First-In-Human Study of Utomilumab, a 4-1BB/CD137 Agonist, in Combination with Rituximab in Patients with Þ Follicular and Other CD20 Non-Hodgkin Lymphomas a C Ajay K

Published OnlineFirst March 6, 2020; DOI: 10.1158/1078-0432.CCR-19-2973

CLINICAL CANCER RESEARCH | CLINICAL TRIALS: IMMUNOTHERAPY

First-in-Human Study of Utomilumab, a 4-1BB/CD137 Agonist, in Combination with Rituximab in Patients with þ Follicular and Other CD20 Non-Hodgkin Lymphomas A C Ajay K. Gopal1, Ronald Levy2, Roch Houot3,4,5, Sandip P. Patel6, Leslie Popplewell7, Caron Jacobson8, Xinmeng J. Mu9, Shibing Deng9, Keith A. Ching9, Ying Chen9, Craig B. Davis9, Bo Huang10, Kolette D. Fly10, Aron Thall9, Adrian Woolfson11, and Nancy L. Bartlett12

ABSTRACT ◥ Purpose: In this phase I study (NCT01307267), we evaluated the utomilumab treatment-related adverse events (AE) were safety, pharmacokinetics, clinical activity, and pharmacodynamics grade 1 to 2; the most common AE was fatigue (16.4%). The of treatment with utomilumab plus rituximab in patients with pharmacokinetics of utomilumab in combination with rituximab þ relapsed/refractory follicular lymphoma (FL) and other CD20 was linear in the 0.03 to 10 mg/kg dose range. A low incidence non-Hodgkin lymphomas (NHL). (1.5%) of treatment-induced antidrug antibodies against Patients and Methods: Primary objectives were to assess treat- utomilumab was observed. The objective response rate was ment safety and tolerability for estimating the MTD, using a 21.2% (95% CI, 12.1%–33.0%) in all patients with NHL, includ- modified time-to-event continual reassessment method, and select- ing four complete and 10 partial responses. Analysis of paired ing the recommended phase II dose (RP2D). biopsies from a relapsed/refractory FL patient with complete Results: Sixty-seven patients received utomilumab (0.03– response showed increased T-cell infiltration and cytotoxic 10.0 mg/kg every 4 weeks) and rituximab (375 mg/m2 weekly) activity in tumors. Biomarker correlations with outcomes in the dose-escalation groups or utomilumab (1.2 mg/kg every suggested that clinical benefit may be contingent on patient 4 weeks) plus rituximab in the dose-expansion cohort. No immune function. patient experienced dose-limiting toxicity. The MTD for Conclusions: Utomilumab in combination with rituximab utomilumab in combination with rituximab was not reached demonstrated clinical activity and a favorable safety profile in þ and estimated to be ≥10mg/kgevery4weeks.Themajorityof patients with CD20 NHLs.

Introduction immunotherapeutic approaches including 4-1BB/CD137, programmed death 1 (PD-1), and PD-ligand 1 (PD-L1) targeted anti- Treatment with anti-CD20 antibodies, such as rituximab, induces þ bodies, as well as bispecific antibodies and chimeric antigen receptor responses in most patients with CD20 non-Hodgkin lymphoma (CAR) T cells are currently under investigation in patients with NHL (NHL) and results in a significant reduction in the risk of death (1). and other hematologic malignancies (4–14). The antitumor activity of rituximab is believed to be mediated in part The anti-epidermal growth factor receptor (EGFR) antibody cetuxi- by antibody-dependent cellular cytotoxicity (ADCC; ref. 1). However, mab has been reported to enable cross-presentation of tumor antigens a substantial proportion of patients develop resistance to treatment, by dendritic cells and enhance EGFR-targeted cytotoxic T lymphocyte underscoring the need for more effective strategies (1–3). A variety of (CTL) responses in patients with head and neck squamous cell carcinoma (HNSCC; ref. 15). Treatment of patients with HNSCC with a combination of cetuximab and the 4-1BB/CD137 agonist 1University of Washington, Fred Hutchinson Cancer Research Center, Seattle antibody urelumab was associated with elevated levels of activated Cancer Care Alliance, Seattle, Washington. 2Stanford Cancer Center, Stanford, California. 31 CHU Rennes, Service Hematologie Clinique Rennes, France. 4Uni- natural killer (NK) cells, myeloid cells, and T cells in circulation (16). versity of Rennes, EFS, Microenvironment, Cell Differentiation, Immunology and These data support the hypothesis that 4-1BB/CD137 agonist anti- Cancer Rennes, France. 5INSERM 0203, Unite d'Investigation Clinique, Rennes, bodies can increase innate and adaptive immune responses arising France. 6University of California at San Diego Moores Cancer Center, San Diego, after treatment with ADCC-competent antibodies. 7 8 California. City of Hope National Medical Center, Duarte, California. Dana- Utomilumab (PF-05082566) is a fully-human IgG2 agonist mAb 9 fi Farber Cancer Institute, Boston, Massachusetts. P zer Oncology, San Diego, that selectively binds human 4-1BB/CD137, resulting in NF-kB California. 10Pfizer Oncology, Groton, Connecticut. 11Pfizer Oncology, New York, New York. 12Washington University School of Medicine, Siteman Cancer Center, activation and downstream cytokine production in cell lines and St Louis, Missouri. primary lymphocytes (17). Utomilumab can induce human leukocyte proliferation and has demonstrated significant antitumor activity as a Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). single agent in human peripheral blood lymphocyte (PBL) SCID xenograft tumor models (17). Corresponding Author: Ajay K. Gopal, University of Washington/Seattle Cancer Utomilumab was well tolerated and active both as a single agent and Care Alliance, SCCA, 825 Eastlake Avenue East, CE3-300, Seattle, WA 98109. – Phone: 206-606-2037; Fax: 206-606-1130; E-mail: [email protected] in combination with the anti PD-1 antibody pembrolizumab, in patients with advanced solid tumors (18, 19). Durable responses Clin Cancer Res 2020;XX:XX–XX (duration >6 months) have been observed following single-agent or doi: 10.1158/1078-0432.CCR-19-2973 combination treatment in patients with Merkel cell carcinoma, non– 2020 American Association for Cancer Research. small cell cancer and small cell lung cancer, renal cell carcinoma,

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product of the diameters >2.25 cm2; Eastern Cooperative Oncology Translational Relevance Group (ECOG) performance status ≤1; absolute neutrophil count Prior studies have shown that 4-1BB/CD137 agonistic antibo- ≥1.0 109/L; platelet count ≥75 109/L; hemoglobin ≥8.0 g/dL; and dies can deliver costimulatory signals, enhancing antibody- adequate renal, hepatic, and cardiac functions. dependent cellular cytotoxicity and potentially eliciting T-cell– Exclusion criteria included prior allogeneic hematopoietic stem cell mediated antitumor immune responses. To evaluate new treatment transplant, any anticancer therapy or hematopoietic growth factors strategies for relapsed/refractory disease after rituximab therapy, within 28 days or systemic corticosteroid therapy or radiation therapy þ patients with follicular lymphoma (FL) and other CD20 relapsed/ within 14 days prior to the first dose of study treatment, prior refractory non-Hodgkin lymphomas (NHL) were treated with the treatment with a 4-1BB/CD137 agonist, an autoimmune disorder or anti–4-1BB/CD137 antibody utomilumab and rituximab. Utomi- an active clinically significant infection, a severe allergic or anaphy- lumab has previously demonstrated a well-tolerated safety profile lactic reaction to antibodies or infused therapeutic proteins, and and antitumor activity as monotherapy or in combination with the known utomilumab and/or rituximab antidrug antibodies (ADA). anti-programmed death 1 (PD-1) antibody pembrolizumab in Furthermore, patients were not included if they had symptomatic patients with advanced solid tumors. In this phase I study, com- brain metastases requiring steroid therapy. Patients were eligible if bined treatment with utomilumab plus rituximab showed a favor- they had completed treatment for their brain metastases and recovered able safety profile and clinical activity in patients with relapsed/ from the acute effects of radiation therapy or surgery, had discontinued þ refractory FL and other CD20 NHLs. corticosteroids for ≥4 weeks, and were neurologically stable. The study was approved by the institutional review board or independent ethics committee of the participating institutions and followed the Declaration of Helsinki and the International Conference squamous cell carcinoma of the head and neck, and thyroid on Harmonization Good Clinical Practice guidelines. All subjects cancer (18, 19). Treatment with utomilumab was associated with a provided written informed consent. The study was sponsored by Pfizer favorable safety profile, with no dose-limiting toxicities (DLT) and no and registered at ClinicalTrials.gov (NCT01307267). treatment-related deaths at the doses evaluated. Treatment-emergent adverse events (AE) were mostly mild or moderate in severity (18, 19). Treatment In this phase I, dose-finding study, we assessed the safety, tolera- Utomilumab was administered as a 1-hour intravenous infusion bility, pharmacokinetics, preliminary clinical activity, and pharmaco- every 4 weeks. In dose escalation, patients were assigned to receive dynamics of treatment with utomilumab in combination with rituxi- utomilumab at a starting dose of 0.03 mg/kg, with the dose being mab in patients with relapsed or refractory follicular lymphoma (FL) escalated/de-escalated in subsequent dose cohorts to: 0.06, 0.12, 0.18, þ and other CD20 NHLs. Potential associations between clinical out- 0.24, 0.30, 0.60, 1.2, 2.4, 5.0, and 10 mg/kg, using the modified time-to- comes and biomarkers were also evaluated in tumor and blood samples event continual reassessment method (TITE-CRM; refs. 20–22). Intra- from treated patients. patient dose escalation was not allowed. In the expansion cohort, patients received utomilumab at 1.2 mg/kg. Patients were allowed to continue treatment with utomilumab for up to 2 years or until Patients and Methods unacceptable toxicity, disease progression, or withdrawal of consent, Study objectives whichever occurred first. Rituximab (375 mg/m2) was administered This multicenter, open-label, multiple-dose, phase I study consisted once weekly for 4 weeks per institutional standards, with the first and of a dose-escalation and a dose-expansion component. The primary second doses given 8 days and 1 day prior to the initial utomilumab objectives were to evaluate safety and tolerability of treatment with infusion, respectively. Rituximab dosing could be repeated according utomilumab in combination with rituximab, to estimate the MTD and to the standard of care and at the investigator's discretion once every select the recommended phase II dose (RP2D) for patients with 8 weeks, starting on or after cycle 4 (day 0) and continuing up to cycle þ relapsed/refractory CD20 NHL. Secondary objectives included over- 10 (day 0). all safety, pharmacokinetics, immunogenicity [antidrug antibodies (ADA) and neutralizing antibodies (NAb) against utomilumab], and Study assessments antilymphoma activity. Assessments of peripheral blood T cells, Safety circulating soluble 4-1BB/CD137 (s4-1BB/CD137) levels, and Safety evaluations included vital signs, physical examination, review immune-related biomarkers in tumors were incorporated as explor- of treatment-emergent AEs and serious AEs (SAE), electrocardio- atory objectives. grams (12 lead), and laboratory assessments. AEs were characterized by type, frequency, timing, seriousness, and relationship to study drug, Patients and graded by National Cancer Institute Common Terminology Patients ≥18 or ≥20 years of age (if required by local, regulatory Criteria for Adverse Events v.4.03. authorities) were eligible if they had histologically confirmed, relapsed/ DLTs were defined as grade 4 neutropenia, febrile neutropenia, þ refractory CD20 NHL or small lymphocytic lymphoma (SLL)/chron- neutropenic infection, grade ≥3 thrombocytopenia with associated ic lymphocytic leukemia (CLL) with nodal disease and ≤10,000 bleeding, grade 4 thrombocytopenia, grade 4 anemia, grade ≥3 hemo- lymphocytes/mL, with no available, effective therapy. Patients were lysis, or grade ≥3 nonhematologic toxicities [with the exception of enrolled into the expansion cohort if they had FL refractory to previous grade 3 nonhematologic AEs (e.g., nausea, vomiting, and diarrhea) rituximab therapy or relapsed/refractory diffuse large B-cell lympho- that responded to treatment with standard supportive therapy within ma (DLBCL). 48 hours], if they were attributable to combination treatment with Patients were required to have measurable disease with at least one utomilumab and rituximab and not to disease progression, and extranodal tumor mass >1 cm in the greatest transverse diameter occurred within the first two treatment cycles (up to 28 days following (GTD) or malignant lymph nodes >1.5 cm in the GTD, and the the second treatment dose).

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Pharmacokinetic and ADA analyses RNA-seq reads were processed by alignment to hg19 UCSC tran- Blood samples were collected for the evaluation of pharmacokinetic scriptome using STAR aligner (v2.4) and gene expression was quan- parameters at multiple, protocol-defined time points and analysed tified using RSEM (v1.2.14; refs. 24, 25). RNA deconvolution was using a validated enzyme-linked immunosorbent assay (18). Standard implemented using the nu-support vector regression (nuSVR) method plasma pharmacokinetic parameters for utomilumab were estimated in Matlab, as described previously (26). Cox proportional-hazards using noncompartmental analysis. Blood samples for the determina- regression model was used to assess the dependence of PFS on gene tion of ADAs against utomilumab were collected at protocol-specified expression. Genes with low expression levels or low variance across time points and analyzed using a validated, electrochemiluminescent samples were filtered out, leaving 6,272 genes. Specifically, a low- bridging assay (18). ADA-positive samples were further evaluated for variance filter was applied using varFilter in the nsFilter R package. In the presence of NAbs using a cell-based assay (18). addition, genes with <3 samples with nonzero expression were excluded. Regression was performed for each gene postfiltering, according Response assessments to its median expression level (coded into a binary variable) stratified Tumor responses were assessed by computed tomography or by dose levels. Multivariate analysis was also carried out adjusting for magnetic resonance imaging scans of chest, abdomen, and pelvis, age, sex, race, stage, and whether patients had progressed on prior approximately every 8 weeks until cycle 10, and subsequently every rituximab-containing therapy. Gene set enrichment analysis (GSEA) 16 weeks until disease progression, end of treatment, or study with- was run preranked by HR from the Cox regressions, using classic drawal (if not done in the previous 6 weeks). Responses were deter- statistics with 1,000 permutations (27). Gene sets evaluated were mined using the Cheson 2007 criteria for NHL (23). from the LM22 signatures and the hallmark gene set collection in the Molecular Signatures Database (MSigDB; excluding size <10; Pharmacodynamics refs. 26, 28). Peripheral blood samples were collected for flow cytometry as described previously (18) with the addition of a draw prior to Statistical analyses administration of the first rituximab dose (on day 7 relative to the The primary study endpoint was DLT in the first two cycles of first utomilumab dose). Absolute numbers and relative proportions of treatment with utomilumab in combination with rituximab. The MTD T, B, and NK cells were assessed using an antibody panel containing: was estimated as the highest dose level associated with a ≤25% CD3, CD9, CD16, and CD56. T-cell phenotypes were determined estimated DLT rate per the modified TITE-CRM (21–23). The using antibody panels containing: CD3, CD4, CD8, CD45RO, CCR7, TITE-CRM design was implemented in this study as described with CD137, Ki67, CD28, PD-1, and CD57. NK phenotypes were assessed cyclical adaptive weight function (22). A dose-escalation steering using antibody panels containing: CD3, CD16, CD56, CD69, CD25, committee was established to facilitate the trial conduct process (23). VCAM/CD106, Ki67, and CD137. Antibodies were obtained from BD As previously described (18), a sample size of 45 patients (with early Biosciences, BioLegend, and Thermo Fisher Scientific. Lymphocyte stopping rules) was estimated to provide an accurate estimate of the subpopulations were assessed using a FACScanto II flow cytometer MTD and to detect unexpected toxicities occurring at a 5% rate with a (BD Biosciences). Serum samples were analyzed for soluble 4-1BB/ probability of 0.90 and at a 10% rate with a probability of 0.99. The CD137 (s4-1BB/CD137) using a Luminex-xMAP LEGENDplex cus- objective response was summarized with objective response rates tom assay (BioLegend), previously validated in the presence of uto- (ORR) and exact two-sided 95% CI calculated using the Clopper– milumab, as described (18). Pearson method. Time-to-event endpoints [duration of response (DOR), PFS] were analyzed using the Kaplan–Meier (KM) method. Biomarker association analyses Point estimates of KM rates and median times were presented with Flow marker data were compared in patients experiencing clinical 95% CIs. The CIs for the KM rates were calculated using log–log benefit, defined as complete or partial response and/or progression- transformation with back transformation to a CI on the untrans- free survival (PFS) >6 months, versus patients with PFS ≤6 months. formed scale. The CIs for the median were calculated according to the The comparison between responders and nonresponders was assessed Brookmeyer and Crowley method. using a Wilcoxon rank-sum test. P values were not corrected for multiple comparisons. The ROC curve was plotted using sensitivity versus specificity when sliding the cut-off on the marker value. The Results 95% confidence interval (CI) was estimated using bootstrapping Patients and treatment methods. All analyses were performed in R software 3.3.3. In this study, a total of 67 patients were treated with rituximab and Tumor and lymph node biopsies were collected for biomarker utomilumab at the dose levels of 0.03 (n ¼ 3), 0.06 (n ¼ 3), 0.12 (n ¼ 4), assessments when clinically feasible. IHC was performed on forma- 0.18 (n ¼ 3), 0.24 (n ¼ 3), 0.30 (n ¼ 3), 0.60 (n ¼ 4), 1.2 (n ¼ 32), 2.4 (n lin-fixed paraffin-embedded sections (Mosaic Laboratories) using anti- ¼ 3), 5.0 (n ¼ 5), and 10.0 (n ¼ 4) mg/kg every 4 weeks. The 1.2 mg/kg bodies specific for PD-L1 (E1L3N; Cell Signaling Technology), CD8 every 4 weeks group included patients treated at this dose level in dose (C8/144B; Dako), FoxP3 (236A/E7; Abcam), pSTAT3 (D3A7, Cell escalation and patients with rituximab-refractory FL enrolled in the Signaling Technology), CD3 (Leica), granzyme B (GrB-7; Dako), and dose-expansion cohort. Rituximab-refractory disease was defined as perforin (5B10; Leica). Assay results were evaluated by image analysis no response to a rituximab-containing regimen or disease progression usingwhole-slidescans(AperioImageScope;Leica)exceptforgranzyme within 6 months following the last dose of rituximab. One patient was B and perforin, which were assessed on manually selected 20 fields. enrolled but discontinued, after a repeat biopsy showed no lymphoma Peripheral blood samples for RNA-sequencing (RNA-seq) analysis recurrence. were collected on day 1, Cycle 1 (prior to the first utomilumab dose) in Thirty-eight (56.7%) patients were men and 29 (43.3%) were RNA PAXgene tubes (PreAnalytiX; Qiagen/BD Bioscience) and main- women, with a mean age of 62.3 (range 38–84) years. Thirty-one tained at 80C until processing. RNA-seq was performed using (46.3%) patients were ≥65 years of age. Primary cancer diagnoses are TruSeq Stranded mRNA kits on a HiSeq 2500 platform (Illumina). listed in Table 1; the majority of patients (70.1%) had FL. More than

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Table 1. Patient demographics and baseline characteristics. Table 2. Utomilumab treatment-related adverse events reported in >2 patients (all cycles). Utomilumab N ¼ 67 AEa, N ¼ 67 All grades Grade 1 Grade 2 Grade 3

Male : female, n (%) 38 (56.7) : 29 (43.3) Any AE 35 (52.2) 25 (37.3) 8 (11.9) 2 (3.0)b Median age, years (range) 63 (38–84) Fatigue 11 (16.4) 8 (11.9) 3 (4.5) 0 ≥65 years, n (%) 31 (46.3) Nausea 4 (6.0) 4 (6.0) 0 0 Race, n (%) Diarrhea 3 (4.5) 2 (3.0) 0 1 (1.5) White 51 (76.1) Black 2 (3.0) aNo utomilumab treatment-related grade 4 to 5 AEs were reported. Asian 6 (9.0) bOne (1.5%) patient developed grade 3 neutropenia. Other 8 (11.9) Primary cancer, n (%)a FL 47 (70.1) DLBCL 7 (10.4) milumab treatment-related AEs of any grade. The majority of the MCL 6 (9.0) treatment-related AEs were grade 1 to 2 and the most common SLL/CLL 3 (4.5) treatment-related AE was fatigue (16.4%; Table 2). One patient each Marginal zone lymphoma 2 (3.0) (1.5%) developed treatment-related grade 3 neutropenia and grade 3 NLPHL 1 (1.5) diarrhea (both in the 1.2 mg/kg dose group). Most of the laboratory Other 1 (1.5) abnormalities were grade 1 to 2. A grade 3 increase in alanine Stage at diagnosis, n (%) aminotransferase (n ¼ 1, 1.5%; 2.4 mg/kg group) was the only grade IA 3 (4.5) 3 to 4 liver function test abnormality reported (Supplementary IB 1 (1.5) IIA 4 (6.0) Table S1). IIIA 14 (20.9) No patient experienced a treatment-related death. Fifty-two (77.6%) IIIB 4 (6.0) patients discontinued treatment with utomilumab, due to disease IVA 29 (43.3) progression (n ¼ 38, 56.7%), global deterioration of health status IVB 10 (14.9) (n ¼ 4, 6.0%), AE (n ¼ 3, 4.5%), unwillingness to continue on the study ECOG PS, n (%) (n ¼ 3, 4.5%), or other reason (n ¼ 4, 6.0%). No patient had a dose 0 44 (65.7) reduction for utomilumab; 5 (7.5%) patients had one or two dose 1 23 (34.3) reductions for rituximab. The median duration of treatment was 5.0 Prior anticancer radiation therapy, n (%) (range, 1–27) cycles for utomilumab across all dose levels and 4.0 Yes 14 (20.9) (range 1–6) cycles for rituximab. No 53 (79.1) Prior systemic anticancer treatment, n (%) No 0 Pharmacokinetics and immunogenicity Yes 67 (100) Utomilumab exposure [area under the serum concentration–time C 1 13 (19.4) curve (AUC) and maximum observed serum concentration ( max)] 2 17 (25.4) increased in a dose-dependent manner across all the doses evaluated 3 8 (11.9) (Supplementary Table S2). Three (4.5%) of 66 patients had positive >3 29 (43.3) ADAs against utomilumab at baseline, likely due to preexisting host Response to prior rituximab therapy, n (%) antibodies that were cross-reactive with utomilumab. One (1.5%) Refractory 43 (64.2) patient developed treatment-induced ADAs and exhibited positive Relapsed 6 (9.0) Other/not reported 18 (26.9) NAbs against utomilumab. None of the patients had treatment- Subsequent anticancer therapy, n (%) boosted ADAs. Yes 24 (35.8) No 2 (3.0) Clinical activity Not reported 41 (61.2) Best overall response (BOR) of complete response (CR) or partial response (PR) was observed in patients with FL (4 CRs and 7 PRs) and Abbreviation: ECOG PS, ECOG performance status score. mantle cell lymphoma (MCL), DLBCL, or nodular lymphocyte- a One patient was enrolled but discontinued, after a repeat biopsy showed no predominant Hodgkin lymphoma (NLPHL; one PR each). All CRs lymphoma recurrence. and most PRs occurred at dose levels of utomilumab ≤1.2 mg/kg (Fig. 1). The ORR across all dose levels of utomilumab evaluated in the half of the patients had stage IV at diagnosis (58.2%), ECOG perfor- study was 21.2% (95% CI, 12.1%–33.0%; n ¼ 66; Table 3). Among mance status 0 (65.7%), and had received ≥3 prior systemic anticancer patients with rituximab-refractory FL, 4 patients achieved a CR and 5 therapies (55.2%). patients had a PR, as BOR (Fig. 1). The median time to response was 2.1 (range, 1.9–7.4) months, median DOR was 20.3 [95% CI, 2.6–not Safety estimable (N.E.)] months, and median PFS for all treated patients with None of the patients experienced a DLT event at the utomilumab NHL (n ¼ 66) was 4.6 (95% CI, 3.9–7.5) months. Overall, 28 (42.4%) doses evaluated (0.03–10 mg/kg). The MTD for utomilumab in patients achieved stable disease (SD) as BOR and 21 (31.8%) had combination with rituximab was not reached and estimated to be progressive disease. Three (4.5%) patients were not evaluable for BOR ≥10 mg/kg per the modiﬁed TITE-CRM design. owing to the lack of post-baseline disease assessments, due to death. Among all patients, 64 (95.5%) patients had at least one treatment- One patient with granuloma (and no active disease at baseline) was emergent all-causality AE and 35 (52.2%) patients experienced uto- excluded from efﬁcacy evaluations.

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Figure 1. A, Waterfall plot of best percentage change from baseline in target lesions A 60 for patients with CD20þ NHL. One patient with granuloma was excluded 40 from the analysis. Utomilumab was administered at the doses indicated 20 in combination with rituximab SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD PR PR PR PR PRCR PR PR PR PR CR 2 (375 mg/m ). B, Duration of treatment 0 SD SD SD SD SD SD in responders with CD20þ NHL. Trian- gles indicate ﬁrst CR, squares indicate –20 ﬁrst PR, and circles indicate progressive disease. White bars indicate –40 patients with CR who completed the planned 8-month treatment. R, ritux- –60 imab-refractory. –80 Best change (%) from baseline –100

Uto 0.24 mg/kg

B 0.03 mg/kg (n = 1) 0.12 mg/kg (n = 1) 0.18 mg/kg (n = 2) 1.2 mg/kg (n = 8) 2.4 mg/kg (n = 1) 5 mg/kg (n = 1)

PR R-FL

PR MCL

PR FL

PR R-DLBCL

PR R-FL

> CR R-FL

CR R-FL

PR R-FL

PR NLPHL Individual patient data PR FL

PR R-FL

> CR R-FL

0 2 4 6 8 101214161820222426283032343638 Duration of treatment in months

þ Pharmacodynamics Fig. S1B). No relationship was noted between CD8 T-cell expansion Increases in circulating s4-1BB/CD137 were observed in individual and treatment dose or tumor response. patients following combination treatment, at utomilumab doses The reported connection between intratumoral CTLs and out- between 0.18 and 10 mg/kg (Supplementary Fig. S1A). Levels of s4- come following rituximab treatment suggested that the combina- 1BB/CD137 typically peaked at 50 to 100 hours after utomilumab tion of utomilumab and rituximab could be associated with dosing, returning to baseline in some but not all cases. An expansion in increased CTL activity in the tumor (29, 30). This possibility could þ circulating CD8 T cells was observed in some patients following be assessed using pre- and on-treatment, paired tumor biopsies combination treatment (utomilumab 0.06–10 mg/kg; Supplementary from a patient (1.2 mg/kg utomilumab plus rituximab group), who

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þ Table 3. Best overall response in patients with CD20 NHL. achieved a CR after 12 treatment cycles. IHC analysis showed þ increased CD8 T-cell infiltration, reduced PD-L1 expression, and a N ¼ 66 destruction of follicular architecture (Fig. 2A). Expression of þ n (%) 4-1BB/CD137 appeared to be high on PD-1 cells at baseline but CR 4 (6.1) was reduced following combination treatment. The staining pattern PR 10 (15.2) of cytotoxic proteins granzyme B and perforin in the baseline SD 28 (42.4) specimen was consistent with primary localization in cytoplasmic Objective progression 21 (31.8) granules (Fig. 2B), whereas it indicated redistribution to the plasma Not evaluable 3 (4.5) membrane in the paired on-treatment specimen. The changes in ORR (CR þ PR) 14 (21.2) staining pattern are consistent with the hypothesis that the objective (95% exact CI) (12.1, 33.0) benefit observed in this patient may have been mediated by increased, antitumor CTL activity. aOne patient with granuloma was excluded from efficacy evaluations.

Figure 2. Associations between clinical outcome and tumor biomarkers. A and B, On-treatment changes in lymphoid architecture and content versus baseline tumor biopsies, assessed by IHC in a patient with CR (utomilumab 1.2 mg/kg plus rituximab). A, CD8, PD-L1, and 4-1BB/PD-1. B, CD3, CD16, granzyme B, and perforin expression. 20 objective, 200 overall magniﬁ- cation. (Continued on the following page.)

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Figure 2. (Continued.) C, Expression of PD-L1, CD8, and other markers of immune activation in baseline tumors from patients with objective response and/or durable SD versus patients with objective progression.

Association of biomarkers with clinical outcome 8 days). Deconvolution analysis demonstrated significant correla- Associations between outcome and biomarkers in baseline tumor tions between gene signatures for lymphocytes and T cells and biopsies were investigated by IHC analysis of PD-L1, CD8, FoxP3, the corresponding subpopulations detected by flow cytometry pSTAT3, granzyme B, and perforin (Fig. 2C). A significant association (Supplementary Fig. S2). GSEA analysis identified gene sets char- þ þ between clinical benefit and biomarker (P < 0.05) was only observed acteristic of CD4 and CD8 T-cell subsets that were associated for elevated FoxP3, although similar trends were observed for the other with better PFS; in contrast, gene sets characteristic of monocytes, biomarkers as well. neutrophils, and some B-cell populations were associated with Prior analyses of circulating lymphocytes in patients with FL treated poorer PFS (Fig. 3C). Broader GSEA analysis identified additional with single-agent rituximab suggested a positive correlation between pathways characteristic of inflammation that were associated with systemic T-cell levels and response to therapy (31). In this study, a poorer PFS (Supplementary Fig. S3). significant, positive association was observed between clinical benefit þ (defined as CR/PR and/or PFS >6 months) and circulating CD8 central memory T cells at baseline [Wilcoxon rank-sum test P ¼ 0.002 Discussion (cycle 1, day 1); Fig. 3A]. ROC analysis with a 95% CI identified a This is a first-in-human study evaluating the 4-1BB/CD137 þ CD8 central memory T-cell cut-off that generated 68% specificity and agonist mAb utomilumab in combination with rituximab for the þ 76% sensitivity (dashed lines in Fig. 3B). The area under the ROC treatment of patients with relapsed/refractory FL and other CD20 curve was 0.80 (95% CI, 0.66–0.94). NHLs. The safety of the combination in this patient population The association between circulating lymphocytes and clinical was favorable and consistent with previous studies of utomilumab benefit was further examined using RNA-seq analysis of peripheral in patients with solid tumors (18,19),includingastudyof blood samples collected immediately prior to utomilumab dosing utomilumab in combination with the PD-1 inhibitor pembrolizu- (after two rituximab treatments administered over the previous mab, in which there were no DLTs or treatment-related grade 5

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Figure 3. Correlation of clinical outcome with circulating immune cell subsets at cycle 1, day 1 in patients with follicular lymphoma. A, Absolute numbers þ (per mL) of CD8 T central memory cells (CD3þCD8þCD45ROþCCR7þ)in patients before receiving the first dose of utomilumab. Clinical benefitis defined as CR, PR, or PFS >6 months. Wilcoxon rank-sum test, P ¼ 0.002. B, ROC curve analysis. The gray zone identifies 95% CI. Dashed lines indicate a cutoff with 68% specificity and 76% sensitivity. C, Association with survival of immune cell subsets from expression signatures. NES were derived from GSEA with genes preranked by HR from multivariate Cox proportional-hazards model stratified by dose levels and adjusting for age, sex, race, stage, and whether patients had progressed on prior rituximab-containing therapy. Gene sets assessed are from the LM22 signatures. Significant cell populations are highlighted in orange (FDR- adjusted q value <0.05).

AEs (19). As the highest dose of utomilumab evaluated in this The pharmacokinetic of utomilumab was linear in the 0.03 to study was 10 mg/kg, with no DLTs observed across the dose levels 10 mg/kg dose range when administered in combination with rituxi- evaluated, the MTD for utomilumab in combination with ritux- mab, which is similar to that previously observed with single-agent imab was estimated to be at least 10 mg/kg every 4 weeks in treatment (18). The 1.2 mg/kg every 4 weeks dose level was selected for patients with NHL. the expansion cohort based on prior ﬁndings that adequate exposure

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Utomilumab Plus Rituximab in CD20þ Non-Hodgkin Lymphoma

was achieved at utomilumab dose levels ≥0.24 mg/kg every 4 weeks and adaptive resistance, but the magnitude and/or location of the target modulation was detected in the 0.24 to 1.2 mg/kg every 4 weeks resistance mechanisms may have been inadequate to protect the dose range (18). tumor (36). Alternatively, as 4-1BB/CD137 can be induced on Administration of utomilumab plus rituximab was associated activated NK cells, utomilumab may have overcome this induced with a very low incidence (1.5%) of treatment-induced ADAs resistance through activation of NK cells, which could be less against utomilumab, different from previous studies with single- susceptible to down-modulatory mechanisms. In the patients with agent utomilumab or combined treatment with pembrolizumab in “hot” tumors that progress on therapy, the resistance mechanisms patients with solid malignancies, in which 42% to 65% of patients may contribute to disable the active immune response; in these developed ADAs against utomilumab (18, 19). In addition, only 1 patients, the addition of antagonists to the PD-1/PD-L1 pathway patient (1.5%) had treatment-induced utomilumab NAbs. Both may be needed for clinical benefit. þ patients with ADAs/NAbs did not receive rituximab past cycle 1 Associations between circulating CD8 T cells and clinical outcome and had grade 1 to 2 IRRs to rituximab. They received utomilumab were noted for single-agent utomilumab and a combination of uto- through cycle 8 and 24, respectively. B-cell depletion induced by milumab and pembrolizumab (18, 19) as well as with single-agent rituximab in the patients included in this study may have been rituximab (31); it is therefore unsurprising that similar associations responsible for the lower ADAs observed, compared with patients were observed with the combination of utomilumab and rituximab. treated with utomilumab in other clinical studies, as rituximab- The mechanistic basis for the inverse association between gene sig- containing regimens have been reported to reduce ADA responses natures of myeloid cell populations and inflammatory gene pathways to other therapeutic proteins (1, 18, 19, 32). requires further investigation. Elevated neutrophil-to-lymphocyte Combination treatment was associated with preliminary evidence ratios have been associated with poor prognosis in multiple cancer þ of clinical activity in patients with CD20 NHL, including relapsed/ settings (37). It remains to be determined whether the observations refractory FL following prior therapy with rituximab-containing regi- reported in this study are reflective of systemic immune dysfunction mens. In six of the nine responders with rituximab-refractory FL, that reduces the ability to maintain an effective antitumor immune response to utomilumab in combination with rituximab exceeded the response, or a generally poor prognosis for nonimmune as well as 6-month period used to define rituximab-refractory status following immune-based therapy. prior rituximab-containing therapy (33). Safety, pharmacokinetic, In other studies, single-agent activity has been observed with the pharmacodynamic, and clinical activity results from the expansion PI3K inhibitors idelalisib, copanlisib, and duvelisib, with ORRs of cohort provided multiple lines of evidence that utomilumab at 1.2 mg/- 50% and PFS of 1 year in relapsed/refractory FL (38–40). However, kg every 4 weeks in combination with rituximab was clinically active treatment was associated with significant toxicity, including hepatitis, and well tolerated. Therefore, this dose was chosen as the RP2D for gastrointestinal toxicity, risk of infections, and cytopenias. The ORR þ utomilumab in patients with CD20 NHL. reported with the Bruton's kinase inhibitor ibrutinib in a phase II trial The first dose of utomilumab was administered after the second of was just 16.7% in patients with refractory FL (41). A similar ORR 4 weekly doses of rituximab based on the hypothesis that 4-1BB/ (20.9%) was observed with single-agent ibrutinib in the larger, single- CD137 agonism would be most effective after a period of time, to allow arm, phase II DAWN study conducted in patients with relapsed/ for innate and adaptive immune activation by rituximab. Pharmaco- refractory FL who had received ≥2 prior lines of chemoimmunother- dymamic changes consistent with 4-1BB/CD137 agonism were apy (42). Dose-finding, phase I studies evaluating a triple combination observed, including increases in s4-1BB/CD137 levels and expansion of the PI3K-d inhibitor idelalisib with lenalidomide and rituximab in þ of CD8 T cells in the circulation as seen in prior studies (17, 18). patients with relapsed/refractory FL or MCL were terminated due to Analysis of paired biopsies from a patient with relapsed/refractory FL, the excessive toxicity observed (43). Other investigational combina- who achieved a CR following combination treatment, showed tions have been explored for the treatment of patients with relapsed/ increased T-cell infiltration and cytotoxic activity in tumors, consistent refractory FL. In a phase Ib study, administration of rituximab and the with findings in murine tumor models following agonistic engagement anti-CD47 antibody Hu5F9-G4 (which enhances antibody-dependent of 4-1BB/CD137 (34). Because only one combination schedule was tumor cell phagocytosis by macrophages) was associated with an ORR evaluated, the relative contributions of rituximab and utomilumab to of 71% (CR 43%) in a small number of patients with FL (44). Treatment these effects cannot be distinguished in this study. with lenalidomide plus rituxumab in phase II to III trials demonstrated Analyses of both tumor and blood biomarkers suggested that T-cell ORRs of 76% to 77% in relapsed/refractory FL and significant immunity is a component of response to the combination but may be improvement in PFS, while combination of lenalidomide with obi- limited by factors in the local tumor microenvironment or in the nutuzumab was associated with an ORR of 63% in an early systemic circulation (35). Tumor biopsies from the study patients study (45–47). could generally be assigned to two categories: immunologically “hot,” Initial results from a phase Ib study of the anti PD-1 antibody þ þ which contained elevated proportions of CD8 and PD-L1 cells, and nivolumab showed a 40% ORR (10% CR rate) in hematologic immunologically “cold,” which did not (36). Objective responses were malignancies and durable responsesinafewpatientswith observed primarily in patients whose tumors had the “hot” phenotype, relapsed/refractory FL (48). Subsequent evaluations estimated a although this phenotype was also observed in patients with progressive response rate of 10% with anti–PD-1 monotherapy in relapsed/ disease. Patients with “cold” tumors generally showed progressive refractory FL (49). Preliminary activity was reported in a phase I/IIa disease. These results are consistent with previous studies of patients combination study of nivolumab and ibrutinib in patients with treated with rituximab monotherapy or rituximab plus IFN-a2a, in relapsed FL (ORR, 33%; ref. 50). which an association between favorable outcome and tumor- In conclusion, the favorable safety and tolerability profile of uto- infiltrating T cells was noted (31). In this study, clinical benefit could milumab in combination with rituximab and the preliminary evidence be observed in some patients even in the presence of immunosup- of clinical activity observed in patients with rituximab-refractory FL pressive biomarkers such as PD-L1 and FoxP3. It is possible that in support further evaluation of this combination, especially in patients these patients an active, antitumor immune response induced requiring treatment regimens with reduced toxicity. A subset of

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Gopal et al.

þ patients with elevated, tumor-associated CD8 T cells that did not Analysis and interpretation of data (e.g., statistical analysis, biostatistics, respond to the combination had elevated PD-L1 expression in the computational analysis): A.K. Gopal, R. Levy, X.J. Mu, S. Deng, K.A. Ching, tumor. It is possible that in these patients PD-L1 is protecting the Y. Chen, C.B. Davis, B. Huang, K.D. Fly, A. Thall, A. Woolfson, N.L. Bartlett Writing, review, and/or revision of the manuscript: A.K. Gopal, R. Levy, R. Houot, tumor from immune attack and that the addition of PD-1/PD-L1 S.P. Patel, L. Popplewell, C. Jacobson, X.J. Mu, S. Deng, Y. Chen, C.B. Davis, B. Huang, inhibitors may restore the immune response and associated clinical K.D. Fly, A. Thall, A. Woolfson, N.L. Bartlett benefit. As utomilumab was well tolerated in combination with a Administrative, technical, or material support (i.e., reporting or organizing data, PD-1 inhibitor, this hypothesis is being tested in an ongoing study constructing databases): R. Levy, K.A. Ching, B. Huang, K.D. Fly with a triplet combination of utomilumab, rituximab, and a PD-L1 Study supervision: A.K. Gopal, R. Levy, B. Huang, K.D. Fly, A. Thall, A. Woolfson inhibitor in patients with FL (NCT03636503). Data Sharing Statement Disclosure of Potential Conflicts of Interest Upon request, and subject to certain criteria, conditions and exceptions (see https://www.pfizer.com/science/clinical-trials/trial-data-and-results for more infor- A.K. Gopal is an employee/paid consultant for Actinium, I-MAB, Asana, Bristol- mation), Pfizer will provide access to individual de-identified participant data from Myers Squibb, Amgen, Takeda, BRIM bio, Seattle Genetics, Janssen, Aptevo, Gilead, Pfizer-sponsored global interventional clinical studies conducted for medicines, Incite, Bayer, Pharmacyclics, and Astra Zeneca, and reports receiving other com- vaccines and medical devices (1) for indications that have been approved in the US mercial research support from Merck, Seattle Genetics, Takeda, Janssen, Pfizer, Agios, and/or EU or (2) in programs that have been terminated (i.e., development for all IgM, Teva, Gilead, and Bristol-Myers Squibb. R. Levy is an employee/paid consultant indications has been discontinued). Pfizer will also consider requests for the protocol, for Five Prime, BeiGene, Teneobio, Sutro, Checkmate, Nurix, Dragonfly, Abpro, data dictionary, and statistical analysis plan. Data may be requested from Pfizer trials Apexigen, Viracta, Forty Seven Inc., Spotlight, XCella, Immunocore, and Walking 24 months after study completion. The de-identified participant data will be made Fish, and reports receiving commercial research grants from Bristol-Myers Squibb available to researchers whose proposals meet the research criteria and other and Janssen. S.P. Patel is an employee/paid consultant for AstraZeneca, Bristol-Myers conditions, and for which an exception does not apply, via a secure portal. To gain Squibb, Lilly, Illumina, Rakuten, Paradigm, and Tempus. C. Jacobson is an employee/ access, data requestors must enter into a data access agreement with Pfizer. paid consultant for Kite, Novartis, Celgene, Bristol-Myers Squibb, Precision Bios- ciences, Nkarta, and Humanigen, reports receiving other commercial research support from Pfizer, speakers bureau honoraria from AXIS and Clinical Care Options, Acknowledgments fi and other remuneration from Weitz Lux. X.J. Mu is an employee/paid consultant for This study was sponsored by P zer. The authors thank the patients and their and reports receiving commercial research grants from Pfizer Inc. K.A. Ching, C.B. families/caregivers, and the investigators, research nurses, study coordinators, and Davis, B. Huang, and A. Thall are employees/paid consultants for Pfizer Inc. S. Deng, operations staff who contributed to this study; C. Deshpande for preparation of the Y. Chen, and K.D. Fly are employees/paid consultants for and hold ownership interest biomarker data included in the association analyses; and E. Negre for data recon- (including patents) in Pfizer Inc. No potential conflicts of interest were disclosed by ciliation and validation efforts. A.K. Gopal received philanthropic support from Frank the other authors. and Betty Vandermeer and from Sonya and Tom Campion. This work is dedicated to the memory of Holbrook Kohrt, MD, PhD. Medical writing and editorial support was provided by S. Mariani, MD, PhD, of Engage Scientific Solutions and was funded by Authors’ Contributions Pfizer. Conception and design: A.K. Gopal, R. Levy, R. Houot, Y. Chen, C.B. Davis, B. Huang, A. Thall, A. Woolfson, N.L. Bartlett The costs of publication of this article were defrayed in part by the payment of page Development of methodology: A.K. Gopal, R. Levy, Y. Chen, C.B. Davis, B. Huang, charges. This article must therefore be hereby marked advertisement in accordance A. Thall, A. Woolfson with 18 U.S.C. Section 1734 solely to indicate this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.K. Gopal, R. Houot, S.P. Patel, L. Popplewell, C. Jacobson, Y. Chen, Received October 17, 2019; revised February 17, 2020; accepted March 3, 2020; C.B. Davis, B. Huang, K.D. Fly, A. Thall, A. Woolfson, N.L. Bartlett published first March 6, 2020.

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First-in-Human Study of Utomilumab, a 4-1BB/CD137 Agonist, in Combination with Rituximab in Patients with Follicular and Other CD20+ Non-Hodgkin Lymphomas

Ajay K. Gopal, Ronald Levy, Roch Houot, et al.

Clin Cancer Res Published OnlineFirst March 6, 2020.

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