1Aside from targeting antigens that are involved in cancer cell proliferation and survival, antibodies can also function to either activate or inactivate immunological pathways that are important in cancer immune surveillance. 2It is now clear that an antigen-specific anti-cancer immune response is the result of a complex dynamic interplay between antigen presenting cells (APCs), T lymphocyte cells (T cells), and the target cancer cells. The criti cal balance of T-cell activity that dictates whether endogenous anti-tumor immune responses will be effective are largely understood to be contro lled by antigen-specific st imuli sensed by the T-cell receptor and by the combined activity of bo th positive (co-stimulatory) and negative (co-inhibitory) T-cell surf ace molecules.3Within the past decade, an tibodies against these key receptors have been designed and evaluated in the clinic with impressive results, heralding the onset of immunotherapy as a key pillar of anti-cancer therapy.4 The most extensively studied immunotherapies in cancer target the negati ve regulatory receptors CTLA-4 or PD-1.5Inhibition of these negative regulatory r eceptors, referred to as immune checkpoint blockade, results in the enhanced activation of T-cell res ponses and potent anti-tumor activity in nonclin ical models. Studies with CTLA-4 blockade provided the first clinical evidence of improvement in overall survival (OS) with immune modulatory anti-cancer therapy in patients with metastatic melanoma.6,7Following that, Topalian et al showed that anti-PD-1 antibody Revised Protocol No.: 04 Date: 31-May-2019 38 Approved 5.0 v Approved 1.0 v Clinical Protocol CA021002 BMS-986226 anti-ICOS therapy resulted in objective responses in patients with melanoma, NSCLC, and renal cell carcinoma (RCC).8 Subsequent to the success of anti-tumor treatmen t with CTLA-4 and PD-1 pathway-blocking agents, the field of tumor immunotherapy is expanding rapidly. In addition to blocking co-inhibitory pathways, activating co-stimulatory pathways to potentiate anti-tumor immune responses is being considered as a promising approach.9With recent emerging clinical evidences of significant anti-tumor activity of single-agent immunotherapies, combination therapies are now explored because they could lead to greater depth of response and OS. This has been observed with the combination therapy of anti-PD-1 and anti-CTLA-4 antibodies in patients with advanced melanoma. It also raises the possibility that co mbining checkpoint blocking antibodies with agonistic antibodies targeting T- cell co-s timulatory molecules could be beneficial. Nonclinical data evaluating this type of treatment combination in mouse tumor models indicate that combination treatment indeed leads to enhanced anti-tumor activity compared with single-agent- treatment. Administration of ipilimumab has been shown to up-regulate ICOS expression on the surface of CD4 and CD8 effector T cells in patients with superficial bladder cancer and advanced melanoma. 10Patients with ≥ 2-fold increase in percentage of circulating CD4+ICOShiexpression that was sustained at Week 12 showed clinical bene fit, while p atients with < 2-fold increase did not, thus suggesting a role for ICOS+ T cells in supporting anti-tum or activity after administration of ipilimumab (n = 14). Nivolumab treatment results in in creased ICOS gene expression in tumor biopsies in patients with both RCC and melanoma.11,12The increase in ICOS expression after both ipilimumab and nivolumab treatment suggests that the ICOS pathway is an appealing target for an agonist antibody in combination with either nivolumab or ipilimumab, alone or as a combination. ICOS costimulation potently indu ces T-cell activation and cytoki ne production, and agonism of ICOS on T-effector cells (Teffs) s hould further enhance antitumor activity. The activity of an ICOS agonist may provide the most benefit in combinat ion with checkpoint inhibitors that induce ICOS expression (eg, ipilimumab or nivolumab) and in patients whose T cells are not receiving an optimal costimulatory signal through ICOS due to low expression of Inducible T-cell COStimulator ligand (ICOS-L) at the tumor site. In this study, participants with advanced tumors will be tr eated with BMS-986226 (an agonistic antibody targeting ICOS) monotherapy or BMS-986226 in combination with nivolumab (a checkpoint blocking antibody [anti-PD-1 an tibody]) or ipilimumab (a checkpoint blocking antibody [anti-CTLA-4 antibody]) to take adva ntage of this enhanced anti-tumor effect. 3.1.1 Research Hypothesis It is anticipated that an anti-ICOS mAb (BMS-986226) administered as a single agent or in combination with nivolumab (BMS-936558/MDX1106, anti-PD-1 antibody) or ipilimumab (BMS-734016/MDX010, anti-CTLA-4 antibody) demonstr ates adequate safety and tolerability, as well as a favorable risk/benefit profile, to support further clinical testing. Revised Protocol No.: 04 Date: 31-May-2019 39 Approved 5.0 v Approved 1.0 v Clinical Protocol CA021002 BMS-986226 anti-ICOS 3.2 Background A detailed description of the chemistry, pharmacology, efficacy, and safety of BMS-986226 is provided in the Investigator’s Brochure (IB). 3.2.1 Indication Background It is hypothesized that tumors with more frequent ICOS-expressing tumor infiltrating immune cells and less frequent or non-proximal ICOS-L-expressi ng cells will re spond to ICOS agonist mAb (BMS-986226) therapy. In addition, the presence of Fc γRII (CD32), particularly the Fc γRIIB (CD32B) isoform, will be required for cross-linking and the anti-tumor activity of BMS-986226. To explore which tumor types may respond better to ICOS mAb therapy, immunohistochemistry (IHC) assessment of ICOS, ICOS-L, and CD32B e xpression using commercial mAbs against these proteins was performed on adjacen t formalin-fixed paraffin-embedded (FFPE) sections. Several tumors were analyzed, including CRC, HNSCC, melanoma, NSCLC-adenocarcinoma type (NSCLC-AD), NSCLC-squamous cell type (NSCLC-SQC), PRC, RCC, and UCC. As expected, specific ICOS staini ng was observed in cells morphologically resembling small lymphocytes. CD32B staining was observed in cells with a m orphology of either a plasma cell or a small lymphocyte. Wh ile ICOS-expre ssing and CD32B-expressing cells were primarily distributed in tumor stroma, ICOS-L staining was seen in cells with the morphology of a small lymphocyte or tumor cells in a subset of cases. Automated image analysis using HALO software on whole slide images revealed a relatively high expression of ICOS and CD32B and low expression of ICOS-L in HNSCC, melanoma, and NSCLC-AD, and relatively high expression of ICOS and low ICOS-L and CD32B expression in UCC, but relatively low ICOS and CD32B expression and high ICOS-L expression in CRC, PRC, and RCC. NSCLC-SQC demonstrated a relatively low ICOS and high ICOS-L and CD32B expression. External evaluation of ICOS gene expression in a variety of epithelial solid tumors demonstrated similar results, with HNSCC, NSCLC, TNBC, gastric carcinoma and CC exhibiting highest ICOS mRNA expression. 13Based on the aforementioned hypothesis, these preliminary datasets support initial development of BMS- 986226 in multiple advanced tumors, including HNSCC, NSCLC, TNBC, CC and MEL. However, ICOS is also know n to be upregulated upon tr eatment with ipilimumab.10Therefore, tumors that have baseline lo w expression of ICOS, may become more favorable to co-stimulation in the presence of anti-CTLA-4 or anti-PD-1 therapy. Given this observation, in this study testing of BMS-986226 will be expanded to all adva nced epithelial solid tumors, including previously identified low-ICOS expressing tumors, such as CRC, PRC, RCC, and UCC. 3.2.2 BMS-986226 3.2.2.1 Pharmacology of BMS-986226The nonclinical pharmacology of BMS-986226 was evaluated in vitro, including binding to cells characterized by flow cytometry and Scatchard analysis and binding to human Fc γRs characterized by surface plasmon resonance. In vitro functional assays elevated activation of T cells, antibody- driven effector function in antibody-dependent cellular cytotoxicity assays, and complement Revised Protocol No.: 04 Date: 31-May-2019 40 Approved 5.0 v Approved 1.0 v Clinical Protocol CA021002 BMS-986226 anti-ICOS binding assays. Cytokine release from whole blood was not observed following BMS-986226 treatment. BMS- 986226 was evaluated for binding to human and cynomolgus tissues and ICOS expression was characterized by flow cytometry on human tumor-infiltrating lymphocytes (TILs). BMS-986226 was fo und to overcome T-regulatory cell (Treg) activity in in vitro suppression assays. In vivo, surrogate mouse binding ICOS agonists were evaluated for anti-tumor activity and PD effects in syngeneic mouse tumor models. Further information on the pharmacology of BMS-986226 is detailed in the IB.14 3.2.2.2 Toxicity of BMS-986226 The nonclinical safety of BMS-986226 was evaluated in vitro in a human tissue cross-reactivity study and in cytokine-release and lymphocyte-activation assays in human cells. In vivo, BMS-986226 was evaluated in exploratory repeat-dose IV toxicity studies in rats and cynomolgus monkeys. In addition, an exploratory single- and repeat-dose (2-week) IV toxicity study was conducted in mice using BMS- 986246, a surrogate anti-mouse ICOS agonistic antibody with identical comp lementarity-determining regions (hamster-derived) as BMS-986226, with mouse Fc domains, and with comparable affinity for mouse ICOS. In a Good La boratory Pract ice (GLP)comp liant tissue cross-reactivity study in normal human tissues, staining was consistent with known ICOS expression on T cells and demonstrated binding of BMS-986226- fluorescein isothiocyanate (FITC) on the membrane of a subset of mononuclear cells (consistent with lymphocytes) in lymphoid tissues and other tissues with m ononuclear infiltrates. BMS-986226 did not indu ce cytokine release or increase the expression of activation markers on human T, B, or natural killer (NK) cells. The mouse, rat, and cynomolgus monkey were evaluated in toxicology studies because BMS-986226 binds to ICOS receptors expresse d on activated T cells from mice, rats, and cynomolgus monkeys with comparable affinity as ICOS expressed on activated human T cells (concentration required for 50% efficacy values of 0.13 nM to 0.87 nM), and is pharmaco logically active. To explore the feasibility of pivotal rodent st udies, single-dose (mouse only) and repeat-dose exploratory studies in rats with BMS-986226 and in mice with BMS- 986246 were conducted. BMS-986226 and BMS-986246 resulted in mortality in rats and mice, respectively, after repeated administration. These mortalities were cons idered secondary to anti-drug antibody (ADA)-mediated hypersensitivity reactions based on the nature and timing of clinical signs, presence of serum ADA in affected animals, and elevated serum cytokine levels and evidence of complement activation in rats. Therefore, rodent sp ecies were considered not suitable for further toxicity assessment in pivotal studies. It should be noted that as described in International Conference on Harmonisation (ICH) S6, the induction of ADA formation in animals is not predictive of a potential for ADA formation in humans. In the GLP 1-month IV toxicity study in cynomolgus monkeys, doses of 0, 1.5 mg/kg (every 3 weeks [Q3W], 2 total doses), and 15 mg/kg or 75 mg/kg (once every week (Q1W), 5 total doses), were administered as a slow bolus injection. BMS-986226 was clinically well tolerated at all doses Revised Protocol No.: 04 Date: 31-May-2019 41 Approved 5.0 v Approved 1.0 v Clinical Protocol CA021002 BMS-986226 anti-ICOS without any adverse effects. No significant irritation or local tolerance issues were observed at the injection sites following repeated IV admin istration of BMS-986226 as a slow bolus in jection at ≤75 mg/kg in cynomolgus monkeys. BMS-986226-related findings were limited to PD and immunomodulatory effects at all doses that included ICOS receptor e xpression (RE)/receptor o ccupancy (RO) changes (for CD4 helper T cells), suppression of T-dependent antigen response to keyhole limpet hemocyanin, decreased levels of T cells (including total T cells, CD4 helper T cells, CD4 Tregs, central memory CD4 T cells, and central memory CD8 T cells), decreased CD4 T-cell activation, and decreased percentages of antigen-specific CD8 T cells. Many of these changes persisted up to the end of the recovery period at ≥15 mg/kg Q1W consistent with continued BMS-986226 exposure throughout the recovery period and the subsequent sustained ICOS receptor downregulation at these doses. In general, ICOS RE levels inversely correlated with serum BMS-986226 concentrations. Based on the lack of adverse findings, the high dose of 75 mg/kg (Q1W, mean sex-combined area under the concentration-time curve (AUC) from t ime zero to 168 hours [AUC(0-168h)=452,000 μg•h/mL]) was considered the no-observed-adverse-effect level (NOAEL). In addition, for determination of the human maximum recommended starting dose ( MRSD), 75 mg/kg was also considered the highest nonseverely toxic dose (HNSTD). The HNSTD/NOAEL of 75 mg/kg in cynomolgus monkeys is 250 ×higher than the proposed starting dose in humans (0.3 mg/kg) with a corresponding plasma AUC multiple of 102 ×the projected human AUC at the starting dose (approximately 4425 μg•h/mL; see Section 5.4.1 ). Overall, the nonclinical toxicology assessment of BMS-986226 has demonstrated an acceptable safety profile, supporting clinical use in oncology patients. Additional details are provided in the BMS-986226 IB. 3.2.2.3 Nonclinical Metabolism and Pharmacokinetics of BMS-986226 This section summarizes the data available from a series of in vivo PK and PD studies that were conducted with BMS-986246 (ICOS.4-mIgG1, an anti-mouse surrogate antibody being used as a rodent surrogate for BMS-986226) and BMS- 986226 in mice and cynomolgus monkeys, respectively. In mice, following a single IV bolus dose of 1 mg/ kg, the serum concentrations of BMS-986246 exhibited a bi-exponential decline. 15The serum clearance was 1.8 mL/h/kg and the apparent terminal half-life in serum (T-HALF) was 0.65 hours. After a single intraperitoneal dose of 0.1, 1, or 10 mg/kg, BMS-986246 was slow ly absorbed, with a time of maximum observed plasma concentration (Tmax) of 6 to 24 hours, and the absolute bioavailab ility wa s 38%, > 100%, and > 100%, respectively; reasons for the higher bioavailability at higher doses are not known. Over this dose range, increases in exposure were more than dose proportional; with a dose ratio of 1:10:10, respectively, and the maximum observe d plasma concentration (Cmax) and AUC from time zero extrapolated to infinite time values increased in the proportion of 1:13:177 and 1:33:599, respectively. Revised Protocol No.: 04 Date: 31-May-2019 42 Approved 5.0 v Approved 1.0 v Clinical Protocol CA021002 BMS-986226 anti-ICOS After IV dosing to cynomolgus monkeys, the sy stemic exposure of BMS-986226 increased in a dose-proportional manner between 1 and 10 mg/kg.15Extracellular distribution was limited in both mice and cynomolgus monkeys. No in vitro or in vivo metabolism studies have been conducted with BMS-986226. In accordance with the regulatory guidelines fo r biotechnology-derived pharmaceut icals,16the expected in vivo degradation of mAbs is to s mall peptides and amino acids via biochemical pathways that are generally understood and independent of typical small-molecule, drug-metabolizing enzymes. In accordance with regulatory guidelines for biotechnology-derived pharmaceut icals,16no mass balance studies with BMS-986226 were conducted in animals. 3.2.2.4 Clinical Pharmacology and Safety BMS-986226 has been administered to 21 partici pants between Octobe r 2017 and 28-March-2019, in doses ranging from 2 mg to 200 mg. All described data are preliminary and subject to change. Nineteen of 21 treated par ticipants received BMS-986226 monotherapy. Monotherapy tr eatment was well tolerated. Grade 1/2 i nfusion reactions have been re ported in 7 of 19 participants, and have been clinically manageable. A Grade 3 drug-r elated hypersensitivity was reported for a participant in the 80 mg cohort during BMS- 986226 infus ion after the above data cut- off date. Two participants have been treated with BMS-986226 in combination with ipilimumab, with no adverse events (AEs) reported as of the 28-March-2019 data cut-off date. Given the small number of participants treated with BMS-986226, the subsequent more detailed evaluation of safety is based on informatio n from nonclinical studies, as described in Section 3.2.2 , as well as preliminary clinical study data.As described in ICH S6, the induction of ADA formation in animals is not predictive of a potential for ADA formation in humans. Since BMS-986226 is a humanized mAb, it is expected to be less immunogenic in humans; however, it has the potential to induce the development of ADAs following administration. Preliminary data suggests that ADAs are formed in participants treated with BMS-986226. The r elationship between the incidence of infusion r eactions and emergence of ADAs is currently unknown based on the small number of treated participants. However, most infusion reactions are occurring during the fi rst infusion of BMS- 986226, wh ile the ADAs are detected 2-4 weeks following the initial BM S-986226 administration. Additionally, because BMS- 986226 will be evaluated in combination with eith er nivolumab or ipilimumab, it is possible that this combination may increase the de velopment of BMS- 986226 ADA versus BMS- 986226 monotherapy. In combination th erapy, BMS-986226 may potentiate immune-related adverse reactions caused by nivolumab or ipilimumab. Po tential safety concerns and recommended management guidelines regarding nivolumab- or ipilimumab-induced AEs are summarized in the nivolumab IB 17and ipilimumab IB.18 Immuno-modulatory antibodies, which provide activation signals to T cells, also have the potential to cause cytokine release. Nonclinical data indicate that BMS-986226 does not induce cytokine release when incubated with human peripheral blood mononuclear cells (PBMC). In addition, administration of nivolumab and/or ipilimumab monotherapy or combination therapy in clinical Revised Protocol No.: 04 Date: 31-May-2019 43 Approved 5.0 v Approved 1.0 v Clinical Protocol CA021002 BMS-986226 anti-ICOS studies did not produce AEs related to cytokine release syndrome. There is no evidence that BMS- 986226 will induce cytokine release syndrome when administered in combination. BMS-986226 did not cause cytokine release or activat ion of lymphocytes when incubated with human PBMCs, and tissue cross- reactivity studies rev ealed staining of only mononuclear cells in various tissues (no off-target binding). Thus, the expected effects are anticipated to be limited to activated CD4 and CD8 T lymphocytes and T regs. 3.2.2.5 Pharmacokinetics of BMS-986226 BMS-986226 has been administered to 12 participants to date, at four different doses; therefore, insufficient data are available to clearly characterize human pharmacokinetics. Based on nonclinical data, the PK of BMS-986226 are expected to be similar to those of other mAbs. The predicted PK estimates for total plasma clearance (CL), volume of distribution, and T-HALF of BMS-986226 following IV administration are 0.079 mL/h/kg, 0.066 L/kg, and 25 days, respectively. All projected human exposures are exp ected to be less than those exposures deemed safe in the nonclinical toxicology studies.