Clinical Pharmacology Considerations for the Development of Immune Checkpoint Inhibitors

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1 Supplement Article Clinical Pharmacology Considerations for the Development of Immune Checkpoint Inhibitors The Journal of Clinical Pharmacology 2017, 57(S10) S26 S42 C 2017, The American College of Clinical Pharmacology DOI: /jcph.990 Jennifer Sheng, PhD 1, Shivani Srivastava, MBBS 1, Kinjal Sanghavi, PhD 1, Zheng Lu, PhD 2, Brian J. Schmidt, PhD 1, Akintunde Bello, PhD 1, and Manish Gupta, PhD, FCP 1 Abstract Immuno-oncology works through activation of the patient s immune system against cancer, with several advantages over other treatment approaches, including cytotoxic agents and molecular-targeted therapies. The most notable feature of immuno-oncology treatments is the nature of the patient responses achieved, which can be more durable and sustained than with other modalities. Increased understanding of immune system complexity has provided a number of opportunities to advance several strategies for the development of immuno-oncology therapies. This review outlines the clinical pharmacology characteristics and development challenges for the 6 approved immunomodulatory monoclonal antibodies that target 2 immune checkpoint pathways: ipilimumab (an anti cytotoxic T-lymphocyte antigen-4 antibody) and, more recently, nivolumab and pembrolizumab (both anti programmed death-1 antibodies) and atezolizumab, avelumab, and durvalumab (all anti programmed death ligand-1 antibodies). These agents have revealed much about the clinical pharmacology features of immune checkpoint inhibitors as a class, as well as the pharmacometric approaches used to support their clinical development and regulatory approval. The development experiences with these pioneering immuno-oncology agents are likely to serve as useful guides in the discovery, progression, and approval of future drugs or combination of drugs in this class. This review includes summaries of the pharmacokinetics and exposure response of the immune checkpoint inhibitors approved to date, as well as an overview of some quantitative systems pharmacology approaches. The ability of immuno-oncology to meet its full potential will depend on overcoming development challenges, including the need for clear strategies to determine optimal dose and scheduling for monotherapy as well as combination approaches. Keywords immunopharmacology, oncology, clinical pharmacology, clinical trials, pharmacology, immunotherapy, checkpoint inhibitors It could be said that if there were a book chronicling the history and progression of cancer treatment, we are now moving on from the chapter describing the time of sole reliance on chemotherapy, the chapter outlining the advent of targeted therapy is partially completed, and a new chapter on immuno-oncology is just beginning. The past few years have seen the regulatory approval of several immuno-oncology agents (Figure 1), including 6 immune checkpoint inhibitors: ipilimumab (Yervoy; Bristol-Myers Squibb), pembrolizumab (Keytruda; Merck), nivolumab (Opdivo; Bristol-Myers Squibb), atezolizumab (Tecentriq; Genentech), avelumab (Bavencio; EMD Serono), and durvalumab (Imfinzi; AstraZeneca). 1,2 Immunotherapy in oncology is a switch from the cell-killing modality using relatively nonspecific cytotoxic methods (chemotherapy) or therapies that target cancer-specific pathways to methods that employ the patients immune system to attack cancer. The immuno-oncology approach calls on the understanding of complex signaling processes of effector and regulatory mechanisms that mediate the immune system and involves the coordination of both stimulatory (promoting immunity) and inhibitory (depressing immunity) factors. 1 The complexity of the immune system and immune response provides enormous scope for different immuno-oncology agents and strategies. 3 The immuno-oncology agents approved so far show that this treatment modality offers distinct and, in many cases, improved activity over traditional chemotherapy and targeted therapeutic methods. This approach also provides the potential for further improvement as strategies for the use of immunotherapeutic drugs are refined. The current 1 Bristol-Myers Squibb, Princeton, NJ, USA 2 Astellas, Northbrook, IL, USA Submitted for publication 22 April 2017; accepted 3 July Corresponding Author: Manish Gupta, PhD, FCP, Bristol-Myers Squibb Company, 3551 Lawrenceville Road, Princeton, NJ Manish.Gupta-LVL@bms.com Fellow of the American College of Clinical Pharmacology: Manish Gupta Correction added after publication 15 September 2017: In Table I, the time to steady state for ipilimumab was changed from 9 weeks of repeated dosing Q4W to 9 weeks of repeated dosing Q3W.

2 Sheng et al S27 Figure 1. Timeline of approved immuno-oncology treatments. AIDS, acquired immunodeficiency syndrome; ALL, acute lymphoblastic leukemia; BiTE, bi-specific T-cell engager; chl, classical Hodgkin lymphoma; CML, chronic myelogenous myeloma; IMiD, immunomodulatory derivative; non- SQ, nonsquamous; NSCLC, non small cell lung cancer; RCC, renal cell carcinoma; SCCHN, squamous cell carcinoma of the head and neck. review describes the clinical pharmacology of the 6 approved immunomodulatory monoclonal antibodies that target 2 immune checkpoint pathways: the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the programmed death-1 (PD-1) pathways. T-cell checkpoint inhibitors have shown marked success since the approval of ipilimumab in 2011, leading to the acceleration of drug development in the field of immuno-oncology (Figure 1). The currently approved checkpoint inhibitors include ipilimumab, an anti-ctla-4 inhibitor; nivolumab and pembrolizumab, which are anti-pd-1 inhibitors; and atezolizumab, avelumab, and durvalumab, which are anti programmed death ligand-1 (PD-L1) inhibitors. The mechanism of action of these agents in harnessing effector T-cell function of the immune system to achieve cancer cytotoxicity has resulted in demonstrated superiority and, in many cases, approval of these drugs for the treatment of multiple tumor types including melanoma, non small cell lung cancer (NSCLC), urothelial carcinoma, renal cell carcinoma (RCC), classic Hodgkin lymphoma, gastric carcinoma, head and neck squamous cell carcinoma, and Merkel cell carcinoma. 2,4 17 Tumors escape immune surveillance in the host body by various methods, including upregulation of suppressor immune cells, coinhibitory receptor ligand expression, and production of immunosuppressive cytokines. 18 CTLA-4 and PD-1/PD-L1 have complementary and synergistic roles in regulating T-cell activation (Figure 2). 19,20 CTLA-4 acts as a coinhibitory receptor of the CD28-B7 axis, which has a critical role in T-cell response and in cancer escape of tumor surveillance; blocking this pathway prevents induction of tolerance and increases activated T-cell number and repertoire. 18,19,21 Anti-CTLA-4 ipilimumab was the first immuno-oncology therapy to show overall survival (OS) benefit in metastatic melanoma compared with the standard of care, 10,22 and in 2011 ipilimumab initiated the rise of checkpoint inhibitors as cancer therapy (Figure 1). Anti-PD-1 biologics, such as nivolumab and pembrolizumab, block PD-1 signaling on T cells, and the PD-L1 inhibitors atezolizumab, avelumab, and durvalumab block PD-L1 on tumor cells and/or tumor-infiltrating immune cells; both methods inhibit the interaction between PD-1 and PD-L1 and/or PD-L2 and release the PD-1/PD-L1 immune pathway. Anti-PD-1 biologics enable T-cell activation and proliferation and may result in subsequent cytokine production, resulting in the restimulation of previously primed T cells for cancer recognition. 18,19,21 In addition, another CTLA-4 checkpoint inhibitor, tremelimumab, is being investigated in phase 3 trials in combination with durvalumab, although it has failed to show superiority as a single agent in randomized studies in melanoma (versus standard of care chemotherapy temozolomide or dacarbazine) and mesothelioma (versus placebo). 23,24 Clinical Pharmacology of Checkpoint Inhibitors Checkpoint inhibitors are a versatile class of immunomodulatory agents and have been demonstrated to provide clinical benefits in the treatment of several cancers. The recent increase in the number of approvals of these agents has accelerated the development of immuno-oncology therapy in general (Figure 1). Here we describe the clinical pharmacology of the 6 agents approved to date; at the time of writing, there is relatively limited information for durvalumab in the public domain. Ipilimumab was the first checkpoint inhibitor to be made commercially available. Ipilimumab received US Food and Drug Administration (FDA) approval in 2011 for the treatment of unresectable or metastatic melanoma and in 2015 as adjuvant therapy for

3 S28 The Journal of Clinical Pharmacology / Vol 57 No S Figure 2. Complementary and synergistic roles of CTLA-4 and PD-1/PD-L1. Top: CTLA-4 immunologic checkpoint is expressed on activated T cells in the lymph node. T cells are activated by antigen-presenting cells such as dendritic cells through 2 interactions. They are the interaction of MHC (presented with tumor antigen) with the T-cell receptor and the interaction of B7 signaling with CD28 on the T cell. To maintain immunologic balance, CTLA-4 downregulates the function of activated T cells by the interaction of B7 signaling with CTLA-4 on the T cell. Tumors may develop tolerance to this inhibitory signaling, thereby enhancing the survival potential of the cancer cells. The aim of anti-ctla-4 immunotherapy is to block the negative signaling and restore (or boost) the immune system s function to destroy the cancer cells. Bottom: PD-1 immunologic checkpoint is expressed on activated T cells in both the lymph node (early stage) and tumors (late stage) in the tumor microenvironment. Interactions between PD-1 and its ligands PD-L1 and PD-L2 are complex because they occur at multiple steps of an immune response. In both early and late stages, PD-1 maintains immunologic balance by downregulating the function of activated T cells. As mentioned, tumors may develop tolerance to this inhibitory signaling, thereby enhancing the survival potential of the cancer cells. As with anti-ctla-4 immunotherapy, the aim is to block the negative signaling and restore (or boost) the immune system s function to destroy the cancer cells. CTLA-4, cytotoxic T-lymphocyte antigen-4; MHC, major histocompatibility complex; PD-1, programmed death-1; PD-L1, programmed death ligand-1, PD-L2, programmed death ligand-2 (figure and legend republished with permission of John Wiley and Sons Inc. from Immune Checkpoint Inhibitors: Lee L, Gupta M, Sahasranaman S. An introduction to the next-generation cancer immunotherapy. J Clin Pharmacol. 2015:56 20 ; permission conveyed through Copyright Clearance Center, Inc. melanoma. 10,22,25 Nivolumab was first approved by the Japan Pharmaceuticals and Medical Devices Agency in July 2014 and by the FDA in December 2014 for the treatment of patients with unresectable or metastatic melanoma, initially for second-line treatment after failure of ipilimumab (and also after a BRAF inhibitor in patients positive for BRAF V600 mutation). Subsequently, nivolumab was approved by the FDA in January 2016 for first-line treatment regardless of BRAF status. 4 Nivolumab has also been approved for the treatment of advanced NSCLC, metastatic RCC, head and neck squamous cell carcinoma (HNSCC), Hodgkin lymphoma, and urothelial cancer as well as in combination with ipilimumab

4 Sheng et al S29 for the treatment of advanced melanoma. 7 9,26,27 Pembrolizumab was approved by the FDA in September 2014 as second-line treatment for metastatic melanoma and then in December 2015 as firstline treatment. 5 Pembrolizumab is also approved for use in advanced NSCLC, HNSCC, Hodgkin lymphoma, urothelial cancer, and microsatellite instabilityhigh cancer. 11,28 30 Atezolizumab, the first commercially available PD-L1 inhibitor, received accelerated approval in May 2016 for locally advanced or metastatic urothelial carcinoma after failure of platinum-containing chemotherapy and is now also approved for NSCLC. 12,13,17 In 2017, 2 further PD- L1 inhibitors received accelerated FDA approval: avelumab for the treatment of adults and pediatric patients 12 years and older with metastatic Merkel cell carcinoma 15,31 and durvalumab for locally advanced or metastatic urothelial carcinoma after failure of platinum-containing chemotherapy. 16,32 Full FDA approval for all agents initially approved under accelerated approval is contingent on further verification of initially promising data. Currently, ipilimumab, nivolumab, pembrolizumab, and atezolizumab have each been shown to prolong OS in at least 1 indication. The FDA accelerated approvals granted for other indications and other agents were based on objective response rate (ORR) and duration of response; in these cases, continued approval may be contingent on verification and description of clinical benefit in confirmatory trials. It is pertinent to note here that the pivotal phase 3 atezolizumab study (IMvigor211) in patients with previously treated advanced urothelial cancer recently failed to meet its primary end point of OS when compared with chemotherapy. 33 This unexpected result serves to highlight that, despite extensive knowledge about the immune checkpoint inhibitors, much remains to be discovered. Dose Selection Determination of first-in-human starting dose for anticancer drugs is generally based on nonclinical toxicology testing in the appropriate, most sensitive species and then is converted to the human-equivalent dose and finally the recommended safe dose. 34,35 Because of differences in target binding, distribution, and catabolism capacities between humans and animals, this approach is challenging for biologics. 36 In general, monoclonal antibodies (mabs) including immunomodulatory mabs have proved to be well tolerated; their toxicity is usually related to exaggerated pharmacology, which usually can be predicted. 37 However, the well-publicized adverse events observed with an immunomodulatory anti-cd28 superagonist mab (TGN1412) in a first-in-human clinical trial has highlighted how, on rare occasions, immunomodulatory mabs can be highly toxic when dose and schedule are not assiduously investigated. 37 An approach accepted and recommended by both the FDA and the European Medicines Agency is to consider the minimum anticipated biological effect level for the first-in-human starting dose to ensure a balance of both safety and pharmacological activity. 38 For dose escalation, instead of the maximum tolerated dose (MTD) approach, adaptive models typically provide a general framework to incorporate and make decisions for dose escalation based on nonclinical data, such as animal efficacy and toxicity data; clinical data, including pharmacokinetic (PK)/pharmacodynamic (PD) data; and dose /exposure response data for efficacy and safety. 39 Although ipilimumab was initially tested over a dose range of 0.3 to 10 mg/kg, the 3 mg/kg dose was administered in phase 3 trials to both previously treated and untreated melanoma patients. 10,40,41 In addition, ipilimumab is approved in the United States at 10 mg/kg as adjuvant therapy for melanoma. 41 In a phase 1 doseescalation study, nivolumab was tested at doses of 0.1 to 10 mg/kg every 2 weeks for up to 96 weeks. 42 Nivolumab was well tolerated up to 10 mg/kg, and no MTD was defined; response across tumor types appeared to plateau at a dose of 3 mg/kg every 2 weeks, which was accordingly chosen for further clinical development. 43 A flat dose of 240 mg every 2 weeks was later approved for nivolumab treatment in patients with RCC, NSCLC, and melanoma based on dose /exposure response and safety analysis. 44 More recently, facilitated by a good understanding of nivolumab clinical pharmacology, the availability of clinical data across multiple tumor types and well-characterized exposure response relationships for efficacy and safety (see below), a model-based exposure response analysis has predicted a comparable benefit-risk profile for nivolumab 480 mg every 4 weeks to nivolumab 3 mg/kg every 2 weeks. 45 Based on this assessment, the 480 mg every-4-week dosing schedule has been included in several ongoing nivolumab clinical trials in different tumor types. Similar to nivolumab, the MTD for pembrolizumab was not reached in phase 1 studies, with doses ranging from to 10 mg/kg. 46 The data from this study, with further translational modeling, suggested that pembrolizumab dosed in the range of 2 mg/kg every 3 weeks to 10 mg/kg every 2 weeks was safe and effective and should be explored in subsequent clinical trials, with a dose of 2 mg/kg every 3 weeks ultimately selected for the pivotal cohorts of the KEYNOTE-001 study, on which registration was based. 47 An exposure response analysis using data from the phase 1b KEYNOTE-001 and

5 S30 The Journal of Clinical Pharmacology / Vol 57 No S phase 2 KEYNOTE-002 studies confirmed the dose and schedule selected for pembrolizumab development, showing similar tumor shrinkage at doses of 2 and 10 mg/kg every 3 weeks. 48,49 In the case of atezolizumab, doses ranging from 0.01 to 20 mg/kg given every 3 weeks were tested, with no dose-limiting toxicity observed. 50 The preclinical target serum drug level was achieved at doses of 10 mg/kg or greater, which, combined with the lack of safety concerns with doses up to 20 mg/kg, led to the selection of a flat dose of 1200 mg (15 mg/kg) for further clinical development. 51 Population PK analysis showed no clinically meaningful difference in exposure after flat and weight-based dosing. 51 The avelumab dose of 10 mg/kg every 2 weeks was selected based on adequate safety and tolerability, as well as PK and target occupancy data. 52 No MTD was reached with doses up to 20 mg/kg. Mean target occupancy was 90% or higher at doses of 3 and 10 mg/kg. However, several patients treated at 3 mg/kg (but none treated at 10 mg/kg) had trough levels below 1 μg/ml, which in vitro studies suggested was the serum level required to ensure 90% target occupancy. 52,53 Durvalumab was tested in a phase 1 dose-escalation study over a dose range of 0.1 to 10 mg/kg every 2 weeks, 15 mg/kg every 3 weeks, and 20 mg/kg every 4 weeks with no dose-limiting toxicity or MTD identified. 16,54 56 PK simulations indicated that >90% of patients treated with 10 mg/kg every 2 weeks were expected to maintain durvalumab level 40 μg/ml, at which >99% target saturation was expected. 57,58 Based on PK and PD data, as well as clinical safety data, the 10 mg/kg every-2-week dosing schedule was selected for further development. Dosing Strategy (Flat Dosing Versus Body Weight Based Dosing) Immunotherapeutic mab clinical development has traditionally used body weight based dosing, as body size was believed to be a major contributor to interindividual PK variability. However, modeling and simulation studies using a range of mabs have suggested that body weight based dosing does not always offer advantages over flat (body weight independent) dosing. 59,60 Flat dosing is particularly suited to agents with a broad therapeutic index 61 and, in addition, offers practical advantages such as easier dose preparation, elimination of drug wastage, and a reduced risk of dosing error. All the currently approved checkpoint inhibitors were initially developed and approved by the FDA as body weight based dosing regimens, apart from atezolizumab, which was approved as a flat dose. 2 Following the initial body weight based approvals, the need for this type of dosing was reassessed for both pembrolizumab and nivolumab based on modeling and therapeutic window information derived from exposure response analyses, as well as demonstrated clinical safety at doses up to 10 mg/kg. 62,63 These assessments showed that 200-mg and 2 mg/kg doses provided similar exposure distributions for pembrolizumab and that the benefitrisk profile of nivolumab 240 mg every 2 weeks was comparable to 3 mg/kg every 2 weeks. Pembrolizumab was approved as a flat dose for all indications subsequent to melanoma, whereas in the case of nivolumab, modification of the initially approved 3 mg/kg every-2- week regimen to 240 mg every 2 weeks was approved by the FDA for melanoma, NSCLC, urothelial cancer, and RCC indications. A recently published population PK analysis showed overall PK exposure with durvalumab using the approved, body weight based dosing (10 mg/kg every 2 weeks) was similar to that with 2 flat-dosing regimens (1500 mg every 4 weeks and 750 mg every 2 weeks). 56 All 3 regimens were expected to maintain target trough exposure of approximately 50 μg/ml in at least 95% of patients, showing the feasibility of switching to a flat-dosing regimen. Pharmacokinetics The PK of the approved checkpoint inhibitors (mabs) is similar to that of endogenous immunoglobulin G (IgG). The typical volume of distribution of mabs is comparable to plasma volume (ie, 2 to 4 L); however, it has been shown that mabs administered by all routes do reach the peripheral tissues. 38 Drugreceptor binding affinity and association-dissociation kinetics have an important role in distribution. 64 Elimination occurs by both specific (target-mediated) and nonspecific (Fc-mediated) routes, accounting for the nonlinear and linear elimination PK, respectively. 38 Following target saturation, the linear, nonspecific route of elimination is predominant, and, accordingly, the half-life of these drugs ranges from 3 to 4 weeks. 38,65 The PK parameters of all 6 agents were typically characterized using 2-compartment models with linear elimination (the exception was durvalumab, for which both linear and nonlinear PK was characterized); a summary for each is shown in Table ,40,41,44,46,51,52,56 58,66 73 PK characteristics listed and described here are not an exhaustive characterization of each agent. Characteristics were linear over at least a part of the dose ranges tested for all 6 agents, although the PK for pembrolizumab, durvalumab, and avelumab was nonlinear at doses of <0.1, 46 <3, 32 and <10 mg/kg, 52 respectively. As expected for these mabs, all had a relatively low distribution (volumes of distribution of 4.72 to 6.9 L).

6 Sheng et al S31 Table 1. Pharmacodynamic and Pharmacokinetic Properties for Approved CTLA-4, PD-1, and PD-L1 Inhibitors a Ipilimumab Nivolumab b Pembrolizumab Atezolizumab Avelumab Durvalumab Dosing characteristics IgG type IgG1 κ IgG4 κ IgG4 κ Nonglycosylated IgG1 κ Dose and dosing schedules tested Approved dose/dosing schedule 0.3, 3, or 10 mg/kg Q3W 4 Unresectable/ metastatic melanoma: 3mg/kg Q3W for a maximum of 4 doses Melanoma adjuvant setting: 10 mg/kg Q3W 4, then 10 mg/kg Q12W for up to 3 years mg/kg as a single dose or as multiple doses Q2W or Q3W Melanoma (nivolumab monotherapy), NSCLC, RCC, and urothelial cancer: 240 mg Q2W Melanoma (combined with ipilimumab): nivolumab 1 mg/kg, then ipilimumab on the same day, Q3W 4, then nivolumab 240 mg Q2W chl and HNSCC: 3mg/kgQ2W 1 10 mg/kg Q2W and 2 10 mg/kg Q3W Melanoma, NSCLC, HNSCC, chl, and urothelial cancer: 200 mg Q3W Microsatellite instability-high cancer: 200 mg Q3W (adults), 2mg/kgQ3W (children) mg/kg and flat dose of 1200 mg Urothelial cancer and NSCLC: 1200 mg Q3W IgG1 λ IgG1 κ 1 20 mg/kg Q2W mg/kg Q2W, 15 mg/kg Q3W, and 20 mg/kg Q4W Merkel cell carcinoma: 10 mg/kg Q2W Urothelial cancer: 10 mg/kg Q2W Administration IV IV IV IV IV IV route Infusion time 90 minutes 60 minutes 30 minutes 60 minutes for the 60 minutes 60 minutes first infusion; if tolerated, 30 minutes for subsequent infusions Pharmacokinetics CL, GM (CV) 403 ml/day (38%) 197 ml/day (53.9%) 212 ml/day (46%) 200 ml/day (CV NR) 590 ml/day (CV NR), 198 ml/day (37.3%) for 10 mg/kg Q2W V ss, GM (CV) 6.0 L (CV NR) 6.8 L (27.3%) 6.1 L (21%) 6.9 L (CV NR) 4.72 L (CV NR) 5.6 L (17%) t 1/2, GM (CV) 15.4 days (34%) 25 days (77.5%) 23 days (30%) 27 days (CV NR) 6.1 days (CV NR) for 17 days (23.2%) 10 mg/kg Q2W Time to steady 9 weeks of repeated 12 weeks of repeated 19 weeks of repeated 6 9 weeks of 4 6 weeks of 16 weeks state dosing Q3W dosing Q2W dosing Q3W repeated dosing Q3W repeated dosing Q2W Accumulation 1.5-fold 3.7-fold 2.2-fold 1.91-fold (AUC) 1.25-fold 4.3-fold Dose proportionality of exposure Drug drug interactions Dose proportional over mg/kg No formal drug drug interaction studies conducted Dose proportional over mg/kg Q2W No formal drug drug interaction studies conducted Dose proportional over 2 10 mg/kg Q3W No formal drug drug interaction studies conducted Dose proportional over 1 20 mg/kg, including 1200-mg flat dose Q3W No formal drug drug interaction studies conducted Pharmacodynamics, exposure-response Mechanism of action/target Anti-CTLA-4 receptor Anti-PD-1 receptor Anti-PD-1 receptor Anti-PD-L1 ligand; blocks interaction with PD-1 and B7.1 receptors Dose proportional over mg/kg Q2W No formal drug drug interaction studies conducted Anti-PD-L1 antibody; binding blocks interaction with PD-1 and B7.1 receptors More than dose proportional at doses <3 mg/kg and dose proportional at doses 3 mg/kg No formal drug drug interaction studies conducted Anti-PD-L1 antibody; binding blocks interaction with PD-1 and B7.1 receptors (Continued)

7 S32 The Journal of Clinical Pharmacology / Vol 57 No S Table 1. Continued Ipilimumab Nivolumab b Pembrolizumab Atezolizumab Avelumab Durvalumab Receptor binding and target trough concentration CTLA-4 binding to its CD80 and CD86 ligands maximally inhibited by 6 20 μg/ml and 1 3 μg/ml of ipilimumab, respectively 66 In a model analysis, ipilimumab levels (C min1 ) exceeded 3 μg/ml by 99% and 100% of patients at 3 and 10 mg/kg doses, respectively 66 In vitro, 0.04 μg/ml sufficient to occupy approximately 70% of PD-1 molecules on T cells; clinical data suggest plateau PD-1 occupancy maintained at serum levels < 1.2 μg/ml (lower limit of detection) 67 Phase 1: mean receptor peak and plateau PD-1 receptor occupancy 85% and 72%, respectively, over dose range mg/kg 67 Maximum serum target engagement reached with trough levels of doses 1 mg/kg Q3W 46 Steady-state dose of 2mg/kgQ3W needed to reach 95% target engagement 72 6 μg/ml, based on nonclinical tissue distribution data in tumor-bearing mice and receptor occupancy in the tumor In pivotal phase 2 study (dose 1200 mg Q3W) steady-state trough serum concentrations exceeded 6 μg/ml in all patients Receptor occupancy >90% at doses of 3 and 10 mg/kg 52 >99% target saturation (soluble and membrane bound) expected at 40 μg/ml 57,58 PK simulations show that >90% of patients expected to maintain PK exposure 40 μg/ml throughout the dosing interval at a dose of 10 mg/kg Q2W 57,58 Exposure efficacy response Exploratory study of pooled data from 498 patients: C min was a significant independent predictor of OS by Cox proportional hazard model Increasing dose from 3to10mg/kg might be associated with increased OS Flat exposure ORR relationship over thedoserange mg/kg in patients with melanoma Flat exposure response relationship for ORR across AUC range with 2 and 10 mg/kg doses No significant exposure efficacy relationship for ORR at 1200 mg Q3W Exposure efficacy analyses in 88 patients identified an apparent relationship between C troughss and BOR, where higher exposure was associated with a higher rate of response No significant exposure efficacy relationship for ORR or best percentage change in tumor size at 10 mg/kg Q2W 73 Exposure safety response Renal impairment (no formal study conducted) Grade 2 and grade No relationship 3 iraes increased between exposure with increasing (Cavg,ss) and time ipilimumab to first grade 3 exposure. At TRAE, AEs leading median C min for 3 to discontinuation, and 10 mg/kg and all grade 3 dosing, a AEsatadoseof 3mg/kgQ2W model predicted grades 2 4 iraes to increase with dose recommendation was made through studies in patients with mild or moderate renal impairment recommendation wasmadethrough studies in patients with mild, moderate, or severe renal impairment Flat safety response relationship (as assessed by AEs grade 3 or serious AEs and by AEs of special interest) across AUC range with doses of 1 10 mg/kg recommendation wasmadethrough studies in patients with mild, moderate, or severe renal impairment No significant exposure safety relationships at 1200 mg Q3W recommendation was made through studies in patients with mild or moderate renal impairment A weak association between increased exposure and increasing irae incidence, but no relationship between exposure and the risk of TEAEs or infusion-related reactions recommendation wasmadethrough studies in patients with mild, moderate, or severe renal impairment No relationship between exposure and either grade 3 AEsorAEs leading to discontinuation 73 No dose reductions are recommended. No clinically significant effect of mild or moderate renal impairment on durvalumab PK (Continued)

8 Sheng et al S33 Table 1. Continued Ipilimumab Nivolumab b Pembrolizumab Atezolizumab Avelumab Durvalumab Hepatic impairment (no formal study conducted) QTc study is recommended for patients with mild hepatic function based on Study in patients with melanoma (n = 82); no large QT prolongation attributed to ipilimumab 3 or 10 mg/kg. No concentrationdependent prolongation in QT by exposure response analysis is recommended for patients with mild hepatic function based on In a QT substudy, no dose response in QT parameters, and no significant elevation in QT profile is recommended for patients with mild hepatic function based on In a QT substudy, no large change (>20 milliseconds) in QTc interval with doses up to 10 mg/kg Q3W is recommended for patients with mild hepatic function based on In a QT substudy, no significant elevation in QT profile is recommended for patients with mild or moderate hepatic function based on No clinically meaningful effect on cardiac repolarization in exposure QTc analysis No dose reductions are recommended. No clinically significant effect of mild hepatic impairment on durvalumab PK Durvalumab is unlikely to prolong the QT/QTc interval AE, adverse event; AUC, area under the curve; BOR, best objective response; C min, minimum drug concentration; chl, classic Hodgkin lymphoma; CL, clearance; CTLA-4, cytotoxic T-lymphocyte antigen-4; CV, coefficient of variation; GM, geometric mean; HNSCC, head and neck squamous cell carcinoma; IgG, immunoglobulin G; irae, immune-related adverse event; IV, intravenous; NR, not reported; NSCLC, non small cell lung cancer; ORR, objective response rate; OS, overall survival; PD-1, programmed death-1; PD-L1, programmed death ligand-1; PK, pharmacokinetics; Q2/3/4/12W, every 2/3/4/12 (as applicable) weeks; RCC, renal cell carcinoma; t 1/2, terminal half-life; TEAE, treatment-emergent adverse event; TRAE, treatment-related adverse event; V ss, volume at steady state. a Table compiled using applicable prescribing information and FDA Clinical Pharmacology and Biopharmaceutics reviews unless otherwise specified. b Data refer to nivolumab given as monotherapy unless otherwise specified. Without exception, clearance of each of the approved PD-1 and PD-L1 immune checkpoint inhibitors has been shown to decrease over time, with a mean maximal reduction (coefficient of variation [CV]) from baseline ranging from 17.1% (40.6%) for atezolizumab to 41.7% (40.0%) for avelumab ,44,70 Although these changes in clearance were not considered clinically relevant (as per comments in each package insert), recent PK modeling analyses in several cases prompted by the regulatory authorities taking into account the time-varying nature of this parameter have shown that clearance changes appear to be associated with response to treatment. 51,56,74,75 Patients who benefited from treatment with nivolumab, pembrolizumab, or atezolizumab with respect to tumor response and improved OS had lower clearance; tumor shrinkage in patients treated with durvalumab was also associated with decreased clearance. One explanation for this observation is that, as disease state improves, a reduction in cachexia would result in lowered catabolism and thus reduced antibody clearance. 74,75 The mabs commonly display modest interpatient variability in PK, and is used to identify intrinsic and extrinsic covariates that can explain part of the observed variability. 60 The covariates chosen for investigation focused primarily on intrinsic factors such as sex and age, as well as those relating to disease state (for example, tumor type, tumor burden, and baseline parameters such as performance status, lactate dehydrogenase [LDH], and albumin), which might rationally be expected to differ between patients and so contribute to interpatient PK variability. Statistically significant covariates identified through analysis for approved immune checkpoint inhibitors are shown in Table 2. 66,76,77 Covariates identified as statistically significant varied between the approved agents, but in at least half of cases for clearance included sex, Eastern Cooperative Oncology Group (ECOG) performance status, body weight, tumor type, tumor burden, baseline LDH, estimated glomerular filtration rate, albumin, and immunogenicity and for central volume of distribution included sex and body weight. Renal and Hepatic Impairment FDA guidance recommends conducting a dedicated study to assess the effect of renal impairment on the PK of drugs with molecular weight <69 kda. Because most mabs are in the range of 100 to 1000 kda, renal impairment has less potential to influence the PK of these large molecules. 65 Generally, the effect of renal impairment is assessed using a approach.

9 S34 The Journal of Clinical Pharmacology / Vol 57 No S Table 2. Covariate Statistical Significance of Pharmacokinetic Parameters for Approved CTLA-4, PD-1, and PD-L1 Inhibitors a PK Parameter Covariate b Ipilimumab c Nivolumab c Pembrolizumab c Atezolizumab Avelumab Durvalumab Clearance VC VP Sex Age Race ECOG PS d Body weight d Tumor type Tumor burden d LDH d Mild hepatic impairment egfr d Creatinine clearance Albumin d Prior ipilimumab therapy Immunogenicity Soluble PD-L1 PD-L1 expression Sex Age Race ECOG PS d Body weight d Tumor type LDH d Creatinine clearance Albumin d Prior ipilimumab therapy Immunogenicity Soluble PD-L1 Sex Body weight d Immunogenicity CTLA-4, cytotoxic T-lymphocyte antigen-4; ECOG PS, Eastern Cooperative Oncology Group performance status; egfr, estimated glomerular filtration rate; LDH, lactate dehydrogenase; PD-1, programmed death-1; PD-L1, programmed death ligand-1; PK, pharmacokinetics; VC, volume of distribution, central compartment; VP, volume of distribution, peripheral compartment. a Table compiled using applicable prescribing information and FDA Clinical Pharmacology and Biopharmaceutics reviews unless otherwise specified. b Only covariates found to be statistically significant for at least 1 immune checkpoint inhibitor per PK parameter are included in the table. c Data for ipilimumab, nivolumab, and pembrolizumab also based on Feng et al (2014), 66 Bajaj et al (2017), 76 and Ahamadi et al (2017), respectively. 77 d At baseline. As described above, renal function was a statistically significant covariate for clearance for several of the approved checkpoint inhibitors; however, the magnitude of impact on PK was <20% to 30%. On this basis, using analyses, no clinically meaningful impact on PK of nivolumab, pembrolizumab, or avelumab was observed in patients with mild, moderate, or severe renal impairment compared with healthy patients, and no dose adjustment for these agents is recommended in patients with renal impairment. 30,31,44,71 Similarly, no ipilimumab dose adjustment is needed for patients with renal impairment on the basis of analyses, which failed to find any clinically important differences in ipilimumab clearance between patients with renal impairment and those with normal renal function. 41 Mild and moderate renal impairment had no effect on either atezolizumab or durvalumab PK, but the effect of severe renal impairment is unknown; nonetheless, no dose adjustment is recommended in patients with renal impairment treated with either agent. 32,56,70 The effect of hepatic impairment on the PK of small-molecule drugs is assessed if hepatic metabolism contributes to >20% of metabolism. Because mabs typically undergo proteolytic degradation and liver CYP enzymes have no role in their metabolism, no dedicated hepatic impairment studies are usually performed. However, the liver has an important role in protein catabolism and so could indirectly influence the PK of mabs. Population PK modeling is often used to compare the PK parameters and exposure in patients with normal and compromised hepatic function, using markers such as aspartate aminotransferase, alanine aminotransferase, albumin, and bilirubin as covariates. However, results from a single marker are unreliable and thus a composite score (such as the Child-Pugh

10 Sheng et al S35 score) is often chosen to estimate the effect of hepatic impairment on the disposition of mabs. 78 Mild hepatic dysfunction did not significantly influence the PK of nivolumab, pembrolizumab, atezolizumab, or durvalumab; therefore, no dosing adjustments are recommended in this situation. The impact of these monoclonal antibodies in patients with moderate to severe hepatic impairment is unknown, as most trials excluded these patients. 30,32,44,70 Similarly, mild hepatic impairment did not alter the PK of ipilimumab and was comparable with PK in patients with normal hepatic function; ipilimumab has not been studied in patients with moderate or severe hepatic function. 41 For avelumab, neither mild nor moderate hepatic function had a clinically meaningful impact on PK, and although the effect of severe hepatic impairment is unknown, no dosing adjustments are recommended for patients with hepatic impairment. 31,71 Drug Drug Interaction Potential Polypharmacy is common in patients with cancer, and therefore potential drug drug interaction (DDI) is routinely tested during drug development. Because mabs are large molecules that are not commonly metabolized by cytochrome P450 (CYP450) enzymes, the potential for coadministered small-molecule drugs (most of which are metabolized by CYP450) to alter the exposure of mabs by the route of metabolism and transport is low. However, some studies have shown an increase in cytokine expression following mab administration; many cytokines in turn are known to modify CYP and transporter activity, with consequential implications for the clearance of small molecules The FDA 2012 draft guidance on DDIs and expert review of this guidance have produced a strategy to assess DDIs with therapeutic proteins. 81,82 A risk assessment for the therapeutic protein is necessary, including its potential to modulate inflammatory or other cytokines, any likely small-molecule comedications, clearance mechanism for all agents that may be coadministered (including the therapeutic protein), and potential for CYP-mediated interaction between the protein and comedications. Options for clinical assessment of DDIs include a approach using data from multiple phase 3 studies, which should suffice to recommend dosing adjustments when the signal of interaction is clear and large. 83 No formal DDI studies have been conducted for nivolumab. However, its DDI potential was indirectly assessed by measuring the expression of a number of cytokines, including those known to affect CYP450 enzyme activity over the dose range of 0.3 to 10 mg/kg every 3 weeks. Following nivolumab treatment, most cytokines evaluated (interleukin [IL] 1A, IL-1B, interferon-γ, tumor necrosis factor [TNF] ɑ, IL-12P, and IL-23M) were below the lower limit of quantification, and there was no meaningful change in levels of quantifiable cytokines in systemic circulation (IL-2 soluble receptor α, IL-6, and IL-10), suggesting that nivolumab has little or no potential to modulate CYP enzyme activity. 84 Similarly, avelumab did not induce cytokines in humans to concentrations needed to affect either transporters involved in the distribution or CYP450 metabolism of small-molecule drugs. 71 To our knowledge, the DDI potential for pembrolizumab and atezolizumab has not been assessed in the same way, and formal DDI studies were considered unnecessary. Although no formal DDI studies have been done for ipilimumab, the effect of ipilimumab on the PK of dacarbazine and carboplatin/paclitaxel and the effect of these agents on ipilimumab PK were investigated in a randomized phase 1 study. 85 Dacarbazine and paclitaxel are both metabolized by various CYP450 enzymes, which might be downregulated by cytokines in the presence of ipilimumab, with the consequential potential for altered systemic exposure of either or both chemotherapy agents. However, no major PK or PD interactions were observed between ipilimumab and either chemotherapy regimen. Immunogenicity Therapeutic mabs have the potential to elicit a general immunogenic response, defined by the detection and quantification of circulating antidrug antibodies (ADAs), thought to be primarily mediated by humoral mechanisms. 86,87 ADAs against therapeutic mabs can appear transiently or throughout the entire treatment period; can cause immune reactions such as anaphylaxis, cytokine release, and hypersensitivity; and may lead to complete or partial loss of efficacy because of target neutralization. The presence of ADAs can alter the PK of the drug, which in turn can influence safety and efficacy. Efforts are under way to standardize methodological approaches for gathering and reporting ADA data, as there is currently little consensus on reporting and analyzing immunogenicity data. 88 The proportion of patients who developed ADAs against the 6 approved immune checkpoint inhibitors cannot be directly compared, as there is no consistency in reporting and evaluation of immunogenicity data. 31,32,40,51,68,69 In fact, immunogenicity assay results are highly dependent on several factors, including assay sensitivity and specificity, assay methodology, sample handling, timing of sample collection, concomitant medications, and underlying disease. Therefore, despite apparent higher incidence noted for ADAs to

11 S36 The Journal of Clinical Pharmacology / Vol 57 No S atezolizumab, it is difficult to make definitive comparisons based on the reported incidence rates. Exposure Response Analyses Exposure Efficacy Relationship Overall survival in a phase 3 study is the gold standard efficacy measure of oncology drug treatment with traditional anticancer therapy. However, longer observation periods are required to determine this response, which can also be confounded by crossover or poststudy treatment. Other measures, such as progression-free survival and ORR assessed by Response Evaluation Criteria In Solid Tumors, are therefore used as primary efficacy end points, particularly in earlier phase studies, and can be evaluated in terms of drug exposure for clinical benefit analysis. 89 These exposure efficacy response analyses are frequently used to assess the appropriate selection of dose and dosing regimen in oncology drug development and in regulatory decision-making. As in the PK analyses, interpatient variability can be estimated for efficacy, and therefore covariates are incorporated as possible confounding factors. Covariates that are typically attributable to the heterogeneity in oncology populations include demographic factors, such as age, race, sex, and body weight, and patient characteristic factors such as performance status, tumor burden, clinical laboratory markers, renal or hepatic impairment, and liver function. Data from multiple doses may be needed to fully tease out the confounding effect of patient heterogeneity, and inclusion of trials testing various dose levels or dosing regimens in the exposure response analysis is accordingly recommended Nonetheless, exposure response analysis based on a single dose is often done because of limited data and can lead to misinterpretation confounded by disease state. 90 In addition, a steep exposure-versus-efficacy relationship, based on a univariate exposure, could be imbalanced, and a case control analysis has been applied recently to address this challenge. 93 Note that comparisons between the approved immune checkpoint inhibitors discussed here are difficult, because a range of doses was tested for ipilimumab, nivolumab, and pembrolizumab, but only a single dosing schedule for each of atezolizumab, avelumab, and durvalumab. Note, too, that dosing schedules for the approved immune checkpoint inhibitors varied both between inhibitor and according to indication. One major difference in scheduling is that ipilimumab monotherapy for metastatic melanoma is dosed for only 4 cycles, rather than continuously, as for the PD-1 and PD-L1 inhibitors. Finally, exposure response data may vary across tumor type, which may also confound interagent comparisons. Bearing in mind the factors limiting comparison, the relationship between drug exposure and ORR was flat or nonsignificant over the dose ranges tested for all approved PD-1 and PD-L1 inhibitors except avelumab (Table 1). A flat dose exposure relationship with ORR was observed in a large phase 3 trial in patients with melanoma treated with nivolumab 3 mg/kg every 2 weeks. 69 In the same study, baseline tumor burden was the only significant covariate identified for efficacy; higher tumor burden was associated with lower ORR. Similarly, the relationship between pembrolizumab exposure (area under the curve [AUC]) and response (probability of ORR) was flat with 2 and 10 mg/kg doses. 68 For patients with metastatic bladder cancer receiving atezolizumab 1200 mg, the probability of ORR was not associated with C min ; by multivariate analysis, the covariates of baseline ECOG performance status, number of metastatic sites at enrollment, and immune cell score were statistically significantly associated with ORR. 51 In patients treated with durvalumab 10 mg/kg every 2 weeks, the probability of objective response was similar in all quartiles of exposure, with no obvious trend between exposure and percentage change in tumor size. 73 In contrast, logistic regression analysis of data from 88 patients with Merkel cell carcinoma treated with avelumab 10 mg/kg every 2 weeks identified an apparent relationship between trough concentration at steady state (C minss ) and best objective response, with higher exposure associated with improved response. 71 Efficacy with ipilimumab varied with dose in the range of 0.3 to 10 mg/kg in patients with advanced melanoma. 94 The probability of achieving an objective response (complete response, partial response) or the probability of longer OS each increased with increasing exposure, as measured by C minss.however,inthefinal model, C minss was significantly associated with only OS; baseline LDH and ECOG performance status were also significantly associated with OS. Exposure Safety Relationship Although unrestrained T-cell activation with immune checkpoint blockade translates into antitumor responses, it is also associated with the development of adverse events (AEs). The relationship between dose/exposure and toxicity of immune checkpoint inhibitors was evaluated during clinical development (Table 1). With ipilimumab treatment in melanoma patients, C minss was identified as a significant predictor for the occurrence of an immune-related AE (irae). 94 The probability of experiencing grade 3 iraes increased with increasing ipilimumab exposure at the median C minss for doses of 0.3, 3, and 10 mg/kg; however, analyses of the relationship between exposure and safety with approved PD-1 and PD-L1 inhibitors have found a flat relationship. In patients with melanoma who received nivolumab 0.1 to 10 mg/kg every 2 weeks, the risk of time to first grade 3 drug-relatedaes

12 Sheng et al S37 and AEs leading to discontinuation did not increase with average concentration at steady state (C avgss ). 69 In patients treated with pembrolizumab, none of the AEs were associated with the tested dose or exposure (AUC) of 1 to 10 mg/kg. 68 Similarly, neither atezolizumab exposure at steady state (AUC ss ) with 1200-mg every-3- week dosing nor durvalumab exposure with 10 mg/kg every-2-week dosing was associated with grade 3 toxicities. 51,73 For avelumab, there was a weak association between increased exposure and increased incidence of iraes, but none with risk of treatmentemergent AEs or infusion-related reactions. 71 QT/QTc Potential Large molecules have less potential to block cardiac herg channels and thereby less influence on the QT interval, making a thorough QTc study unnecessary. Instead, extensive electrocardiogram (ECG) monitoring is highly recommended during the early phase of clinical testing. None of the approved checkpoint inhibitors have been shown to have a significant effect on the QT profile (Table 1). At doses of 3 and 10 mg/kg, ipilimumab did not produce clinically meaningful changes in ECG intervals. 95 A nivolumab QTc substudy in patients with solid tumors collected ECGs in triplicate at baseline and during treatment with 0.3, 2, or 10 mg/kg every 3 weeks. 96 A linear mixed-effects model was used to analyze nivolumab concentrations versus change from baseline in ECG parameters and found no clinically meaningful influence of nivolumab on the QTc interval. Similarly, the influence of pembrolizumab concentrations at dose levels up to 10 mg/kg every 3 weeks was studied but did not detect large changes in the QT interval (>20 milliseconds). 68 An atezolizumab QTc substudy also found no significant changes in the QT interval with doses up to 20 mg/kg or 1200 mg every 3 weeks. 51 An exposure QTc analysis using data from 689 patients treated with avelumab showed a minimal effect of avelumab on the QTc and change in QTc from baseline, suggesting that avelumab has no clinically relevant effect on cardiac repolarization. 71 Immune Checkpoint Inhibitor Combination The first FDA approval of combination therapy with 2 checkpoint inhibitors was nivolumab plus ipilimumab, indicated for unresectable or metastatic melanoma. This combination demonstrated remarkable clinical benefits over nivolumab or ipilimumab monotherapy. 6,97 In this particular nivolumab-ipilimumab combination, nivolumab was dosed at 1 mg/kg, followed by ipilimumab on the same day at 3 mg/kg every 3 weeks for 4 doses; nivolumab was then administered at 240 mg every 2 weeks. The clearance of both nivolumab and ipilimumab given in combination was assessed using a approach, demonstrating that nivolumab clearance increased by 24%, but no effect on ipilimumab clearance. 44,98 In addition, the immunogenicity of nivolumab plus ipilimumab was evaluated, with higher incidence of nivolumab ADAs seen in combination compared with nivolumab as a single agent. In the presence of anti-nivolumab antibodies, nivolumab clearance increased by 42%; there was no effect of antiipilimumab antibodies on ipilimumab clearance. Even with an increased incidence of ADAs or neutralizing antibodies, there was no evidence of impact on efficacy or safety in patients with advanced melanoma. 44 In addition, in a recent study of IL-21 and ipilimumab, higher incidence of IL-21 immunogenicity was seen when combined with ipilimumab (unpublished data on file). Drug drug interaction assessment, typically between mabs (perpetrator) and small molecules (victim), may also be necessary with immunotherapy agent combinations depending on the impact of individual agents on in vitro and/or in vivo cytokine modulation, which can have potential downstream effects on CYP regulation pathways. Pharmacometric Strategies: Application of Systems Modeling A quantitative systems pharmacology (QSP) model can assist in exploring biological factors influencing patients response to provide insight in predicting a first- or best-in-class profile and to optimize the dose and schedule of immuno-oncology treatment. In addition, because they enable a network-centric systemic view and analysis of biological systems and interacting processes (instead of a one-gene, one-receptor, onemechanism approach), 99 QSP models are also well suited for guiding combination therapy development. QSP models may describe intracellular pathways and cell dynamics, distribution of key players, drug PK, and clinically measured biomarkers and end points, as required to meet project goals, 100 and often employ a system of ordinary differential equations. The scope of a QSP model is an important decision made early in model development: whether to begin initial development by focusing the model on a few key pathways or to develop a broader disease platform model. 101 In immuno-oncology, alternative models of the cancerimmunity cycle describing interactions between tumor, immune cells, cytokines, and molecular players including the process of antigen presentation, priming and trafficking of T cells, infiltration of immune cells, killing of tumor cells, and release of cancer immunotherapy cycle cancer antigen have been described, with the primary aim being to impact translational and