytokine target may be substantially lower in comparison to the diseased condition. In addition, many adverse effects associated with cytokine therapeutics may have a low incidence or occur several years after treatment, which cannot be captured in animal toxicology studies. Thus, there is the potential that the nonclinical safety assessment studies for a cytokine biotherapeutics may not detect certain adverse effects that can occur in patients. Case examples of clinical adverse effects that were or were not detected in general repeat-dose toxicology studies will be reviewed for certain cytokine therapeutics in this section. Adalimumab (Humira) is a recombinant IgG1 mAb specific for human TNF-a. Adalimumab inhibits biological responses regulated by TNF-a and is indicated for different conditions such as rheumatoid arthritis, juvenile idiopathic arthritis, plaque psoriasis, and ulcerative colitis, ankylosing spondylitis, and adult and pediatric Crohn disease. A major clinical adverse event of adalimumab is the reactivation of latent tuberculosis (TB) or hepatitis B (which is considered to be a class effect of TNF-a inhibitors such as Enbrel and Remicade). The toxicology studies for adalimumab were conducted in cynomolgus monkeys as the NHP was the only pharmacologically relevant animal species for the nonclinical safety assessment program. In the chronic repeat-dose study, adalimumab was administered once weekly by IV injection at doses of 0 (vehicle), 32, 82.9, or 214.8 mg/kg (3 animals/sex/group) for 39 weeks followed by a 20-week recovery period.5 The study design is presented in Table 2. The primary treatment-related findings observed in this study were considered to be due to the pharmacological activity of adalimumab and consisted of deceased thymus weights and decreased cellularity in splenic follicular centers. No evidence of infection, however, was detected in this study.5 As mentioned previously, toxicology studies are typically performed in normal healthy animals, and the reactivation of latent infections, such as TB or hepatitis B, was not observed in this monkey study. However, in a host-resistance study, the potential of adalimumab administration to increase the susceptibility

to TB infection was evaluated in cynomolgus monkey infected with Mycobacterium tuberculosis.6 In monkeys with active TB infection, persistent growth of M tuberculosis (in the bronchoalveolar lavage fluid or gastric aspirate) and higher bacterial burden were observed in monkeys administered adalimumab in comparison to controls. In monkeys that had latent TB infection, administration of adalimumab resulted in reactivation of the latent infection (in comparison to control) as evidenced by increased bacterial burden, miliary disease throughout the lungs, liver, and spleen, and lung granulomas. Thus, the data from this study indicate that the reactivation of latent TB observed in adalimumab patients can in fact be detected in a nonclinical test when an appropriate animal model that mimics the patient population (eg, monkeys with latent TB infection) is used. Ustekinumab (Stelara) is an IgG1 mAb that specifically binds to the p40 subunit of IL-12 and IL-23. Ustekinumab disrupts IL-12- and IL-23-mediated signaling by blocking the interaction of IL-12 and IL-23 with a shared cell-surface receptor chain, IL-12Rb1.7 Ustekinumab was approved by the Food and Drug Administration (FDA) in 2009 and is indicated for the treatment of psoriasis and psoriatic arthritis. Major adverse events associated with ustekinumab observed in patients include serious infections and the reactivation of latent infections, such as TB. The general, repeat-dose toxicology studies were conducted in cynomolgus monkeys where ustekinumab was administered twice weekly at doses update to 45 mg/kg by SC injection for 26 weeks in duration.7 In this study, 2 groups of main study animals were included to evaluate the potential effects of ustekinumab with subchronic exposure (13 weeks) and chronic exposure (26 weeks). The study design is presented in Table 2. The treatment-related findings observed in this study were limited to changes observed in a single monkey in the highdose (45.0 mg/kg) group. This animal began to show clinical signs of low food consumption, weight loss, and diarrhea around week 22. Increased neutrophil counts and hyperplasia of myeloid cells in the bone marrow were also noted in this animal, and histologically, bacteria associated with severe inflammation were observed in the ileum, indicating the animal had bacterial enteritis. Although the bacterial enteritis was observed only in a single ustekinumab-treated animal, this finding did indicate the potential risk of infection associated with the disruption of IL-12- and IL-23-mediated signaling by ustekinumab. In summary, cytokines are a unique class of regulatory proteins that play an important role in maintaining and regulating immune system functions, and an imbalance in cytokines and/ or cytokine receptors can lead to different pathological disorders. Over the years, cytokines have been recognized to serve as excellent therapeutic targets for the treatment of various disease conditions. As with any therapeutic medicine, adverse events can occur with cytokine biotherapeutics, and due to the complex biologic of cytokines, several factors need to be considered in the design of nonclinical safety assessment studies for cytokine biotherapeutics. In some cases, certain aspects of

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toxicology studies and animal models used can limit the ability of these studies to predict the potential risk to patients. One such example (as described previously) is evaluating the potential of reactivation of latent infections associated with adalimumab in a toxicology study conducted in healthy animals versus a study where the monkeys have latent TB infection. Thus, understanding the mechanism of action of the cytokine therapeutic (eg, inhibition or activation of a cytokine and/or cytokine receptor) is critical in predicting the potential toxicities (or lack thereof) observed in the nonclinical studies, and furthermore, understand how the nonclinical safety data translates to the patient population.

Targeting Cytokine Pathways to Treat Disease (Patricia C. Ryan. MedImmune, LLC) Biologics that target cytokine pathways are promising therapies for a variety of diseases including cancer, autoimmune and immune-mediated diseases, and inflammatory diseases. Special considerations for designing and interpreting nonclinical safety studies for biologics are required. For example, the majority of toxicities from biologics arise from on-target excessive pharmacologic effects. As a result, consideration of expected pharmacology is important to appropriately interpreting nonclinical safety results. This symposium describes the application of translational approaches for predicting safety profiles of biologics in clinical development. In each case, special considerations were required for designing and interpreting nonclinical safety studies for biologics that either induce cytokines or block a key cytokine pathway as part of their mechanisms of action. These considerations will be detailed. All animal studies adhered to high ethical standards and were conducted at AAALAC-accredited facilities with protocols approved by the Institutional Animal Care and Use Committee. The first case example, MEDI-565 (AMG 211), is a novel bispecific single-chain antibody of the bispecific T-cell engager class in development to treat gastrointestinal cancers known to overexpress carcinoembryonic antigen (CEA). The MEDI-565 transiently links CEA (also called CEACAM5, CD66e) on cancer cells as well as CD3 on T cells. The CEA is a well-characterized tumor-associated antigen with low expression on normal tissues of epithelial origin8 and is frequently overexpressed in carcinomas including colorectal, gastric, lung, breast, pancreas, and ovarian cancer.8,9 Binding of MEDI-565 to CEA on tumor cells and CD3 on T cells results in activation and proliferation of T cells and release of proinflammatory cytokines. Because of the mechanistic impact on T cells and cytokines, adoption of a broader approach to dosage justification was employed. This approach uses all available information, including determination of the minimum anticipated biologic effect level (MABEL), prior to selecting a starting dose for first-in-human (FIH) clinical studies. MEDI-565 binds to human CD3 but does not bind to CD3 of cynomolgus monkey or mouse. Consequently, no pharmacologically relevant animal species exists for testing the toxicity of

MEDI-565. In an effort to introduce a pharmacologically relevant model, 2 surrogate antibodies were produced, cyS111 and hyS111, with specificity for monkey or mouse CD3, respectively. Binding affinity and in vitro pharmacologic effects of MEDI-565 were compared with those of hyS111 and cyS111 using analogous model systems. Results revealed that nonspecific activities (T-cell activation independent of CEA binding) of both surrogates would likely misrepresent the specific activity and effects of MEDI-565 in humans, thereby limiting their utility in nonclinical toxicity studies. For this reason, a nonclinical strategy was implemented without hyS111 or cyS111 and no in vivo toxicology studies were conducted in a relevant animal model with either MEDI-565 or with the 2 surrogate antibodies. Rather, a strategy that employed an in vitro approach to assessing nonclinical safety instead of performing in vivo toxicity studies was implemented. The nonclinical safety of MEDI-565 was assessed in a cell-based system using cocultures of human peripheral blood mononuclear cell (PBMC) and CEA-positive target cells to establish dose response for activity. The MEDI-565-induced in vitro lysis of tumor cells was determined to be the most sensitive measure of MABEL. A nonterminal PK study was performed in cynomolgus monkeys, and human PK parameters of MEDI-565 were predicted using allometric scaling based on the monkey PK data. The human dose that would result in serum concentrations around the identified MABEL concentration was determined based on simulated human PK profiles. Results from these studies were used to select an appropriate starting dose for the FIH study of MEDI-565 for the treatment of patients with cancers expressing CEA. Mavrilimumab (CAM-3001) is fully human IgG4 mAb targeting GM-CSF-a receptor. MedImmune is currently being developed for the treatment of rheumatoid arthritis. The GM-CSF plays a central role in the pathogenesis of rheumatoid arthritis through the activation, differentiation, and survival of macrophages and neutrophils. To support clinical development, we evaluated the nonclinical safety of mavrilimumab in several studies in cynomolgus monkeys, a pharmacologically relevant species. The dosage- and time-related accumulation of foamy macrophages in lung following exposure to mavrilimumab observed in several NHP studies was expected based on the known role of GM-CSFR-a signaling in the function of alveolar macrophages.10-12 Adverse chronic changes observed in the lungs of some animals exposed to high dosages of mavrilimumab were characterized as an inflammatory reaction (granulomatous) to foreign material and may reflect impaired ability of macrophages to clear foreign material or endogenous cell debris. These exaggerated pharmacologic effects in lungs from prolonged GM-CSF blockade shares some similarities to lung lesions observed in human pulmonary alveolar proteinosis and GM-CSF knockout mice. Mavrilimumab is immunogenic in cynomolgus monkeys, and the development of ADA is associated with substantially reduced exposures. To formally demonstrate the no-effectlevel (NOEL) dosage of mavrilimumab in a long-term setting, a low SC 26-week repeat-dose study was performed in

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cynomolgus monkeys. As expected, the immunogenicity of mavrilimumab led to a high rate of ADAs with insufficient toxicokinetic exposures in a majority of animals. Nevertheless, exposure was maintained in 4 animals at 10 mg/wk (approximately 4 mg/kg/wk), which represented NOEL. The human exposures in phase IIb studies were below the exposure concentrations anticipated to induce foamy macrophages in human lungs and therefore, are anticipated to confirm the adequacy of the safety margin to support the continued clinical development of mavrilimumab. Finally, anifrolumab (MEDI-546) is a fully human mAb directed against the type I IFN receptor. Anifrolumab is under development to treat systemic lupus erythematosus. Because the IFN-a pathway is not upregulated in healthy NHP, nonclinical safety studies in cynomolgus monkeys used a unique approach to assess PD by measuring receptor occupancy (RO). The PD biomarkers can measure target engagement and downstream target modulation. Using these nonclinical data, we developed a translational PK-PD model to facilitate early clinical study designs and dosage finding. Whole blood samples were collected, a various time points postadministration of anifrolumab in cynomolgus monkeys, and RO was measured by flow cytometry. Simultaneous modeling of PK exposure and RO using a target-mediated drug disposition model was used to select a human starting dosage and a dosage-increase scheme based on target engagement. In each of these case examples, consideration of the expected pharmacology and employing translational approaches were important to appropriately interpreting nonclinical safety results and support clinical development activities.

Beyond TeGenero: Development of Targeted Therapeutics Without Deadly Consequences (Theodora W. Salcedo, Bristol Myers Squibb Company) What is cytokine release syndrome and can we reliably predict it? Cytokine release syndrome (CRS) results from sustained activation of large numbers of immune cells, which release inflammatory cytokines in an unregulated manner. Immune cells that may be affected include lymphocytes (B cells, T cells, and natural killer cells) as well as myeloid cells (macrophages, dendritic cells, and monocytes), and cytokine release from these cell types may be enhanced by multiple cellular interactions (ie, FcgR; endothelial cells). Cytokine release syndrome has been associated with therapeutic mAb infusions, including monospecific as well as bispecific-targeted therapies. A very unfortunate incident of severe CRS occurred in 2006 in a phase 1 clinical trial following administration of the novel humanized CD28 superagonist (CD28-SA) mAb, TNG 1412. In that trial, TGN 1412 was administered as a single IV dose to 6 healthy volunteers. Unexpectedly within 90 minutes following administration, TGN 1412 induced a systemic inflammatory response in all 6 patients characterized by headache, myalgias, nausea, diarrhea, erythema, vasodilatation, and

hypotension.13 The condition of the 6 patients continued to deteriorate and within 12 to 16 hours of the initial infusion, all were critically ill and required intensive care including cardiopulmonary support, dialysis, high-dose methylprednisolone, and anti-IL-2 receptor antagonist antibody.13 Following the TGN 1412 incident, a large number of investigations were conducted to determine what went wrong in the trial and subsequently, regulatory recommendations were developed that focused on improving the safety of FIH clinical trials. Most notably, in 2007 the Committee for Medicinal Products for Human Use at the European Medicines Agency (EMEA) issued ‘‘Guidelines on strategies to identify and mitigate risks for first-in-human clinical trials with investigational medicinal products.’’14 In particular, this guideline recommends the use of an MABEL approach for estimation of the FIH starting dose for high-risk medicinal products.14 Another result of the CD28-SA (TGN1412) ‘‘cytokine storm’’ incident was that a number of groups began optimizing methods for in vitro CRAs that might predict the potential in vivo clinical toxicity of therapeutic mAbs, including TGN 1412.15-17 In 2009, the EMEA sponsored a 1-day Workshop with regulatory, academic, and industry scientists to discuss the state of the art in CRA.18 More recently in 2014, a survey and postsurvey follow-up of current CRA practices used by pharmaceutical companies, independent testing facilities, and academic institutions was conducted by the ILSI-Health and Environmental Sciences Institute Immunotoxicology Committee Cytokine Release Assays Working Group.19 From these efforts, it was recommended that CRAs be considered hazard identification tools (rather than an accurate and reliable risk quantification tool) for screening novel biotherapeutics directed against targets having a potential risk for eliciting adverse proinflammatory clinical infusion reactions.18,19 However, there is no standard approach or strategy for assay formats or reporting and interpretation of the data from CRAs. Instead given the varying therapeutic mechanisms of actions for novel biotherapeutics, different approaches should be considered that account for the pharmacotoxicological profile of the therapeutic.18,19 In this regard, novel approaches including engineered mouse models are being used to supplement in vitro CRA assessments for certain therapies including CD28.20-22 Progress is also being made in defining risk mitigation options that can be used clinically. In the event of a positive CRA, potential risks may be managed by adequate risk mitigation strategies rather than discontinuation of the product development.18 In some cases, CRS may be predictable based on the mechanism of action of a therapeutic and/or previous clinical experience with other therapeutics. For instance, T-cell-engaging therapies including bispecific T-cell-engaging antibodies such as blinatumomab and CAR-modified T-cell therapies used in the oncology setting can induce high levels of T-cell activation. The T-cell activation and CRS response to therapy often correlates with both toxicity and efficacy.23,24 Novel strategies are being explored to define new grading systems of CRS severity in individual patients with the intent of developing treatment algorithms to maximize toxicity control without compromising efficacy.23,24 Such approaches

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may include direct cytokine-targeted approaches using inhibitors of IL-6, IL-2R, IL-1R, TNF-a, and/or potentially other cytokine antagonists.23,24

What are we learning from studies with CD28 superagonists? An area of intense research associated with CRS is focused on increasing our understanding of the mechanisms of action of TGN 1412/CD28-SAs. This research is providing valuable insights into the pathophysiology of CRS that may be applicable to other therapeutic targets. In particular, there have been a number of learnings related to (1) Why studies with cynomolgus monkeys failed to predict the severe cytokine storm that occurred in humans? (2) Why are human T cells differentially stimulated by TGN 1412 in vitro? (3) What is the potential role of cell–cell interactions between T cells and other cell types in CD28-SA cytokine release? (4) Is there a role for FcgRs in mediating cross-linking the Fc domain of TGN 1412? (5) Why did normal physiologic negative feedback mechanisms fail? One of the most important questions was why toxicity studies of TGN 1412 in monkeys did not show the severe adverse effects observed in humans. This is of particular interest because the extracellular domain of CD28 is entirely conserved between humans and macaques, and TGN 1412 binds CD28 from cynomolgus monkeys.15 However, in contrast to humans, CD28 is not expressed on CD4þ effector memory T cells of several NHP species used for preclinical safety testing, including cynomolgus and Rhesus monkeys.22,25-28 Consequently, this species difference in CD28 expression on the CD4þ effector memory Tcell subset provided an explanation for the differential effects of TGN 1412 in humans relative to cynomolgus monkeys.28 Interestingly, more recent evaluations of other NHP species have revealed that in contrast to macaques, but like humans, the majority of baboon CD4þ T lymphocytes express CD28 in their effector memory cell compartment.22 In both in vitro and trans vivo models, baboon PBMC are activated, proliferate, and secret cytokines in response to agonist or superagonist anti-CD28 mAb stimulation.22 These findings highlight the importance of evaluating not only binding but also potential expression and functional differences in nonclinical species. Another question was why do fresh human T cells fail to respond to soluble TGN 1412 in vitro. Stebbings et al demonstrated that incubating human whole blood or PBMCs with soluble TGN 1412 failed to elicit a strong cytokine release in vitro unless immobilized on plastic or cross-linked.15 Ro¨mer et al propose that CD28þ T cells in peripheral blood are at low density and CD28 is not competent to transmit signals.29 In contrast, in secondary lymphoid organs, CD28þ T cells are at higher density and competent. Competency may be due to cell–cell interactions and/or MHC class I/II signals. Further, it is suggested that human CD4þ effector memory cells (the main responding cell to TGN 1412) reside mainly in peripheral tissues such as the lungs and gastrointestinal mucosa.28 Thus a protocol has been described and designated RE-Setting T cells to Original Reactivity (RESTORE),

designed to mimic more closely tissue-like conditions for use in CRAs. In this model, preculturing PBMCs at high density resulted in increased sensitivity of T cells to cytokine release stimulated by TGN 1412.29,30 Further, using T cells presensitized via the RESTORE protocol, recent studies have demonstrated that FcgR-mediated cross-linking of TGN 1412 (in particular by CD32-expressing B cells) may enhance T-cell responses relative to those obtained using TGN 1412 alone.31 A further role for other cell types and/or FcgR-independent mechanisms is suggested by findings that ICOS–LICOS interactions between T cells and cytokine-activated HUVECs are sufficient to induce proliferation and cytokine expression of TGN 1412-stimulated T cells.32 The ICOS is an important costimulatory molecule that is upregulated after T-cell activation and can enhance T-cell proliferation and cytokine secretion.32 A positive feedback mechanism was described showing that while cytokine stimulation enhances LICOS expression on HUVECs, the ICOS–LICOS interaction upregulates ICOS expression on TGN 1412-treated T cells. Thus, as suggested by Weissmu¨ller et al, TGN 1412-mediated T-cell activation may be a multifactorial event involving FcgR-dependent and FcgR-independent mechanisms.32 Given that TGN 1412 is an IgG4 antibody with weak interactions predicted for FcgRs, the physiologic role of TGN 1412 Fc/FcgR interactions is not completely understood and continues to be investigated. Overall, these findings highlight the importance of modeling tissue-like compartments in in vitro systems and may suggest a potentially more complex mode of action for TGN 1412. Recently the role of feedback mechanisms involved in controlling physiologic T-cell activation was also explored in CD28-SA-stimulated T cells. In this regard, Thaventhiran et al33 investigated whether the uncontrolled activation of CD28SA-stimulated T cells was due to both enhanced expression of activation molecules and the lack of or reduced expression of inhibitory signals.33 They found that while anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation, and IL-2 secretion that is accompanied by PD-1 expression, CD28-SA-activated T cells demonstrate uncontrolled activation. This was characterized by enhanced expression of LFA-1 and CCR5 and a failure to express PD-1 on the cell surface. These data suggest a molecular explanation for the dysregulated activation of CD28-SAstimulated T cells. The value of evaluating the functionality of the PD-1 pathway as a potential safety biomarker while suggested requires further investigation and validation.33

CD28 Therapeutics in Development The learnings from the TGN 1412 story over the past decade are being applied toward the development of safer biotherapeutics including those targeting CD28. Currently both CD28 superagonists and antagonists are in development. It is clear from the TGN 1412 incident that superagonist therapeutics targeting CD28 require appropriate dose selection and risk mitigation procedures to prevent severe cytokine storm. For

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CD28 antagonists, therapeutic blockade of CD28 requires use of monovalent therapeutics to prevent T-cell activation driven by CD28 cross-linking, as well as appropriate risk mitigation strategies.34 TGN 1412 was initially developed based on data from preclinical rodent models demonstrating that CD28-SA treatment is efficacious in various autoimmune diseases such as adjuvantinduced arthritis, diabetes, and experimental autoimmune encephalomyelitis. Recently, TGN 1412, now called TAB08 (theralizumab), was evaluated in vitro and in vivo to determine whether CD28-SA titration can identify a dose range where proinflammatory cytokine secretion from conventional T cells is absent, while T regulatory (T-reg) cell activation is maintained, even under effective blockade of proinflammatory cytokine release by corticosteroid treatment.35,36 In vitro, TAB08 potently expanded T-reg cells from healthy human donors and patients with rheumatoid arthritis. Further, slow infusion of low doses of TAB08 (1000-fold lower starting dose [0.1 mg/kg] than during the FIH study in 2006 [0.1 mg/kg]) in healthy humans was associated with the T-reg-cell cytokine IL-10, in the absence of other proinflammatory cytokines or adverse clinical events.35 The authors suggest these data support a potential role for TAB08 in mobilizing T-reg cells for treatment of autoimmune and inflammatory conditions.35 Additional studies are needed to further characterize the clinical safety profile of low-dose TAB08 and the potential for aberrant cytokine release from conventional T cells. CD28 antagonists are also being evaluated as therapies for autoimmune and inflammatory conditions based on their ability to suppress conventional T-cell activation. Relative to therapeutic approaches using CTLA 4Ig (ie, Orencia, Nulojix), it is proposed that a specific and selective CD28 antagonist may be more efficacious in certain disease contexts.21,37-39 Monovalent antihuman CD28 domain antibodies (dAbs) have been generated using phage display and affinity maturation and conjugated to polyethylene glycol (PEG).37 These dAbs are potent inhibitors of T-cell proliferation and cytokine production, while not interfering with T-reg function.37 A main concern with CD28 antagonists is to ensure they are devoid of any agonist activity. To evaluate the anti-CD28 dAbs for potential agonist activity, a comprehensive panel of in vitro assessments was conducted including assays to evaluate the potential of the anti-CD28 dAbs to provide a costimulatory signal to T cells when combined with anti-CD3, and assays to evaluate the potential for anti-CD28 dAb antibodies to cross-link the dAb and subsequently lead to T-cell activation. These later studies were conducted using either an anti-Vk or anti-PEG Ab to capture the anti-CD28 dAb in the presence or absence of anti-CD3. In all of these studies, the anti-CD28 dAbs were completely devoid of agonist or coagonist activity.37 Another anti-CD28 antagonist antibody in development is called FR104, which is a monovalent humanized Fab’ antibody fragment PEGylated to prolong its half-life.21,38 The immunological safety profile of this molecule was investigated in vitro and trans vivo using human T cells in NOD/SCID mice adoptively transferred with human PBMC. In addition, a novel

approach was used to evaluate FR104 in an NHP, specifically the baboon (Papio anubisis). While PBMCs from macaques do not express CD28 on their effector memory T cells, Poirier et al found that the majority of baboon CD4þ memory T cells express CD28, similar to humans.22 Using in vitro and trans vivo (baboon T cells in NOD/SCID mouse model) models, baboon PBMC were shown to be activated by CD28-SA, but not FR104. Furthermore, there was no evidence of FR104-related cytokine release syndrome (based on hematology, body temperature, blood pressure, heart rate, and oxygen saturation assessments) following FR104 IV administration to baboons at doses resulting in 100% RO that was maintained over 2 months.22 The FR104 was also evaluated in vitro to evaluate the potential for cross-linking of the monovalent anti-CD28 Ab by ADA. For this purpose, IgG was purified from sera obtained from ADApositive baboons that were previously exposed to FR104 or naive animals. When included in the in vitro assays, FR104 had no agonistic activity in the presence of purified IgG ADAþ even in the presence of activation with anti-CD3.21,22 Since the TGN 1412 incident, significant progress has been made in increasing our understanding of CRS and the mechanisms of action of TGN 1412/CD28-SA. This research is providing valuable insights into the pathophysiology of CRS and is applicable to both current anti-CD28 therapies and other highrisk-targeted therapeutics. Experimental approaches to optimize and tailor in vitro CRAs to address questions related to specific therapeutic mechanisms of action and the use of novel in vivo approaches are being pursued. Although much progress has been achieved, future work is needed to reach the overall goal of maximizing therapeutic benefit of novel immunotherapeutics while preventing and miniziming toxicities associated with CRS.

Cytokines as Biomarkers for Immunotoxicity (Gregory Bannish, Huntingdon Life Sciences) In addition to targeting cytokines for use as therapeutic agents to block or enhance immune responses, cytokine expression has also been evaluated to assess the immune response in order to determine potential drug toxicities.40 Cytokines are most well known for their role in the inflammatory response, including the proinflammatory cytokines IL-1, IL-6, and TNF-a. These cytokines are expressed early and sequentially and serve to amplify inflammatory event. Other cytokines, such as IL-10 and TGF-b, help to downregulate the response repair injured tissues. There remains a need for improved biomarkers of toxicity. Some disease conditions, such as interstitial pneumonitis, idiosyncratic liver injury, and testicular toxicity, lack useful blood biomarkers. When biomarkers for toxicity are present, that can allow drugs to fail faster, and lead to better candidate selection. The Critical Path (FDA) acknowledges gaps in toxicity assessment and encourages biomarker discovery (ie, renal toxicity biomarker panel). In addition, improved blood biomarkers of toxicity could reduce animal usage compared with biomarkers available only at term.

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Any biomarker that is useful for evaluation of toxicity should have many of the following attributes listed subsequently: 1. 2. 3. 4. 5. 6.

Readily accessible in body fluids or tissues. Stable enough to measure. Sensitive, dose-dependent response to injury. Specificity to correlate biomarker level with toxicity. Low interanimal variability. Recovery of biomarker to baseline levels following removal of toxic agent. 7. Premonitory to allow prediction of toxicity. 8. Species conservation to correlate toxicity in animal species to that in humans. Cytokine measurements must be performed carefully in order to generate meaningful data. The choice of analytical platform is one such consideration, and whether one or multiple cytokines are to be analyzed. Multiplex analysis of several cytokines from 1 sample can help to detect a trend (ie, inflammatory response) but can be difficult to optimize buffers and concentrations. It is important to validate the cytokine of interest to include stability, precision, and accuracy parameters in conjunction with a fit-for-purpose approach.41 In addition, usage of a purified, species-specific cytokine is important to verify the kit’s performance. If not available, ex vivo stimulation of PBMCs may generate endogenous cytokine expression that can be used as evidence for species specificity. High background may arise and be due to high endogenous levels of the cytokine being measured or to other cytokines that could cross-react with the assay. Because platelets contain many cytokines such as IL-1 and TGF-b, care must be taken in generation of sera and/or plasma for sample analysis. Freeze–thaw cycles in samples containing platelets may lead to increased levels of cytokines due to release upon their lysis. Cytokines often pose challenges for use as safety biomarkers. Many have a short serum half-life that requires careful timing for sample collection and careful sample handling during analysis. Many have low to undetectable levels in naive animals, which can create challenges for the design of speciesspecific assays. Cytokines are used for multiple purposes and may not reflect drug toxicity within the animal or may not be expressed systemically. For example, cytokines participate in maintenance of organ structure and function by tissue resident macrophages and in restoration of homeostasis, and the contribution of these processes to systemic cytokine levels is unclear.42 IL-13 is a Th2 cytokine that has been shown to be an important mediator of airway inflammation contributing to asthma lesions. IL-13 was evaluated in sera from human asthmatic patients by using a very sensitive assay on a Singulex platform that can detect 0.07 pg/mL and was determined to exhibit a 10-fold variability in expression and no change in expression between naive patients, asymptomatic, or symptomatic

Table 3. Cytokine Biomarkers of Toxicity. Disease/ condition

Cytokine

Matrix Notes

Th1

IFN-g, IL-2, and IL-12 Th2 IL-4, IL-5, IL-6, IL-10, and IL-13 Sera Cytokine storm IL-2, IL-6, IL-8, IL-10, IFN-g, TNF-a, and TGF-b Multiple IL-17a Sera sclerosis Coronary heart EN-RAGE disease Prostate cancer IFN-g, IL-6, IL-1, and IL-2 Angiogenesis

IL-6, IL-10, and IL-13

Sera

Biomarker of IFN-b treatment response45 Risk factor for CHD46

Sera

Biomarkers for radiation therapyinduced toxicity47 Plasma Toxicity to aflibercept for treatment in glioblastoma48

Abbreviations: CHD, coronary heart disease; IFN, interferon; IL, interleukin; Th, T helper; TNF-a, tumor necrosis factor a.

asthmatic patients.43 Therefore, systemic evaluation of IL-13 was not useful as a biomarker for disease progression. Cytokine evaluation in preclinical safety toxicity studies need to be considered carefully to minimize nondrugmediated modulation. This includes the impact of stress (can increase IL-6), diurnal variations, and removal of large amounts of blood. One large blood removal was determined to result in the induction of IL-1b, IL-6, IL-10, and TNF-a mRNA in liver and lung tissue in mice.44 Study designs often contain blood draw and time points to evaluate PK but need to also consider the temporal expression of cytokine expression. Single-organ toxicity can result in more subtle modulation of systemic cytokines, as systemic cytokines may not be reflective of local events at the relevant site. Therefore, care must be taken to ensure that the change in cytokine results from the drug rather than other conditions on study. Study designs should include several factors. Baseline measurement of blood cytokines should be taken. Vehicle control groups, which are age and sex matched, should be present. Care must be taken for long studies where age and ovarian cycles may affect cytokine levels. Finally, normalization should occur wherever possible, such as blood collection time points (time of day), dosing, feeding, collection sites, and anesthetics. Some toxicities can affect cytokine expression. For example, a decrease in leukocytes will result in lowered amounts of cytokine secretion. Liver injury can impair synthesis of binding proteins and affect clearance of cytokines. Similarly, renal dysfunction can affect clearance of cytokines for circulation. Finally, compromising the intestinal barrier can lead to endotoxin entry and stimulation of inflammatory cytokines. Some examples of cytokines for use as biomarkers in disease progression and/or drug treatment are included in Table 3.

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Patients with multiple sclerosis have a large heterogeneity in response to drug treatment and increased IL-17a baseline serum levels correlated with good responders to IFN-b treatment.45 In an evaluation of 16 inflammatory biomarkers for coronary heart disease (CHD), EN-RAGE showed the strongest association with risk of CHD (P value 2.0  103).46 The cytokines IFN-g, IL-6, IL-1, and IL-2 were determined to be useful predictive biomarkers for toxicity outcomes in patients with prostate cancer undergoing intensity-modulated radiotherapy.47 Finally, plasma levels of IL-6, IL-10, and IL-13 were correlated with toxicity in patients with glioblastoma.48 In summary, there currently exists a need for more biomarkers in the assessment of toxicity during drug development and treatment. Validation of biomarkers is essential to providing meaningful data. Toxicity biomarkers are most useful when they are sensitive, specific, robust, and predictive. Unfortunately, few biomarkers achieve all of these criteria. However, a large amount of knowledge has been generated in the field of cytokines, including general biology, pleiotropy, redundancy, species differences, and variability. Although few cytokines have emerged as predictive measures of toxicity, the growing information in the field is likely to identify new cytokine biomarkers in the near future. Author Contributions T. Ramani, C. S. Auletta, D. Weinstock, B. Mounho-Zamora, P. C. Ryan, T. W. Salcedo, and G. Bannish contributed to conception and design; drafted the article, critically revised the article, gave final approval, and agreed to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Cytokines: The Good, the Bad, and the Deadly.

Over the past 30 years, the world of pharmaceutical toxicology has seen an explosion in the area of cytokines. An overview of the many aspects of cyto...
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