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Supplementary Figure 10 shows the selectivity of sasanlimab binding to human and cynomolgus monkey PD-1 using HEK-293T transiently transfected cells and flow cytometry

Development of antagonistic mAbs that specifically target the immune checkpoint receptor, programmed cell death protein-1 (PD-1), is of great interest for cancer immunotherapy. Here, we report the biophysical characteristics and nonclinical antagonistic activities of sasanlimab (PF-06801591), a humanized anti-PD-1 antibody of IgG4 isotype. We show that sasanlimab binds selectively and with similar high potency to human and cynomolgus monkey PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, with no detectable Fc-dependent effector function. The binding of sasanlimab to human and cynomolgus PD-1 is associated with the formation of a stable complex, which is likely to be the main driver of this high-affinity interaction. In vitro, sasanlimab significantly augmented T-cell proliferation and cytokine production in mixed lymphocyte reaction and superantigen stimulation assays. In vivo, sasanlimab accelerated the incidence of GvHD by enhancing T-cell proliferation and cytokine secretion in a xenogeneic model of acute GvHD and halted the growth of MC-38 colon adenocarcinoma tumors in human PD-1 knock-in mice. Pharmacokinetic and toxicokinetic findings from cynomolgus monkey showed that sasanlimab was active and well-tolerated. Taken together, the data presented here support the clinical development of sasanlimab for the treatment of patients with advanced cancers as a single agent or in combination with other immunotherapies.

CCR Translation on this Article from C19orf48 Encodes a Minor Histocompatibility Antigen Recognized by CD8+ Cytotoxic T Cells from Renal Cell Carcinoma Patients

Purpose: Tumor regression has been observed in some patients with metastatic renal cell carcinoma (RCC) after nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Cellular and molecular characterization of antigens recognized by tumor-reactive T cells isolated from responding patients could potentially provide insight into the mechanisms of tumor regression.Experimental Design: CD8+ CTL clones that recognized a novel RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201 were isolated from two patients with metastatic RCC who experienced tumor regression or stable disease following nonmyeloablative allogeneic HCT. These clones were used to screen a cDNA library and isolate the unique cDNA encoding the antigen.Results: An alternative open reading frame in the C19orf48 gene located on chromosome 19q13 encodes the HLA-A*0201–restricted minor H antigen recognized by the RCC-reactive T cells. The differential T-cell recognition of donor- and recipient-derived target cells is attributable to a nonsynonymous single-nucleotide polymorphism within the nucleotide interval that encodes the antigenic peptide. Assays for gene expression and CTL recognition showed that the C19orf48-encoded peptide is widely expressed in renal tumors and solid tumors of other histologies. The antigenic peptide can be processed for CTL recognition via both TAP-dependent and TAP-independent pathways.Conclusions: Donor T-cell responses against the HLA-A*0201–restricted minor H antigen encoded by C19orf48 may contribute to RCC regression after MHC-matched allogeneic HCT.

Purpose:Stimulation of effector T cells is an appealing immunotherapeutic approach in oncology. OX40 (CD134) is a costimulatory receptor expressed on activated CD4+ and CD8+ T cells. Induction of OX40 following antigen recognition results in enhanced T-cell activation, proliferation, and survival, and OX40 targeting shows therapeutic efficacy in preclinical studies. We report the monotherapy dose-escalation portion of a multicenter, phase I trial (NCT02315066) of ivuxolimab (PF-04518600), a fully human immunoglobulin G2 agonistic monoclonal antibody specific for human OX40.Patients and Methods:Adult patients (N = 52) with selected locally advanced or metastatic cancers received ivuxolimab 0.01 to 10 mg/kg. Primary endpoints were safety and tolerability. Secondary/exploratory endpoints included preliminary assessment of antitumor activity and biomarker analyses.Results:The most common all-causality adverse events were fatigue (46.2%), nausea (28.8%), and decreased appetite (25.0%). Of 31 treatment-related adverse events, 30 (96.8%) were grade ≤2. No dose-limiting toxicities occurred. Ivuxolimab exposure increased in a dose-proportionate manner from 0.3 to 10 mg/kg. Full peripheral blood target engagement occurred at ≥0.3 mg/kg. Three (5.8%) patients achieved a partial response, and disease control was achieved in 56% of patients. Increased CD4+ central memory T-cell proliferation and activation, and clonal expansion of CD4+ and CD8+ T cells in peripheral blood were observed at 0.1 to 3.0 mg/kg. Increased immune cell infiltrate and OX40 expression were evident in on-treatment tumor biopsies.Conclusions:Ivuxolimab was generally well tolerated with on-target immune activation at clinically relevant doses, showed preliminary antitumor activity, and may serve as a partner for combination studies.

Supplementary Data from A Phase I, Open-Label, Dose-Escalation Study of the OX40 Agonist Ivuxolimab in Patients with Locally Advanced or Metastatic Cancers

Supplementary Figures S1-S2 from C19orf48 Encodes a Minor Histocompatibility Antigen Recognized by CD8+ Cytotoxic T Cells from Renal Cell Carcinoma Patients

Background Sasanlimab is a monoclonal antibody that binds to the programmed cell death receptor 1 (PD-1). Anti-PD-1 monoclonal antibodies have improved patient clinical outcomes; however, not all treated patients derive clinical benefit. Further insights on potential biomarkers beyond PD-L1 expression levels would help to identify the patients most likely to respond to treatment. Objective This study evaluated tumor biopsies from patients treated with intravenous or subcutaneous sasanlimab to identify biomarkers of response and characterize pharmacodynamic activity. Methods Anti-PD-1/PD-ligand 1 (PD-L1)-naive patients with advanced solid tumors received sasanlimab intravenously at 1, 3, or 10 mg/kg every 3 weeks (n = 23) or subcutaneously at 300 mg every 4 weeks (n = 15). Best tumor percentage change from baseline was determined by RECIST. Whole-exome DNA and RNA sequencing were performed in tumor samples collected from treated patients at protocol-defined timepoints. PD-L1 and CD8 protein expression were evaluated in tumor biopsies by immunohistochemistry. Associations with response were assessed by linear regression analysis. Results Baseline tumor mutational burden (TMB), as well as PD-L1 and CD8 expression, were significantly associated with response to sasanlimab across the multiple dose levels, routes of administration, and range of tumor types evaluated. TMB is an independent biomarker from the various tumor inflammatory genes and signatures evaluated. Gene set enrichment analysis showed that higher baseline expression levels of genes related to the interferon-γ and PD-1 signaling pathways and the cell cycle were significantly associated with response to sasanlimab across tumor types. No differences were observed between routes of administration with regard to response to sasanlimab for the biomarkers of interest (TMB, PD-L1, CD8, and interferon-γ signature). Evaluation of pharmacodynamic changes showed increased tumor expression of genes enriched in adaptive immune response pathways. Conclusions Our findings indicate an active, immunomodulatory mechanism for the anti-PD-1 antibody sasanlimab across different tumor types and routes of administration. Trial Registration ClinicalTrials.gov identifier NCT02573259; registered October 2015. Supplementary Information The online version contains supplementary material available at 10.1007/s11523-021-00833-2.

BackgroundIvuxolimab (PF-04518600) and utomilumab (PF-05082566) are humanized agonistic IgG2 monoclonal antibodies against OX40 and 4-1BB, respectively. This first-in-human, multicenter, open-label, phase I, dose-escalation/dose-expansion study explored safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of ivuxolimab+utomilumab in patients with advanced solid tumors.MethodsDose-escalation: patients with advanced bladder, gastric, or cervical cancer, melanoma, head and neck squamous cell carcinoma, or non-small cell lung cancer (NSCLC) who were unresponsive to available therapies, had no standard therapy available or declined standard therapy were enrolled into five dose cohorts: ivuxolimab (0.1–3 mg/kg every 2 weeks (Q2W)) intravenously plus utomilumab (20 or 100 mg every 4 weeks (Q4W)) intravenously. Dose-expansion: patients with melanoma (n=10) and NSCLC (n=20) who progressed on prior anti-programmed death receptor 1/programmed death ligand-1 and/or anti-cytotoxic T-lymphocyte-associated antigen 4 (melanoma) received ivuxolimab 30 mg Q2W intravenously plus utomilumab 20 mg Q4W intravenously. Adverse events (AEs) were graded per National Cancer Institute Common Terminology Criteria for Adverse Events V.4.03 and efficacy was assessed using Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1 and immune-related RECIST (irRECIST). Paired tumor biopsies and whole blood were collected to assess pharmacodynamic effects and immunophenotyping. Whole blood samples were collected longitudinally for immunophenotyping.ResultsDose-escalation: 57 patients were enrolled; 2 (3.5%) patients with melanoma (0.3 mg/kg+20 mg and 0.3 mg/kg+100 mg) achieved partial response (PR), 18 (31.6%) patients achieved stable disease (SD); the disease control rate (DCR) was 35.1% across all dose levels. Dose-expansion: 30 patients were enrolled; 1 patient with NSCLC achieved PR lasting >77 weeks. Seven of 10 patients with melanoma (70%) and 7 of 20 patients with NSCLC (35%) achieved SD: median (range) duration of SD was 18.9 (13.9–49.0) weeks for the melanoma cohort versus 24.1 (14.3–77.9+) weeks for the NSCLC cohort; DCR (NSCLC) was 40%. Grade 3–4 treatment-emergent AEs were reported in 28 (49.1%) patients versus 11 (36.7%) patients in dose-escalation and dose-expansion, respectively. There were no grade 5 AEs deemed attributable to treatment. Ivuxolimab area under the concentration–time curve increased in a dose-dependent manner at 0.3–3 mg/kg doses.ConclusionsIvuxolimab+utomilumab was found to be well tolerated and demonstrated preliminary antitumor activity in selected groups of patients.Trial registration numberNCT02315066.

Recently approved therapies have contributed to a significant progress in the management of ovarian cancer; yet, more options are needed to further improve outcomes in patients with advanced disease. Here we review the rationale and ongoing clinical trials of novel combination strategies involving chemotherapy, poly ADP ribose polymerase, programmed death 1 (PD-1)/PD-ligand 1 immune checkpoint and/or vascular endothelial growth factor receptor inhibitors. Further, we discuss novel agents aimed at targets associated with ovarian cancer growth or progression that are emerging as potential new treatment approaches. Among them, agents targeted to folate receptor α, tissue factor, and protein kinase-mediated pathways (WEE1 kinase, phosphatidylinositol-3 kinase α, cell cycle checkpoint kinase 1/2, ATR kinase) are currently in clinical development as mono- or combination therapies. If successful, findings from these extensive development efforts may further transform treatment of patients with advanced ovarian cancer.

BACKGROUND: Previously, we showed that abnormal levels of bioactive lipids in bronchoalveolar lavage fluid (BALF) from infants with cystic fibrosis (CF) correlated with early structural lung damage. METHOD: To extend these studies, BALF bioactive lipid measurement by mass spectrometry and chest computed tomography (CT, combined with the sensitive PRAGMA-CF scoring method) were performed longitudinally at 2-year intervals in a new cohort of CF children (n=21, aged 1–5 yrs). RESULTS: PRAGMA-CF, neutrophil elastase activity, and myeloperoxidase correlated with BALF lysolipids and isoprostanes, markers of oxidative stress, as well as prostaglandin E2 and combined ceramide precursors (Spearman’s Rho >0.5; P

The increasing use of multiple immunomodulatory (IMD) agents for cancer therapies (e.g. antibodies targeting immune checkpoints, bispecific antibodies, and chimeric antigen receptor [CAR]-T cells), is raising questions on their potential immunogenicity and effects on treatment. In this review, we outline the mechanisms of action (MOA) of approved, antibody-based IMD agents, potentially related to their immunogenicity, and discuss the reported incidence of anti-drug antibodies (ADA) as well as their clinical relevance in patients with cancer. In addition, we discuss the impact of the administration route and potential strategies to reduce the incidence of ADA and manage treated patients. Analysis of published reports indicated that the risk of immunogenicity did not appear to correlate with the MOA of anti-programmed death 1 (PD-1)/PD-ligand 1 monoclonal antibodies nor to substantially affect treatment with most of these agents in the majority of patients evaluated to date. Treatment with B-cell depleting agents appears associated with a low risk of immunogenicity. No significant difference in ADA incidence was found between the intravenous and subcutaneous administration routes for a panel of non-oncology IMD antibodies. Additionally, while the data suggest a higher likelihood of immunogenicity for antibodies with T-cell or antigen-presenting cell (APC) targets versus B-cell targets, it is possible to have targets expressed on APCs or T cells and still have a low incidence of immunogenicity. Electronic supplementary material The online version of this article (10.1186/s40425-019-0586-0) contains supplementary material, which is available to authorized users.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of various cancers by reversing the immunosuppressive mechanisms employed by tumors to restore anticancer immunity. Although ICIs have demonstrated substantial clinical efficacy, patient response can vary in depth and duration, and many do not respond at all or eventually develop resistance. ICI resistance mechanisms can be tumor-intrinsic, related to the tumor microenvironment or patient-specific factors. Multiple resistance mechanisms may be present within one tumor subtype, or heterogeneity exists among patients with the same tumor type. Consequently, designing effective combination treatment strategies is challenging. This review will discuss ICI resistance mechanisms, and summarize findings from key preclinical and clinical trials of ICIs, to identify potential treatment strategies or pathways to overcome ICI resistance.

Development of antagonistic mAbs that specifically target the immune checkpoint receptor, programmed cell death protein-1 (PD-1), is of great interest for cancer immunotherapy. Here, we report the biophysical characteristics and nonclinical antagonistic activities of sasanlimab (PF-06801591), a humanized anti-PD-1 antibody of IgG4 isotype. We show that sasanlimab binds selectively and with similar high potency to human and cynomolgus monkey PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, with no detectable Fc-dependent effector function. The binding of sasanlimab to human and cynomolgus PD-1 is associated with the formation of a stable complex, which is likely to be the main driver of this high-affinity interaction. In vitro, sasanlimab significantly augmented T-cell proliferation and cytokine production in mixed lymphocyte reaction and superantigen stimulation assays. In vivo, sasanlimab accelerated the incidence of GvHD by enhancing T-cell proliferation and cytokine secretion in a xenogeneic model of acute GvHD and halted the growth of MC-38 colon adenocarcinoma tumors in human PD-1 knock-in mice. Pharmacokinetic and toxicokinetic findings from cynomolgus monkey showed that sasanlimab was active and well-tolerated. Taken together, the data presented here support the clinical development of sasanlimab for the treatment of patients with advanced cancers as a single agent or in combination with other immunotherapies.

Background: Clinicians have observed preterm infants in the neonatal intensive care unit growing disproportionally; however, the only growth charts that have been available were from preterm infants born in the 1950s which utilized the ponderal index. Prior to creating the recently published BMI curves, we found only 1 reference justifying the use of the ponderal index. Objectives: To determine the best measure of body proportionality for assessing growth in US preterm infants. Methods: Using a dataset of 391,681 infants, we determined the body proportionality measure that was most correlated with weight and least correlated with length. We examined the sex-specific overall correlations and then stratified further by gestational age (GA). We then plotted the body proportionality measures versus length to visualize apparent discrepancies in the appropriate measure. Results: The overall correlations showed weight/length3 (ponderal index) was the best measure but stratification by GA indicated that BMI (weight/length2) was the best measure. This seeming inconsistency was due to negative correlations between ponderal index and length at each GA. BMI, on the other hand, had a correlation with length across GAs, but was uncorrelated with length within GAs. Both ponderal index and BMI were positively correlated with weight. Conclusions: BMI is the appropriate measure of body proportionality for preterm infants, contrary to current practice.

Introduction: PF-06863135 (PF-3135) is a full-length, humanized, bispecific monoclonal antibody (mAb, IgG2a) targeting BCMA, which is highly expressed on multiple myeloma cells, and CD3 expressed on T cells. Intravenous (IV) dosing of PF-3135 has been previously evaluated at 0.1 - 50 μg/kg weekly with preliminary evidence of anti-myeloma activity in RRMM, without reaching the maximum tolerated dose (MTD). SC dose escalation was subsequently initiated given the potential to reduce the maximum concentration (Cmax), which is believed to be associated with cytokine release syndrome (CRS) and inflammatory response, with the intent of achieving a more favorable therapeutic window. Methods: Patients with RRMM previously treated with immunomodulatory drugs, proteasome inhibitors, and anti-CD38 mAb received SC dosages of PF-3135 at 80, 130, 215, and 360 μg/kg weekly. A modified toxicity probability interval method was used for dose escalation, with dose-limiting toxicities (DLTs) monitored in the first cycle (21 days). AEs were graded by CTCAE criteria except CRS, which was graded by Lee et al. 2014 criteria. Responses were assessed at the end of each cycle by the International Myeloma Working Group (IMWG) criteria. Peripheral blood was evaluated for soluble BCMA, cytokines, and immunophenotyping. PF-3135 pharmacokinetics and immunogenicity were also characterized. Results: As of April 15, 2020, 18 patients had received PF-3135 SC at 80 (n=6), 130 (n=4), 215 (n=4) and 360 (n=4) μg/kg weekly. The median number of prior anticancer treatment regimens was 7; all patients had received prior daratumumab therapy; 4 (22%) patients had received prior BCMA-targeted antibody-drug conjugate or chimeric antigen receptor T-cell therapy. All patients completed the DLT-monitoring period and none were observed. At cut-off date, dose escalation was ongoing with MTD not reached. All patients experienced treatment-emergent AEs (TEAEs); the most common were CRS reported in 11 (61%) patients, anemia in 9 (50%), thrombocytopenia in 7 (39%), and injection site reaction and lymphopenia in 6 (33% each). Grade (G) 3-4 TEAEs were observed in 12 (67%) patients, including G3 anemia in 8 (44%), G3 thrombocytopenia, lymphopenia, neutropenia, hypoalbuminemia, and bone pain in 2 (11% each), G4 lymphopenia in 4 (22%), G4 thrombocytopenia in 3 (17%), and G4 neutropenia in 2 (11%) patients. G5 TEAEs occurring in 3 (17%) patients (80 μg/kg group) were considered not treatment-related (disease progression [n=2] and sepsis [n=1]). CRS, the most common treatment-related AE, was observed across all dose levels in 11 patients (61% overall; 50% at 80 and 130 µg/kg; 75% at 215 and 360 µg/kg). Of these, 9 (50%) patients experienced G1 CRS and 2 (11%) had G2 CRS. CRS began within the first 2 days of treatment, with a median duration of 2 days (range 0-4). Per IMWG criteria, the objective response rate was 33% in the overall SC-dosed population and 75% at the top 2 dose levels (215 and 360 μg/kg). Two patients each achieved a best response of partial response (PR), very good PR (VGPR), and stringent complete response (sCR). Seven patients had best response of stable disease. Exposure to PF-3135 increased with dose in an approximately dose-proportional manner. SC administration resulted in a lower dose-normalized Cmax relative to IV administration and a prolonged absorption phase, with median time to Cmax ranging from 3 to 7 days. Consistent with the observed CRS, cytokine increases were the highest following the first dose and diminished significantly at subsequent doses. Levels of free, soluble BCMA decreased with increasing doses and demonstrated sustained decreases throughout each dose interval. Conclusions: AEs were generally manageable; CRS reported in the majority of patients was grade 1-2. Responses were achieved with SC dosing of PF-3135 in 6 of 8 (75%) patients at the 2 highest dose levels evaluated. Compared to IV, SC dosing did modulate the Cmax, so higher doses could be administered without observing increased severity of CRS. SC dose escalation is ongoing and additional results, including biomarkers and data from higher dose cohorts, will be presented. Clinicaltrials.gov identifier: NCT03269136. Funding: Pfizer. Disclosures Lesokhin: Janssen: Research Funding; Genentech: Research Funding; Takeda: Consultancy, Honoraria; Juno: Consultancy, Honoraria; GenMab: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Serametrix Inc.: Patents & Royalties. Levy:Baylor University Med Center: Current Employment; Seattle Genetics: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Bristol Meyers Squibb: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Research Funding, Speakers Bureau. Bahlis:Sanofi: Consultancy, Honoraria; BMS/Celgene and Janssen: Consultancy, Honoraria, Other: Travel, Accomodations, Research Funding; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm Therapeutics: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Solh:Amgen: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Speakers Bureau; Partner Therapeutics: Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding. Sebag:Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Research Funding. Jakubowiak:AbbVie, Amgen, BMS/Celgene, GSK, Janssen, Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive, Juno: Consultancy, Honoraria. Costello:Takeda: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Poseida Therapeutics: Research Funding; Janssen: Research Funding. Chu:BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Teva: Consultancy, Honoraria. Savona:Boehringer Ingelheim: Patents & Royalties; Ryvu: Consultancy; Astex: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Karyopharm: Consultancy, Current equity holder in publicly-traded company; Gilead: Consultancy; AbbVie: Consultancy; BMS: Consultancy; TG Therapeutics: Consultancy, Research Funding. Trudel:Sanofi: Honoraria; Takeda: Honoraria; Amgen: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; GSK: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; AstraZeneca: Honoraria; Karyopharm: Honoraria. Chou:Pfizer: Current Employment, Current equity holder in publicly-traded company. Udata:Pfizer: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Basu:Pfizer: Current Employment, Current equity holder in publicly-traded company. Krupka:Pfizer: Current Employment, Current equity holder in publicly-traded company. Raje:Immuneel: Membership on an entity's Board of Directors or advisory committees; Caribou: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Astrazeneca: Consultancy; Takeda: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Karyopharm: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Amgen: Consultancy. OffLabel Disclosure: BCMA/CD3 bi-specific antibody in development for the treatment of patients with relapsed/refractory multiple myeloma

More oncology biologics are becoming available for subcutaneous (sc.) administration and are expected to provide useful therapeutic options. We evaluated evidence published in the past 5 years to assess the humanistic and economic impact of sc. versus intravenous administration of approved cancer therapies and identify outcomes favoring either administration route. These publications focused predominantly on healthcare resource utilization and economic outcomes, demonstrating resource and cost savings with sc. administration. Patients reported a better health-related quality of life and preference for sc. formulations. Time-and-motion study analyses confirmed the convenience of sc. administration. These findings suggest that future availability of sc. oncology biologics, especially anti-PD-1/PD-ligand 1 antibodies due to their increased utility in various malignancies, may be beneficial for patients, healthcare providers and payers.

Purpose Combining immunotherapeutic agents with different mechanisms of action may enhance efficacy. We describe the safety and efficacy of talimogene laherparepvec (T-VEC; an oncolytic virus) in combination with ipilimumab (a cytotoxic T-lymphocyte–associated antigen 4 checkpoint inhibitor) in patients with advanced melanoma. Methods In this open-label, multicenter, phase Ib trial of T-VEC in combination with ipilimumab, T-VEC was administered intratumorally in week 1 (106 plaque-forming units/mL), then in week 4 and every 2 weeks thereafter (108 plaque-forming units/mL). Ipilimumab (3 mg/kg) was administered intravenously every 3 weeks for four infusions, beginning in week 6. The primary end point was incidence of dose-limiting toxicities. Secondary end points were objective response rate by immune-related response criteria and safety. Results Median duration of treatment with T-VEC was 13.3 weeks (range, 2.0 to 95.4 weeks). Median follow-up time for survival analysis was 20.0 months (1.0 to 25.4 months). Nineteen patients were included in the safety analysis. No dose-limiting toxicities occurred, and no new safety signals were detected. Grade 3/4 treatment-related adverse events (AEs) were seen in 26.3% of patients; 15.8% had AEs attributed to T-VEC, and 21.1% had AEs attributed to ipilimumab. The objective response rate was 50%, and 44% of patients had a durable response lasting ≥ 6 months. Eighteen-month progression-free survival was 50%; 18-month overall survival was 67%. Conclusion T-VEC with ipilimumab had a tolerable safety profile, and the combination appeared to have greater efficacy than either T-VEC or ipilimumab monotherapy.

Introduction: PF-06801591 is a humanized IgG4 monoclonal antibody that binds to the programmed cell death (PD-1) receptor and blocks its interaction with PD-1/2 ligands (PD-L1/2). In a phase 1 dose expansion study (NCT02573259), 106 patients with locally advanced or metastatic non-small cell lung cancer (NSCLC; n=68) or urothelial carcinoma (UC; n=38) were treated with PF-06891591 300 mg subcutaneously monthly. 12 patients with NSCLC (17.9%) and 7 with UC (18.4%) had a best overall response of partial response (PR). Response rates were higher in patients with high (≥1%) vs low ( Citation Format: Byoung Chul Cho, Konstantin Penkov, Igor Bondarenko, Joanna Pikiel, Hee Kyung Ahn, Alison Forgie, Ira Jacobs, Vinicius Bonato, Xinmeng Jasmine Mu, Keith Ching, Jeffrey Chou, Melissa L. Johnson. Molecular markers in a phase I dose expansion study of PF-06801591 in locally advanced or metastatic non-small-cell lung cancer and urothelial carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT138.

BCMA-CD3 bispecific antibody PF-06863135 is currently under evaluation in an ongoing Phase 1 clinical trial in relapsed/refractory (RR) multiple myeloma (MM) patients (NCT03269136). We showed previously that BCMA-CD3 bispecific has potent single agent activity in RR MM human bone marrow aspirates in vitro and in preclinical models of human myeloma (Panowski et al, MCT 2019). Herein we used in vitro assays, ex vivo MM patient 3D cultures, and in vivo efficacy models to study therapeutic combinations of PF-06863135 with immune checkpoint inhibitors, immunomodulatory drugs (IMiD), and gamma secretase inhibitors (GSI). Upregulation of immune checkpoints, such as programmed cell death protein-1 (PD-1) or programmed death ligand 1 (PD-L1) has been demonstrated downstream of response to CD3 bispecifics. While PF-06863135 is effective as a single agent in ex vivo 3D primary MM cultures (EC50 0.2 nM), PD-L1 was upregulated on myeloma cells. We combined PF-06863135 with anti-PD-1 in subcutaneous and orthotopic myeloma models MM.1S and MM.1S-PD-L1 in NSG mice engrafted with human T cells. PF-06863135 demonstrated single agent anti-tumor activity in the MM.1S models; however, in the MM.1S-PD-L1 model, PF-06863135 activity was blunted. When given in combination with an anti-PD-1 blocking antibody, the full thrust of the single agent anti-tumor activity of PF-06863135 was restored. Dysfunctional, senescent like T cells may emerge downstream of T cell immunotherapies. IMiDs, such as lenalidomide, are a standard of care therapy in MM and result in immunomodulatory effects on T cells leading us to hypothesize PF-06863135 mediated tumor growth inhibition could be enhanced by combining with lenalidomide. When PF-06863135 and lenalidomide were given in combination in the human T cell engrafted established orthotopic MM.1S and MOLP8 models, an even greater anti-tumor activity was observed as compared to either agent alone. These studies suggest that PF-06863135 activity can be augmented by combinations with immunomodulatory agents such as anti-PD-1 or lenalidomide. BCMA is shed from the surface of myeloma cells by gamma secretases, not only reducing cell surface target density but contributing to a soluble sink. When treated with GSI in vitro, myeloma cell surface BCMA expression increased concurrent with reduction in soluble/shed BCMA across a panel of myeloma and lymphoma cell lines expressing a range of BCMA. In cytotoxic T lymphocyte co-culture assays, the activity of PF-06863135 was potentiated by combining with GSI. Studies of the combination of PF-06863135 and GSI are ongoing in preclinical in vivo models of myeloma. Taken together, our preclinical studies provide insights into mechanisms of action and resistance for PF-06863135, which has potential for profound single agent activity that can be enhanced with therapeutic combinations. Citation Format: Katarzyna Karwacz, Andrea T. Hooper, Chao-Pei Betty Chang, Heike Krupka, Jeffrey Chou, Viola Lam, Ivana Djuretic, Javier Chaparro-Riggers, Puja Sapra. BCMA-CD3 bispecific antibody PF-06863135: Preclinical rationale for therapeutic combinations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4557.

523 Background: PF-04518600 (PF-8600) is a humanized agonist IgG2 monoclonal antibody to the tumor necrosis factor superfamily receptor OX40. PF-8600 was given to patients (pts) with advanced/metastatic hepatocellular carcinoma (HCC) in dose expansion of a phase 1 study (NCT02315066). Safety and tolerability were primary endpoints and exploratory endpoints included biomarker analyses. Methods: Pts received either 30 mg (Arm 1) or 250 mg (Arm 2) of PF-8600 intravenously Q2W. Pts had pathologic diagnosis of advanced HCC, a Child-Pugh score of A or B7, and had ≤2 prior lines of therapy, or if treatment naïve, had declined standard of care. Radiological tumor assessments were conducted Q8W. Biopsy samples collected at baseline and wk 6 were analyzed by immunohistochemistry and RNA sequencing for pharmacodynamic (PD) analyses. Whole blood samples were collected longitudinally for DNA extraction for high-throughput sequencing of the T cell receptor β-chain. Results: Arm 1 enrolled 16 pts (mean age 65.6 yrs; range 54-81 yrs; prior PD-1/PD-L1: 5 pts) and Arm 2 enrolled 19 pts (mean age 61.7 yrs; range 26-79 yrs; prior PD-1/PD-L1: 3 pts). All grade treatment related adverse events (TRAEs) occurred in 69% of pts in Arm 1 and 58% of pts in Arm 2. The rate of ≥Grade 3 TRAEs was 31% and 16% in Arms 1 and 2, respectively. For both arms combined, the most common (≥10%) TRAEs were rashes and pruritus. In Arm 1, 8 pts (50%) and in Arm 2, 10 pts (53%) achieved stable disease (SD), with a mean duration of 18.4 (range: 14.0-30.3 wks) and 17.4 wks (range: 8.0-31.9 wks), respectively. PD effects were more evident in Arm 1 than Arm 2, including increased OX40 tumor expression and positive changes in gene signatures, reflecting an active anti-tumor immune response. Conclusions: PF-8600 was generally well tolerated and provided meaningful disease control. While safety and efficacy were not significantly different between the 2 doses, there were potential differences in the PD data. The safety and relative durability of SD in HCC pts may provide a rationale for exploration of combination therapy in this pt population. Clinical trial information: NCT02315066.

Background The first-in-human, ongoing phase I study of PF-06801591, a humanized IgG4 antibody to programmed cell death 1 (PD-1) receptor, consisted of a Part 1 dose escalation and Part 2 dose expansion. In Part 1, PF-06801591 was administered intravenously or SC with an acceptable safety profile and antitumor activity in several solid tumor types. Herein, we report on Part 2 where we further evaluated SC PF-06801591 in patients (pts) with NSCLC and UC. Methods Anti-PD-1/L1-naive pts with NSCLC (n = 68) and UC (n = 38) received SC PF-06801591 300 mg q4w. NSCLC pts had ≤1 prior line of chemotherapy (CT) ± ≥1 ALK/EGFR mutation-directed therapies (if applicable) and UC pts had ≤2 lines of prior CT. Tumors were evaluated q8w by RECIST 1.1. Blood samples were collected for pharmacokinetic (PK) and immunogenicity assessments. Archival or baseline biopsies were evaluated by immunohistochemistry for PD-L1, RNA-seq, and whole exome sequencing. Results As of 27Dec2018, with median duration of treatment of 2.8 mos. in all 106 treated pts (55 still receiving treatment), 50.9% had treatment-related AEs (TRAEs) with 8.5% grade 3 or higher. Most common TRAEs were hyperthyroidism (9.4%), pruritus (6.6%), and increased amylase/lipase, anemia, hypothyroidism, pneumonitis, and rash (4.7% each). Treatment-emergent anti-drug antibodies were detected in 3 pts. With median follow-up of 5.1 mos. in 67 modified intent to treat (mITT) NSCLC pts, 11 (16.4%) pts achieved a partial response (PR) and 26 (38.8%) had stable disease (SD); the objective response rate (ORR) was 16.4% with 95% confidence interval (CI) 8.5-27.5% (25% in pts with ≥1% tumor cell PD-L1 expression). With median follow-up of 3.0 mos. in 37 mITT UC pts, 6 (16.2%) pts had a PR and 13 (35.1%) had SD; ORR was 16.2% (95% CI 6.2-32.0%). PK and biomarker data will be presented. Conclusions SC PF-06801591 given monthly was well tolerated, with minimal rates of injection site reactions and low incidence of immunogenicity. SC PF-06801591 demonstrated antitumor activity in NSCLC and UC pts at an early time point. The study is ongoing to further assess the durability of responses and long-term safety profile. Clinical trial identification NCT02573259. Editorial acknowledgement S. Mariani MD PhD of Engage Scientific Solutions, Southport, CT, funded by Pfizer and L. Chen. Legal entity responsible for the study Pfizer Inc. Funding Pfizer Inc. Disclosure B.C. Cho: Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): MOGAM Institute; Research grant / Funding (institution): Dong-A ST; Research grant / Funding (institution), Licensing / Royalties: Champions Oncology; Advisory / Consultancy, Research grant / Funding (institution): Janssen; Advisory / Consultancy, Research grant / Funding (institution): Yuhan; Advisory / Consultancy, Research grant / Funding (institution): Ono; Research grant / Funding (institution): Dizal Pharma; Advisory / Consultancy, Research grant / Funding (institution): MSD; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Roche; Advisory / Consultancy: BMS; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Takeda; Shareholder / Stockholder / Stock options: TheraCanVac Inc; Shareholder / Stockholder / Stock options: Gencurix Inc; Shareholder / Stockholder / Stock options: Bridgebio Therapeutics. K. Penkov: Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Prestige Biopharma; Research grant / Funding (institution): Regeneron; Research grant / Funding (institution): Tanvex. M. Korozan: Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): Regeneron. Y. Kulyaba: Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Azaya Therapeutics; Research grant / Funding (institution): Celltrion; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Janssen; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Regeneron; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Synta Pharmaceuticals; Research grant / Funding (institution): Tanvex. H.K. Ahn: Honoraria (self): AstraZeneca; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Eisai; Honoraria (self): Ono; Honoraria (self): Roche. S. Odintsova: Full / Part-time employment: Complete Medical Technologies. J. Davda: Shareholder / Stockholder / Stock options, Full / Part-time employment: Pfizer. A. Forgie: Shareholder / Stockholder / Stock options, Full / Part-time employment: Pfizer. X. Wang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Pfizer. R. Li: Shareholder / Stockholder / Stock options, Full / Part-time employment: Pfizer. I.A. Jacobs: Shareholder / Stockholder / Stock options, Full / Part-time employment: Pfizer. F. Kazazi: Shareholder / Stockholder / Stock options, Full / Part-time employment: Pfizer. J. Chou: Shareholder / Stockholder / Stock options, Full / Part-time employment: Pfizer. M.L. Johnson: Honoraria (institution), Research grant / Funding (institution): AstraZeneca; Honoraria (institution), Research grant / Funding (institution): Genentech/Roche; Honoraria (institution), Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (institution), Research grant / Funding (institution): Loxo; Honoraria (institution), Research grant / Funding (institution): Merck; Honoraria (institution), Research grant / Funding (institution): Mirati; Honoraria (institution), Research grant / Funding (institution): Sanofi; Honoraria (institution): Calithera; Honoraria (institution): Celgene; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): EMD Serono; Research grant / Funding (institution): Janssen; Research grant / Funding (institution): GenMab; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): StemCentrix; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Array BioPharma; Research grant / Funding (institution): Regeneron; Research grant / Funding (institution): Adaptimmune; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Apexigen; Research grant / Funding (institution): BeiGene; Research grant / Funding (institution): BerGenBio; Research grant / Funding (institution): Birdie; Research grant / Funding (institution): Checkpoint Therapeutics; Research grant / Funding (institution): Corvus; Research grant / Funding (institution): Cytomx; Research grant / Funding (institution): Daiichi–Sankyo; Research grant / Funding (institution): Dynavax; Research grant / Funding (institution): G1 Therapeutics; Research grant / Funding (institution): Genocea; Research grant / Funding (institution): Gritstone; Research grant / Funding (institution): Hengrui Therapeutics; Research grant / Funding (institution): Lycera; Research grant / Funding (institution): Neovia; Research grant / Funding (institution): Syndax; Research grant / Funding (institution): Tarveda; Spouse / Financial dependant, contract lobbyist : Astellas; Spouse / Financial dependant, contract lobbyist : Otsuka Pharmaceuticals. All other authors have declared no conflicts of interest.

Adoptively transferred tumor-specific cells can mediate tumor regression in cancers refractory to conventional therapy. Autologous polyclonal tumor-specific cytotoxic T cells (CTL) generated from peripheral blood and infused into patients with metastatic melanoma show enhanced persistence, compared to equivalent numbers of more extensively expanded monoclonal CTL, and are associated with complete remissions (CR) in select patients. We applied high-throughput T cell receptor Vβ sequencing (HTTCS) to identify individual clonotypes within CTL products, track them in vivo post-infusion and then deduce the pre-adoptive transfer (endogenous) frequencies of cells ultimately responsible for tumor regression. The summed in vivo post-transfer frequencies of the top 25 HTTCS-defined clonotypes originally detected in the infused CTL population were comparable to enumeration by binding of antigen peptide-HLA multimers, revealing quantitative HTTCS is a reliable, multimer-independent alternative. Surprisingly, the polyclonal CTL products were composed predominantly of clonotypes that were of very low frequency (VLF) in the endogenous samples, often below the limit of HTTCS detection (0.001%). In patients who achieved durable CRs, the composition of transferred CTLs was dominated (57-90%) by cells derived from a single VLF clonotype. Thus, HTTCS now reveals that tumor-specific CTL enabling long-term tumor control originate from endogenous VLF populations that exhibit proliferative/survival advantages. Along with results indicating that naive cell populations are most likely to contain cells that exist at VLF within the repertoire, our results provide a strong rationale for favoring T cells arising from VLF populations and with early-differentiation phenotypes when selecting subset populations for adoptive transfer.

Introduction: PF-06863135 (PF-3135) is a bispecific, humanized, monoclonal antibody (mAb) consisting of BCMA- and CD3-targeting arms paired on an IgG2a backbone by hinge-mutation technology. PF-3135 binds BCMA+ myeloma cells and CD3+ T cells with affinities of 20 pM and ~40 nM, respectively (Panowski et al. Blood 2016). We report here findings from the dose-escalation portion of an ongoing, multi-center, open-label, phase I study (NCT03269136) of PF-3135 in patients with RRMM. Methods: Adult patients (≥18 years of age) with RRMM, previously treated with a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 mAb, received escalating, intravenous (IV) doses of PF-3135, once weekly. Prior BCMA-targeted bispecific T-cell engager or chimeric antigen receptor T-cell (CART) treatment was allowed by protocol. Patients had measurable disease per the International Myeloma Working Group (IMWG) updated criteria 2014. A modified toxicity probability interval method (mTPI), targeting a dose-limiting toxicity (DLT) rate of 25% (equivalence interval ± 5%) was used for dose escalation. The primary study objectives are to assess PF-3135 safety and tolerability, to determine the maximum tolerated dose (MTD) and select the recommended phase II dose (RP2D). Secondary objectives include evaluation of anti-myeloma activity, pharmacokinetics (PK), and immunogenicity of PF-3135. Results: As of April 9, 2019, 17 patients had received once weekly, non-continuous, IV infusion of PF-3135 in 6 dose-escalation groups. The majority were men (71%). The median age was 61 yrs (range, 47-82 yrs) and median disease duration since onset was 7 yrs (range, 1.1-13.3 yrs). Ten (59%) patients had ≥1 chromosomal abnormality and 5 (29%) had a normal karyotype (status not known for 2 [12%] patients). The median number of prior anti-myeloma therapies was 11; 5 (29%) patients had received prior BCMA-targeted therapy. Eight (47%) patients had relapsed MM and 8 (47%) had refractory disease (recurrence type not known for 1 [6%] patient). Ten (59%) patients experienced treatment-related (TR) AEs of any grade. Most TRAEs were grade 1-2, including cytokine release syndrome (CRS, 24%), thrombocytopenia (24%), anemia (18%), and pyrexia (18%). Three (18%) patients had grade 3 TRAEs (increased alanine aminotransferase/aspartate aminotransferase, leukocytopenia, neutropenia, and lymphopenia). One patient treated at the highest dose level, who had received prior BCMA CART therapy, developed treatment-related febrile neutropenia, a DLT, which may have been related to CRS and borderline/low neutrophil count at baseline. None of the patients had grade 4-5 TRAEs or discontinued treatment due to a TRAE. The median duration of treatment was 4 (range, 2-12) actual dosing days. Sixteen of the 17 patients were evaluable for response. At the time of data cut-off, one (6%) patient had a minimal response and 6 (35%) patients had stable disease (SD) across dose levels, as best response by investigator IMWG assessment; 9 (53%) patients experienced disease progression. The clinical benefit rate (defined as best response ≥SD) was 41% (95% CI: 18.4%, 67.1%). Conclusions: Treatment with IV PF-3135 was well tolerated at the dose levels evaluated. The observed CRS events were moderate and dose-dependent. Additional dose cohorts are accruing. The latest clinical, biomarker, and PK data will be presented for this ongoing study. Disclosures Raje: Medscape: Honoraria; Research to Practice: Honoraria; Takeda: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AstraZeneca: Research Funding. Jakubowiak:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; SkyLineDx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; KaryoPharm Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Gasparetto:Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Cornell:KaryoPharm: Consultancy; Takeda: Consultancy. Krupka:Pfizer: Employment, Equity Ownership. Navarro:Pfizer: Employment, Equity Ownership. Forgie:Pfizer: Employment, Equity Ownership. Udata:Pfizer: Employment, Equity Ownership. Basu:Pfizer: Employment, Equity Ownership. Chou:Pfizer: Employment, Equity Ownership. Leung:Pfizer: Employment, Equity Ownership. Lesokhin:BMS: Consultancy, Honoraria, Research Funding; Serametrix Inc.: Patents & Royalties; Takeda: Consultancy, Honoraria; Genentech: Research Funding; Juno: Consultancy, Honoraria; GenMab: Consultancy, Honoraria; Janssen: Research Funding. OffLabel Disclosure: PF-06863135, investigational agent

Introduction: PF-06801591 is a humanized IgG4 monoclonal antibody that binds to programmed cell death (PD-1) receptor, blocking its interaction with PD-1 ligands. In a phase I study (NCT02573259; dose-escalation) of 40 patients (pts) with multiple solid tumor types treated with PF-06891591 intravenously (IV) or subcutaneously (SC), 7 pts showed partial response. PF-06801591 was well tolerated at all doses. Biopsy samples were evaluated for predictive biomarkers and pharmacodynamic effects. Methods: Biopsy samples at baseline and on-treatment (IV:day 29; SC:day 36) were collected from 4 dose cohorts (IV:1, 3 and 10 mg/kg; SC:300 mg). Whole-exome sequencing (WES) and RNA-seq of biopsy tissue were used to estimate tumor mutation burden (TMB) and gene expression. Regression analysis was used to identify TMB/genes/pathways potentially associated with response in baseline tissue. Differential analysis of baseline and on-treatment biopsies identified genes/pathways potentially upregulated by PF-06801591. PD-L1 (clone SP-263; Ventana) expression was evaluated in tumor biopsies by immunohistochemistry (IHC) and pathologist scoring. Results: In a combined WES analysis of samples from baseline biopsies (all doses, n=24 pts), higher TMB was significantly associated with improved objective response (R^2=0.215, P=0.013). From RNA-seq analysis, genes positively associated with response were involved in interferon-gamma (IFNG) and PD-1 signaling, and cell cycle; CTLA4 was among the genes most significantly associated with response (P Conclusion: Based on a mixed set of tumor types with possible sensitivity to anti-PD-1, high TMB, IFNG signaling, and PD-L1 were associated with response to PF-06801591, as reported for other anti-PD-1 antibodies. Albeit based on a small sample size and diverse indication profile, baseline expression of CTLA4 appears to be highly associated with response and PD-L1 RNA levels may be better associated with response than protein expression by IHC. Increased expression of genes/pathways associated with adaptive immune activation in on-treatment biopsies indicates an active, immunomodulatory mechanism with anti-PD-1 therapy. This ongoing study will evaluate predictive biomarkers in dose-expansion cohorts of PF-06801591 in non-small cell lung cancer and urothelial cancer. Acknowledgments Study sponsor: Pfizer. Medical writing support: Chu Kong Liew (Engage Scientific Solutions), funded by Pfizer. Citation Format: Siwen Hu-Lieskovan, Fadi Braiteh, Juneko E. Grilley-Olson, Shobha Potluri, Xiao Wang, Alison Forgie, Vinicius Bonato, Jeffrey Chou, Melissa L. Johnson. Predictive biomarkers and pharmacodynamic changes in tumor RNA expression in a phase I study of anti PD-1 monoclonal antibody PF-06801591 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT046.

Importance We assessed feasibility of monthly subcutaneous administration of PF-06801591, a humanized immunoglobulin G4 monoclonal antibody that binds to the programmed cell death (PD-1) receptor and blocks its interaction with PD-1 ligands. Objective To evaluate the safety, efficacy, and pharmacokinetics of PF-06801591 administered intravenously vs subcutaneously. Design, Setting, and Participants Ongoing phase 1, open-label, multicenter, dose-escalation study of 40 patients, 18 years or older, with locally advanced or metastatic solid tumors, enrolled between March 8, 2016, and March 5, 2018, from 4 US medical centers. Interventions An intravenous dose of 0.5, 1, 3, or 10 mg/kg of PF-06801591 was administered every 3 weeks or a subcutaneous dose of 300 mg was administered every 4 weeks. Dose escalation occurred after 2 to 4 patients were enrolled per dose level, with additional patients enrolled in each cohort for further assessment. Main Outcomes and Measures The primary end points were dose-limiting toxic effects and safety. Secondary end points included pharmacokinetics, immunogenicity, PD-1 receptor occupancy, and efficacy. Results Of 40 enrolled patients (12 men and 28 women; mean [SD] age, 61 [13] years) in this phase 1 dose-escalation trial, 25 received PF-06801591 intravenously at escalating dose levels (0.5, 1, 3, or 10 mg/kg) and 15 patients received the monoclonal antibody subcutaneously at a single dose level. No dose-limiting toxic effects were observed. Grade 3 or higher treatment-related adverse events occurred in 4 (16%) patients treated intravenously and 1 (6.7%) patient treated subcutaneously. Immune-related adverse events occurred in 10 (40%) patients treated intravenously and 3 (20%) treated subcutaneously. No dose–adverse event associations were observed during intravenous dose escalation, and no serious skin toxic effects occurred with subcutaneous delivery. Responses were seen in 5 patients receiving PF-06801591 intravenously and in 2 patients treated subcutaneously for an overall objective response rate of 18.4%. Median overall survival was not reached with intravenous dosing vs 10.7 months with subcutaneous administration. Exposure to PF-06801591 increased in a dose-proportional manner over the range of intravenous doses. Median time to maximum observed serum concentration was 8 days after subcutaneous administration. Full PD-1 receptor occupancy was seen in all dose cohorts. Conclusions and Relevance Anti–PD-1 antibody PF-06801591 was tolerable and showed antitumor activity in a variety of tumor types across all dose levels of intravenous and subcutaneous administration. Monthly subcutaneous administration of PF-06801591 offers a convenient, effective alternative to currently available intravenously administered checkpoint inhibitors. Trial Registration ClinicalTrials.gov identifier:NCT02573259

Application of high-throughput DNA sequencing to the analysis of B- and T-lymphocyte antigen receptors has great potential for improving the monitoring of lymphoid malignancies, assessing immune reconstitution after hematopoietic cell transplantation, and characterizing the composition of lymphocyte repertoires. Current technology can define the number and frequency of immunoglobulin heavy, T-cell receptor (TCR)α, TCRβ, or TCRγ chains expressed in a population of lymphocytes; techniques for determining the number of antigen receptor heterodimers, such as TCRαβ pairs, expressed in the population are under development.

Posttransplantation cyclophosphamide (PTCy) effectively prevents graft-versus-host disease (GVHD), but its immunologic impact is poorly understood. We assessed lymphocyte reconstitution via flow cytometry (n = 74) and antigen receptor sequencing (n = 35) in recipients of myeloablative, HLA-matched allogeneic BM transplantation using PTCy. Recovering T cells were primarily phenotypically effector memory with lower T cell receptor β (TRB) repertoire diversity than input donor repertoires. Recovering B cells were predominantly naive with immunoglobulin heavy chain locus (IGH) repertoire diversity similar to donors. Numerical T cell reconstitution and TRB diversity were strongly associated with recipient cytomegalovirus seropositivity. Global similarity between input donor and recipient posttransplant repertoires was uniformly low at 1-2 months after transplant but increased over the balance of the first posttransplant year. Blood TRB repertoires at ≥3 months after transplant were often dominated by clones present in the donor blood/marrow memory CD8+ compartment. Limited overlap was observed between the TRB repertoires of T cells infiltrating the skin or gastrointestinal tract versus the blood. Although public TRB sequences associated with herpesvirus- or alloantigen-specific CD8+ T cells were detected in some patients, posttransplant TRB and IGH repertoires were unique to each individual. These data define the immune dynamics occurring after PTCy and establish a benchmark against which immune recovery after other transplantation approaches can be compared.

The tumor antigen 5T4 is frequently expressed at high levels on renal cell carcinoma (RCC) and other epithelial carcinomas. Surveys of normal tissues demonstrate abundant 5T4 expression on placental trophoblast cells with limited expression elsewhere. 5T4 is the target for a therapeutic cancer vaccine (MVA-5T4) that elicits 5T4-specific serological, proliferative, and cytotoxic T lymphocyte (CTL) responses. However, the antitumor activity of 5T4-specific CTL has not been extensively characterized. CD8 T cells from HLA-A2 healthy donors (n=4) or RCC patients (n=2) were stimulated in vitro with the HLA-A2-binding nonamer peptides 5T417-25 or 5T497-105 and screened by flow cytometry with specific tetramers (TET). CD8/TET T-cell clones specific for 5T417-25 or 5T497-105 peptide were isolated from 4/6 and 1/4 donors, respectively. A subset of clones specific for 5T417-25 was cytolytic for MVA-5T4-infected HLA-A2 EBV-transformed lymphoblastoid cell line target cells and for constitutively HLA-A2-expressing and 5T4-expressing RCC tumor cell lines (including A498 RCC). In a xenoengraftment assay, the coinoculation of a representative 5T417-25-specific CTL clone with A498 RCC tumors cells into immune-deficient mice completely prevented growth of A498 tumors. Taken together, these data demonstrate high-avidity CD8 CTL able to recognize the naturally processed 5T417-25 epitope on RCC tumor cells including putative tumor-initiating cells are present in peripheral blood of both healthy donors and RCC patients. CD8T-cell immunity targeting 5T417-25 is therefore of substantial interest both as a potential target for further development of vaccination or adoptive cellular immunotherapy and for immune monitoring studies in association with nonspecific immunotherapies.

Introduction: PF-8600 is a fully human IgG2 agonistic mAb against the tumor necrosis factor superfamily receptor OX40. OX40 improves T cell survival, proliferation, and activation and may enhance anti-tumor immunity. In an ongoing phase I study (NCT02315066) of 52 patients (pts) with melanoma, hepatocellular carcinoma, head and neck carcinoma, or renal cell carcinoma treated with PF-8600 monotherapy in dose escalation, 2 pts had best overall response of partial response (PR) and 27 of stable disease. PF-8600 was well-tolerated at all doses. Peripheral blood flow cytometry had previously shown increased proliferation and activation of CD4 central memory T cells at certain dose levels, suggesting a PD effect. RNAseq analysis of tumor biopsy samples and TCR sequencing of peripheral blood were used to further support proof of mechanism. Methods: Biopsy samples at baseline and wk 6 were collected from 5 dose cohorts [0.1 (4), 0.3 (3), 1.5 (4), 3.0 (3) and 10.0 (2), dose in mg/kg (n)]. Biopsy tissue was analyzed by RNAseq and gene ranking-based gene set enrichment analysis (FGSEA) to identify immune pathways potentially up-regulated by OX40. CD4/CD8 cells were isolated from peripheral blood samples from 4 dose cohorts (0.1, 0.3, 1.5, and 3.0 mg/kg). DNA was extracted for sequencing of the TCR-β chain complementarity-determining region 3 (CDR3). Results: In a combined analysis of samples from pts dosed with ≥1.5 mg/kg, the top 3 gene sets showing enrichment at wk 6 of therapy were associated with inflammatory response, interferon-γ response and allograft rejection. These gene sets were identical to the top 3 most enriched in tumor from a syngeneic mouse tumor model exposed to a murine OX40 agonist. This pattern of enrichment was not observed if samples from lower doses were included in the analysis. TCR sequencing revealed clonal expansion of CD4/CD8 T cells at all dose levels at wk 6 [CD4: mean = 8 expanded clones per 100,000 clones (range = 1 - 80), CD8: mean = 56 (range = 1 - 500)]. The 2 patients with PR had among the lowest numbers of expanded CD4 and CD8 clones (CD4: 4 and 2; CD8: 7 and 4). Conclusion: Enrichment of gene sets associated with immune activation in tumor tissue from patients dosed with PF-8600 provides evidence supporting an active, immunomodulatory mechanism. Peripheral CD4/CD8 T cell populations exhibited clonal expansion in response to dosing with PF-8600 at all dose levels further suggesting a PD effect. However, clinical response did not necessarily correlate with a high number of expanded T cell clones, suggesting that clinical response to OX-40 agonism may be driven by the expansion of select anti-tumor T cell clones rather than a broad expansion of T cell clones in the peripheral blood. The phase I study will continue to evaluate PD changes in the tumor and peripheral blood in dose-expansion cohorts of PF-8600 ± utomilumab. Citation Format: Adi Diab, Omid Hamid, John A. Thompson, Willeke Ros, Fredericus A. L. M. Eskens, Toshihiko Doi, Siwen Hu-Lieskovan, Hua Long, Tenshang Joh, Shoba Potluri, Xiao Wang, Catherine Fleener, Carrie Turich Taylor, Bishu J. Ganguli, Jeffrey Chou, Anthony B. El-Khoueiry. Pharmacodynamic (PD) changes in tumor RNA expression and the peripheral blood T cell receptor (TCR) repertoire in a phase I study of OX40 agonistic monoclonal antibody (mAb) PF-04518600 (PF-8600) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT010.

Meeting abstracts T-VEC is a herpes simplex virus-1-based oncolytic immunotherapy designed to selectively replicate in tumors, produce GM-CSF, and stimulate antitumor immune responses. OPTiM, a Phase III trial of T-VEC vs GM-CSF in unresectable stage IIIB-IV melanoma, improved the primary endpoint

Cell motility is actuated by a host of intracellular signaling cascades that result in movement of the cell in one direction, even without an external gradient. Phospholipase C-gamma (PLCgamma) has been shown to be important for growth factor-induced lamellipodial protrusion at the front of the cell while Cdc42 has been implicated in both filopodium formation at the leading edge and control of polarity of migrating cells. We asked whether these asymmetries in effector molecules may be linked. When we overexpressed either constitutively active, dominant negative, or GFP-tagged Cdc42, wild-type NR6 fibroblasts lost directionality, as expected. On epidermal growth factor (EGF) exposure these cells produced multiple, transient protrusions in every direction; these extensions failed to result in productive motility. GFP-tagged Cdc42 appeared transiently at edges of newly formed protrusions in EGF-stimulated cells while they moved haphazardly. While PLCgamma is distributed throughout the cell, the ratio of active, tyrosyl-phosphorylated PLCgamma was increased at the leading edge, where phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis is concentrated. This co-localization of activities may be due to Cdc42 directing PLCgamma to the cell front, as PLCgamma associated with Cdc42 in an EGF-dependent manner. We conclude that Cdc42 controls cell polarity, likely in part, through its binding to active PLCgamma.

3027 Background: PF-8600 is a novel fully human IgG2 agonistic mAb against human OX40, a TNF receptor superfamily member expressed primarily on activated T cells. This ongoing phase 1 study (NCT02315066) is investigating the safety, efficacy, PK and PD of PF-8600 in patients (pts) with solid tumors. Methods: PF-8600 (0.01–10 mg/kg) was given IV every 14d. Expression of free/total OX40 receptor, proliferation marker ki67 and activation markers HLA-DR/CD38 were measured in T cell subsets in peripheral blood by flow cytometry in all pts. CD4, CD8, OX40 and FOXP3 were evaluated in paired tumor biopsies (bx), collected from a subset of pts (≥0.1 mg/kg) at baseline (BL) and Wk6, by immunohistochemistry. Results: As of 21Sep2016, 48 pts with melanoma (n = 14), hepatocellular carcinoma (HCC, n = 19), head and neck squamous cell (n = 6) or renal cell carcinoma (n = 9) enrolled in the dose-escalation cohorts (0.01-3 mg/kg). No immune related adverse events (AE) were reported. The most frequent treatment related AEs in > 3 pts were fatigue (27.1%) and nausea and vomiting (8.3% each); all Gr 1-2. 2 pts had a partial response: melanoma at 0.1 (Pt1) and HCC at 0.3 (Pt2) mg/kg. 25 pts had best ORR (BOR) of stable disease (SD; 3 pts ≥24 wks). A majority of pts at 0.1, 0.3, and 3 mg/kg, including Pt1 and Pt2, had increases in ki67 and HLA-DR/CD38 expression in peripheral CD4+ central memory T cells. Pt1, Pt2 and all pts at ≥0.3 mg/kg had full receptor occupancy. Paired bx were only available from pts with BOR of SD or progressive disease. In 10 pts with available paired tumor bx and defined date of radiographic progression (rPD), longer time to rPD correlated with increases in %OX40+ in bx from BL to Wk6, regardless of dose level, tumor type or prior immunotherapy (R2= 0.52, p = 0.0188); no correlation between rPD and CD4+, CD8+ or FOXP3+ expression changes was observed. Updated efficacy, safety, PK and PD data will be presented. Conclusions: PF-8600 is well tolerated with evidence of single agent efficacy. Initial observations of PD markers that change with treatment and correlate with rPD support efforts to confirm these findings as more clinical outcomes and larger sample sizes become available. Clinical trial information: NCT02315066.

Adoptive immunotherapy of tumors with T cells specific for the cancer-testis antigen NY-ESO-1 has shown great promise in preclinical models and in early stage clinical trials. Tumor persistence or recurrence after NY-ESO-1-specific therapy occurs, however, and the mechanisms of recurrence remain poorly defined. In a murine xenograft model of NY-ESO-1+ multiple myeloma, we observed tumor recurrence after adoptive transfer of CD8+ T cells genetically redirected to the prototypic NY-ESO-1157-165 peptide presented by HLA-A*02:01. Analysis of the myeloma cells that had escaped from T cell control revealed intact expression of NY-ESO-1 and B2M, but selective, complete loss of HLA-A*02:01 expression from the cell surface. Loss of heterozygosity in the Major Histocompatibility Complex (MHC) involving the HLA-A locus was identified in the tumor cells, and further analysis revealed selective loss of the allele encoding HLA-A*02:01. Although loss of heterozygosity involving the MHC has not been described in myeloma patients with persistent or recurrent disease after immune therapies such as allogeneic hematopoietic cell transplantation (HCT), it has been described in patients with acute myelogenous leukemia who relapsed after allogeneic HCT. These results suggest that MHC loss should be evaluated in patients with myeloma and other cancers who relapse after adoptive NY-ESO-1-specific T cell therapy.

Xenografts of human colorectal cancer (CRC) in immune-deficient mice have great potential for accelerating the study of tumor biology and therapy. We evaluated xenografts established in NOD/scid/IL2Rγ-null mice from the primary or metastatic tumors of 27 patients with CRC to estimate their capacity for expanding tumor cells for in vitro studies and to assess how faithfully they recapitulated the transcriptional profile of their parental tumors. RNA-seq analysis of parental human CRC tumors and their derivative xenografts demonstrated that reproducible transcriptional changes characterize the human tumor to murine xenograft transition. In most but not all cases, the human stroma, vasculature, and hematopoietic elements were systematically replaced by murine analogues while the carcinoma component persisted. Once established as xenografts, human CRC cells that could be propagated by serial transplantation remained transcriptionally stable. Three histologically atypical xenografts, established from patients with peritoneal metastases, contained abundant human stromal elements and blood vessels in addition to human tumor cells. The transcriptomes of these mixed tumor/stromal xenografts did not closely resemble those of their parental tumors, and attempts to propagate such xenografts by serial transplantation were unsuccessful. Stable expression of numerous genes previously identified as high priority targets for immunotherapy was observed in most xenograft lineages. Aberrant expression in CRC cells of human genes that are normally only expressed in hematopoietic cells was also observed. Our results suggest that human CRC cells expanded in murine xenografts have great utility for studies of tumor immunobiology and targeted therapies such as immunotherapy but also identify potential limitations.

TPS9598Background: Talimogene laherparepvec, an oncolytic viral immunotherapy, was designed to selectively replicate in tumors resulting in lytic cell death, antigen release, and production of GM-C...

9568Background: T-VEC is a herpes simplex virus (HSV)-1 -based oncolytic immunotherapy designed to selectively replicate in tumors, produce GM-CSF and stimulate antitumor immune responses in melanoma. T-VEC significantly improved durable response rate vs GM-CSF in stage IIIB-IV melanoma patients (pts) with injectable tumors. Pembro inhibits programmed cell death protein 1 and improves survival in advanced melanoma. The combination may further improve clinical benefit. Here we report phase 1b efficacy, safety and biomarker data from a phase 1b/3 study of T-VEC+pembro in unresectable stage IIIB-IV melanoma (NCT02263508) with all pts having started on T-VEC+pembro ≥ 6 mo prior. Methods: Key inclusion criteria: unresectable stage IIIB-IV melanoma, injectable lesions; no prior systemic tx; and ECOG PS 0-1. T-VEC: ≤ 4 mL in (sub)cutaneous/nodal lesions, 106 PFU/mL d1, 108PFU/mL d22 then Q2W; pembro: IV, 200 mg d36 then Q2W. Tx until first occurrence of: complete response (CR); no injectable tumors (for T-VEC); ...