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PURPOSE This phase I/II study evaluated tremelimumab (anticytotoxic T-lymphocyte–associated antigen-4 monoclonal antibody) and durvalumab (antiprogrammed death ligand-1 monoclonal antibody) as monotherapies and in combination for patients with unresectable hepatocellular carcinoma (HCC), including a novel regimen featuring a single, priming dose of tremelimumab (ClinicalTrials.gov identifier: NCT02519348 ). PATIENTS AND METHODS Patients with HCC who had progressed on, were intolerant to, or refused sorafenib were randomly assigned to receive T300 + D (tremelimumab 300 mg plus durvalumab 1,500 mg [one dose each during the first cycle] followed by durvalumab 1,500 mg once every 4 weeks), durvalumab monotherapy (1,500 mg once every 4 weeks), tremelimumab monotherapy (750 mg once every 4 weeks [seven doses] and then once every 12 weeks), or T75 + D (tremelimumab 75 mg once every 4 weeks plus durvalumab 1,500 mg once every 4 weeks [four doses] followed by durvalumab 1,500 mg once every 4 weeks). Safety was the primary end point. Secondary end points included objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors v1.1 and overall survival; exploratory end points included circulating lymphocyte profiles. RESULTS A total of 332 patients were enrolled (T300 + D, n = 75; durvalumab, n = 104; tremelimumab, n = 69; and T75 + D, n = 84). Tolerability was acceptable across arms, with grade ≥ 3 treatment-related adverse events occurring in 37.8%, 20.8%, 43.5%, and 24.4%, respectively. Confirmed ORRs (95% CI) were 24.0% (14.9 to 35.3), 10.6% (5.4 to 18.1), 7.2% (2.4 to 16.1), and 9.5% (4.2 to 17.9), respectively. An early expansion of CD8+ lymphocytes was associated with response across arms, with highest proliferating CD8+ lymphocyte levels occurring in the T300 + D arm. The median (95% CI) overall survival was 18.7 (10.8 to 27.3), 13.6 (8.7 to 17.6), 15.1 (11.3 to 20.5), and 11.3 (8.4 to 15.0) months in the T300 + D, durvalumab, tremelimumab, and T75 + D arms, respectively. CONCLUSION All regimens were found to be tolerable and clinically active; however, the T300 + D regimen demonstrated the most encouraging benefit-risk profile. The unique pharmacodynamic activity and association with ORR of the T300 + D regimen further support its continued evaluation in HCC.

Mutations in the STK11 (LKB1) gene regulate resistance to PD-1/PD-L1 blockade. This study evaluated this association in patients with nonsquamous non–small cell lung cancer (NSCLC) enrolled in three phase I/II trials. STK11 mutations were associated with resistance to the anti–PD-L1 antibody durvalumab (alone/with the anti-CTLA4 antibody tremelimumab) independently of KRAS mutational status, highlighting STK11 as a potential driver of resistance to checkpoint blockade. Retrospective assessments of tumor tissue, whole blood, and serum revealed a unique immune phenotype in patients with STK11 mutations, with increased expression of markers associated with neutrophils (i.e., CXCL2, IL6), Th17 contexture (i.e., IL17A), and immune checkpoints. Associated changes were observed in the periphery. Reduction of STAT3 in the tumor microenvironment using an antisense oligonucleotide reversed immunotherapy resistance in preclinical STK11 knockout models. These results suggest that STK11 mutations may hinder response to checkpoint blockade through mechanisms including suppressive myeloid cell biology, which could be reversed by STAT3-targeted therapy. Significance: Patients with nonsquamous STK11-mutant (STK11mut) NSCLC are less likely than STK11 wild-type (STK11wt) patients to respond to anti–PD-L1 ± anti-CTLA4 immunotherapies, and their tumors show increased expression of genes and cytokines that activate STAT3 signaling. Preclinically, STAT3 modulation reverses this resistance, suggesting STAT3-targeted agents as potential combination partners for immunotherapies in STK11mut NSCLC. This article is highlighted in the In This Issue feature, p. 2659

Immune checkpoint inhibitors (ICI) are designed to activate exhausted tumor-reactive T cells thereby leading to tumor regression. Durvalumab, an ICI that binds to the programmed death ligand-1 (PD-L1) molecule, is approved as a consolidation therapy for treatment of patients with stage III, unresectable, non-small cell lung cancer (NSCLC). Immunophenotypic analysis of circulating immune cells revealed increases in circulating proliferating CD4 + and CD8 + T cells earlier after durvalumab treatment. To examine durvalumab’s mechanism of action and identify potential predictive biomarkers, we assessed the circulating T cells phenotypes and TCR genes of 71 NSCLC patients receiving durvalumab enrolled in a Phase I trial (NCT01693562, September 14, 2012). Next-generation sequencing of TCR repertoire was performed on these NSCLC patients’ peripheral blood samples at baseline and day 15. Though patients’ TCR repertoire diversity showed mixed responses to the treatment, patients exhibiting increased diversity on day 15 attained significantly longer overall survival (OS) (median OS was not reached vs 17.2 months for those with decreased diversity, p = 0.015). We applied network analysis to assess convergent T cell clonotypes indicative of an antigen-driven immune response. Patients with larger TCR clusters had improved OS (median OS was not reached vs 13.1 months for patients with smaller TCR clusters, p = 0.013). Early TCR repertoire diversification after durvalumab therapy for NSCLC may be predictive of increased survival and provides a mechanistic basis for durvalumab pharmacodynamic activity. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-020-02833-z.

Background Emerging data suggest poor outcome to anti- PD(L)1 blocking agents in patients with STK11mut tumors1. In the current study, we undertook in-depth translational evaluations of three Ph1/Ph2 independent studies of Durvalumab ± Tremelimumab to elucidate the biology associated with STK11 mutations leading to reduced clinical response in patients with non-squamous NSCLC. Methods Mutational status was evaluated by ctDNA or Foundation One CDx as previously described1. RNA sequencing was conducted on baseline frozen biopsies (N=70). Selected proteins (N=66) were measured by Myriad RBM multiplexed immunoassays on baseline serum (n=91). Screening and longitudinal whole blood was assessed for circulating quantities of T, B or NK cells and activated or memory T cell subsets using bioanalytically-validated, flow cytometry-based immunophenotyping assays. Exploratory analyses of translational endpoints according to STK11 mutational status were conducted by Wilcoxon rank-sum test. Results In the periphery, a reduced number (> 2-fold decreased in median quantities) of NK cells, CD4+ effector memory, CD4+ HLA-DR+, CD8+ effector memory and CD8+ HLA-DR+ T cells was observed at baseline and following treatments in patients with STK11mut vs. STK11wt tumors. At baseline, increased levels of IL6 (p=0.002) and the neutrophil-attracting cytokine IL8 (p=0.02) were found in serum of patients with STK11mut tumors. In the tumor microenvironment, significantly increased expression (p 2) of markers associated with neutrophils, (i.e. CXCL2, IL6, CSF3), Th17 contexture (i.e. IL17A) and immune checkpoints (i.e. KIRs, PD-L1) was found in STK11mut vs. STK11wt tumors. Conclusions The poor outcomes to immunotherapy observed in NSCLC patients with STK11mut tumors might be determined by a compromised peripheral and intra-tumoral immune phenotype. These results might help the development of novel therapeutic interventions able to unleash response to immune checkpoints in NSCLC patients harboring STK11 mutations. Trial Registration NCT01693562, NCT02087423, NCT02000947 Reference Jure Kunkel Met al, JCO. 2018.36.15_suppl.3028.

Background Despite the encouraging successes of immune checkpoint inhibitors, many patients do not benefit and are either refractory or relapse. The mechanisms of refractory or relapsed disease following PD-(L)1 blockade are largely unknown. To identify characteristics associated with refractory or relapsed disease we explored the immune and genomic landscape of samples derived from NSCLC patients who previously received PD-(L)1 blockade and had blood and fresh tumor biopsies collected at the time of progression. Methods Patient response categories were defined prospectively; ‘refractory’ defined as progression within 16 weeks of initiating PD-(L)1 and ‘relapse’ defined as initial clinical benefit (CR, PR, SD) followed by progression. RNAseq (n=52) and PD-L1 IHC (n=22) were performed on tumor tissue. Immune profiling of whole blood was assessed using flow cytometry or Biomark HD (Fluidigm) gene expression panel (n=54 and n=62, respectively). Differential gene expression was defined as unadjusted p 1.5. Pathways analysis was conducted by David tool. Patient samples were collected during screening for clinical trial of second line immunotherapy. Written informed consent was obtained from the patients for publication of this abstract. Results In patients with NSCLC previously treated with PD-(L)1 blockade, tumors of relapsed patients were characterized by increased expression of genes associated with interferon signaling (e.g. CXCL9, SPIC, IFNg), immune suppression (e.g. ARG1, TGFB), immune exhaustion (e.g. ADORA2A), and increased PD-L1 expression (by gene expression and IHC). Refractory disease was associated with increased cadherin signaling and calcium-dependent-cell-adhesion gene expression pathways. In the periphery, reduced quantities of B cells and activated (HLA-DR+ or CD38+) or proliferating (Ki67+) CD8+ T cells were observed in refractory patients. Conclusions The tumor and peripheral compartments of patients with NSCLC previously treated with PD-(L)1 blockade differ based on prior response. Relapsed patients tend to have signals of sturdy immune activation and chronic inflammation thus ultimately leading to immune exhaustion. These results may help inform rational therapeutic strategies to overcome resistance to PD-(L)1 blockade in NSCLC. Trial Registration NCT02000947 Ethics Approval Research on human samples here analyzed have been performed in accordance with the Declaration of Helsinki. Consent Written informed consent was obtained from the patient for publication of this abstract.

In the development of therapeutic compounds that bind cell surface molecules, it is critical to demonstrate the extent to which the drug engages its target. For cell-associated targets, flow cytometry is well-suited to monitor drug-to-target engagement through receptor occupancy assays (ROA). The technology allows for the identification of specific cell subsets within heterogeneous populations and the detection of nonabundant cellular antigens. There are numerous challenges in the design, development, and implementation of robust ROA. Among the most difficult challenges are situations where there is receptor modulation or when the target-antigen is expressed at low levels. When the therapeutic molecules are bi-specific and bind multiple targets, these challenges are increased. This manuscript discusses the challenges and proposes best practices for designing, optimizing, and validating ROA.

Purpose: The safety and preliminary efficacy of MEDI1873, an agonistic IgG1 fusion protein targeting glucocorticoid-induced TNF receptor–related protein (GITR), were evaluated in an open-label, first-in-human, phase I, dose escalation study in previously treated patients with advanced solid tumors. Patients and Methods: Two single-patient cohorts at 1.5 and 3 mg i.v. were followed by 3+3 dose escalation in six cohorts at 7.5, 25, 75, 250, 500, and 750 mg, all every 2 weeks, for up to 52 weeks. Primary endpoints were safety and tolerability, dose-limiting toxicities (DLT), and MTD. Secondary endpoints included antitumor activity, pharmacokinetics, immunogenicity, and pharmacodynamics. Results: Forty patients received MEDI1873. Three experienced DLTs: grade 3 worsening tumor pain (250 mg); grade 3 nausea, vomiting, and headache (500 mg); and grade 3 non-ST segment elevation myocardial infarction (750 mg). An MTD was not reached and treatment was well tolerated up to 500 mg. Most common treatment-related adverse events were headache (25%), infusion-related reaction (17.5%), and decreased appetite (17.5%). MEDI1873 exposure was dose proportional. Antidrug–antibody incidence was low. MEDI1873 increased peripheral CD4+ effector memory T-cell proliferation as well as cytokines associated with effector T-cell activation at dose levels ≥75 mg. The best response was stable disease (SD) in 17 patients (42.5%), including 1 unconfirmed partial response. Eight patients (20.0%) had SD ≥24 weeks. Conclusions: MEDI1873 showed acceptable safety up to 500 mg i.v. every 2 weeks with pharmacodynamics activity, and prolonged SD in some patients. However, further development is not planned because of lack of demonstrated tumor response.

Background:T-follicular helper (TFH) cells are a novel CD4+ T-cell subset that participates in germinal center maintenance/proliferation. Proliferation of TFH is a postulated mechanism of pathogenesis for T-cell lymphomas (AITL, PTCL and others). The inducible T-cell costimulator (ICOS) is highly expressed in TFHand hence in AITL, PTCL FH type, and some cutaneous T cell lymphomas (CTCL) and follicular lymphomas (FL). MEDI-570 is a human afucosylatedIgG1 kappa monoclonal antagonistic antibody directed against ICOS, which binds to and eliminates ICOS expressing cells in preclinical in vivo models. We investigate the safety, pharmacokinetics (PK) and clinical activity of ICOS blockade by MEDI-570 in T-cell lymphomas. Methods: NCI-9930 is a Phase I study of MEDI-570 in R/R malignant lymphomas. It is a 3+3 study design that evaluated 5 dose levels . MEDI-570 was administered intravenously (IV) every 3 weeks for 12 cycles. Eligibility criteria included: age >18 years, ECOG 200 cells/uL (>100 for AITL). Primary endpoints are safety of MEDI-570, dose limiting toxicities (DLT)s and its recommended phase 2 dose (RP2D). Secondary endpoints include: pharmacokinetics (PK), overall response rate (ORR) based on Lugano classification and progression-free survival (PFS). Exploratory endpoints include various correlative studies. This study is supported by the National Cancer Institute Experimental Therapeutics Clinical Trials Network (ETCTN) and Early Drug Development Opportunity Program (EDDOP) (NCT02520791). Results: As of June 2020, the dose escalation phase has been completed. Patients were enrolled and evaluable for safety and efficacy. Median age is 63 (range: 29-80), female/male ratio=5/13, histologic types consisted in AITL (n= 12, 71%), PTCL NOS (n=3, 18%) and CTCL (n=2, 12%). The median number of prior therapies were 7.5 (1 - 16), stage III/IV in 83%, prior autologous HCT in 18%. There were 4 partial remissions (PR) and 7 with stable disease (SD), all patients with AITL. Two patients (1 PR and 1 SD) remain on treatment. One patient completed treatment and remains in a stable PR for over a year without further treatment. One patient in PR underwent allogeneic hematopoietic transplantation (HCT) and remains in remission. The most common grade 3/4 AEs were decreased CD4+ T-cells as expected anemia (12%), hypophosphatemia (12%), thrombocytopenia (6%), infusion related reactions (6%). No DLTs were reported, and the maximum tolerated dose was not established. Initial PK analyses demonstrated that MEDI-570 systemic exposure increased in a dose-dependent manner, and a RP2D was determined. Peripheral blood flow cytometry analysis of T-cell subsets showed that MEDI-570 caused a rapid and sustained decrease in CD4+ T-cells. It also resulted in reductions in circulating of certain ICOS+ T-cells, especially on days 7-21 post treatment. Conclusion: MEDI-570 was safe, well tolerated and showed promising clinical activity in poor-risk refractory and heavily pretreated AITL. A RP2D was established. MEDI-570 results in sustained reduction of the targeted ICOS+T lymphocytes. The study continues enrolling in the expansion phase. Disclosures Chavez: Morphosys: Consultancy, Speakers Bureau; Merck: Research Funding; Bayer: Consultancy; BeiGene: Speakers Bureau; Karyopharm: Consultancy; AbbVie: Consultancy; Genentech: Speakers Bureau; AstraZeneca: Speakers Bureau; Gilead: Consultancy; Verastem: Consultancy; Epizyme: Speakers Bureau; Celgene: Consultancy; Novartis: Consultancy; Kite, a Gilead Company: Consultancy, Speakers Bureau; Pfizer: Consultancy. William:Dova: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; Merck: Research Funding; Kyowa Kirin: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Guidepoint Global: Consultancy. Brammer:Celgene Corporation: Research Funding; Seattle Genetics, Inc.: Speakers Bureau. Smith:Pharmacyclics: Research Funding; Genentech/Roche: Consultancy, Other: Support of parent study and funding of editorial support, Research Funding; Acerta: Research Funding; BMS: Consultancy; Karyopharm: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; FortySeven: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy. Prica:astra zeneca: Honoraria; seattle genetics: Honoraria; Gilead: Honoraria. Zain:Kyowa Kirlin: Research Funding; Mundai Pharma: Research Funding; Seattle Genetics: Research Funding. Glenn:Genentech: Research Funding. Mehta-Shah:Verastem: Research Funding; Karyopharm Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Innate Pharmaceuticals: Research Funding; Celgene: Research Funding; Genetech: Research Funding; C4 Therapeutics: Consultancy; Bristol Myers-Squibb: Research Funding. Boutrin:Astrazeneca: Current Employment, Current equity holder in private company. Zhao:Astrazeneca: Current Employment, Current equity holder in private company. Cheng:Astrazeneca: Current Employment, Current equity holder in private company. Standifer:Astrazeneca: Current Employment, Current equity holder in private company. Carlesso:Astrazeneca: Current Employment, Current equity holder in private company. Siu:Shatthucks: Research Funding; Symphogen: Consultancy, Research Funding; Tessa: Consultancy; Treadwell Therapeutics: Consultancy, Current Employment; Pfizer: Research Funding; Voronol: Consultancy; Rubius Therapeutics: Consultancy; Abbvie: Research Funding; Agios: Current equity holder in publicly-traded company; Arvinas: Consultancy; Astellas: Research Funding; AstraZeneca: Consultancy, Research Funding; Bayer: Research Funding; Boerhinger-Ingelheim: Research Funding; Bristol-Myers Squibb: Research Funding; Glaxo Smith Kline: Research Funding; Intensity Therapeutics: Research Funding; Navire: Consultancy; Novartis: Research Funding; Oncorus: Consultancy; Mirati: Consultancy, Research Funding; Roche/Genentech: Consultancy; Relay Therapeutics: Consultancy; Roche: Consultancy; Merck: Consultancy.

AIMS: To characterize the pharmacokinetics (PK) of moxetumomab pasudotox, an anti‐CD22 recombinant immunotoxin, in adults with relapsed or refractory hairy cell leukaemia, we examined data from a phase 1 study (Study 1001; n = 49) and from the pivotal clinical study (Study 1053; n = 74). METHODS: Data from both studies were pooled (n = 123) to develop a population PK model. Covariates included demographics, disease state, liver and kidney function, prior treatment, and antidrug antibodies (ADAs). Exposure–response and exposure–safety were analysed separately by study. A 1‐compartment model with linear elimination from the central compartment and 2 clearance (CL) rates was developed. RESULTS: Moxetumomab pasudotox was cleared more rapidly after cycle 1, day 1 (CL(1) = 24.7 L/h) than subsequently (CL(2) = 3.76 L/h), with high interindividual variability (116 and 109%, respectively). In Study 1053, patients with ADA titres >10 240 showed ~4‐fold increase in CL. Higher exposures (≥median) were related to higher response rates, capillary leak syndrome and increased creatinine (Study 1053 only), or grade ≥3 adverse events (Study 1001 only). Clinical benefits were still observed in patients with lower exposure or high ADA titres. CONCLUSION: Despite a high incidence of immunogenicity with increased clearance, moxetumomab pasudotox demonstrated efficacy in hairy cell leukaemia.

313 Background: In a phase II study in uHCC (Study 22, NCT02519348), a novel, priming combination regimen of tremelimumab (T; anti-CTLA-4) and durvalumab (D; anti-PD-L1) (T300+D) has shown favorable clinical activity vs each agent as monotherapy or vs another combination (T75+D). The analyses presented here assess the pharmacokinetics (PK) and relationships between tremelimumab exposure, as monotherapy or in combination, and safety, efficacy, and pharmacodynamics (PD) in this study. Methods: Overall, 216 pts were included in these analyses (T, n=65; T300+D, n=72; T75+D, n=79). Safety, antitumor activity, PK, PD, and immunogenicity were analyzed using standard pharmacometrics methods. A previously developed population PK model for T across solid tumors was validated using T monotherapy and combination therapy data from Study 22, including a post-hoc covariate analysis to assess the impact of covariates on individual PK parameters. Population PK and PD models related individual T exposures to safety parameters, PD, and efficacy (overall survival, OS; progression-free survival, PFS; and objective response rate, ORR). The E-R relationships for time-to-event variables OS and PFS were explored by Kaplan-Meier estimates and analyzed by Cox proportional-hazards models (CPHM). Results: For T monotherapy and T+D combinations, no significant E-S relationships were observed for grade 3/4 treatment-related adverse events (TRAEs), grade 3/4 TRAEs of special interest, and AEs leading to treatment discontinuation. Analyses of each quartile of T exposure suggest pts with higher exposure (3rd and 4th quartile) may have longer OS vs lower quartiles. The CPHManalysis showed that after accounting for prognostic factors (baseline albumin and neutrophil-to-lymphocyte ratio), neither AUC nor Cmin appeared to be a significant factor for OS hazard. There was no significant relationship between response and ORR, PFS and any T PK exposure metric in T-treated pts. Saturable relationships (described by Emax) were observed for maximum change from baseline for proliferating T-cell counts as functions of exposure. Conclusions: The observed PK data are generally consistent with predictions based on a historical population PK model, suggesting the PK of T in uHCC pts is consistent with pts with other solid tumors. No significant relationships were observed between E-S and E-R; therefore, PK is not a significant predictor to evaluate for T efficacy or safety. Considering the small sample size limitation, still the saturable relationships observed in proliferating T-cells appear to support a dose of T300. Future studies of pooled data from Study 22 and the larger phase III HIMALAYA study (NCT03298451) will be conducted to further the characterization of E-R relationships and the development of the T300+D regimen. Clinical trial information: NCT02519348.

Background Receptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell-surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface. Methods We developed both a flow cytometry-based free RO assay to detect free surface CXCR4, and a total surface CXCR4 assay. In an effort to evaluate potential displacement interference, we performed in vitro experiments to compare on-cell affinity with the IC50 values from in vitro and in vivo from the free CXCR4 assay. We determined free and total surface CXCR4 on circulating blood cells in cynomolgus monkeys dosed with MEDI3185, a fully human monoclonal antibody to CXCR4. Results We devised an approach to evaluate displacement interference during assay development and showed that our free assay demonstrated little to no displacement interference. After dosing cynomolgus monkeys with MEDI3185, we observed dose-dependence in the magnitude and duration of receptor occupancy and found CXCR4 to increase on lymphocytes, monocytes, and granulocytes. In a multiple dose study, we observed time points where surface CXCR4 appeared fully occupied but MEDI3185 was not detectable in serum. These paradoxical results represented a type of assay interference, and by comparing pharmacokinetic, ADA and total CXCR4 results, the most likely reason for the free CXCR4 results was the emergence of neutralizing anti-drug antibodies (ADA). The total CXCR4 assay was unaffected by ADA and provided a reliable marker of target modulation in both in vivo studies. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published byWiley Periodicals, Inc.

There has been significant interest in combining anti-PD-1/PD-L1 agents with other clinically active anticancer agents. Gefitinib, a first-generation inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for non-small cell lung cancer (NSCLC) patients with sensitizing EGFR mutations. Durvalumab has demonstrated clinical activity in NSCLC and is approved for Stage III, unresectable NSCLC that has not progressed following platinum-based chemotherapy and radiotherapy. A phase I trial (NCT02088112) combining gefitinib and durvalumab was initiated to establish the safety profile of this combination in tyrosine-kinase inhibitor (TKI)-naive patients with NSCLC containing EGFR-positive sensitizing mutations. As part of this trial, paired tumor biopsies and multiple blood samples were collected for biomarker evaluation. Peripheral blood samples were analyzed for gene expression, cytokine production, immunophenotyping, and circulating DNA (ctDNA). Paired tumor biopsies were evaluated for expression of multiple proteins by immunohistochemistry, including phosphorylated EGFR (pEGFR), PD-L1, CD73, and CD8. We saw ≥50% inhibition of pEGFR in 8 of 17 paired biopsies but no consistent directional changes with PD-L1. We observed known durvalumab-mediated effects such as increases in tumor CD8+ cells (3.7 fold, p=0.011), proliferating CD8+ cells in circulation (2.4 fold, p=0.074), and increased interferon gamma (10.3 fold, p=0.01). A cohort of patients (n=10) was treated with a 28-day gefitinib run-in prior to addition of durvalumab. Sample collection during this period allowed us to evaluate the effects of gefitinib alone on immunologic readouts. While treatment with gefitinib alone resulted in ≥50% reduction of pEGFR in 5 of 8 paired tumor biopsies, its effect on expression of immune-related genes, interferon-related cytokines, and immune subpopulations was minimal. Interestingly, we saw an association between higher baseline tumor PD-L1, pEGFR or CD73 expression and a numerical trend for longer progression-free survival. Finally, analysis of ctDNA showed robust decreases in mutant EGFR could be observed as early as Day 5 of gefitinib treatment, either alone or in combination with durvalumab. Altogether, these results demonstrate that each agent had its expected downstream biologic effects and there was no antagonism between the two agents. We did not observe any novel biologic effects nor any striking synergy for known readouts. New hypotheses (i.e., high PD-L1/pEGFR/CD73) for patient selection biomarkers were identified, although more follow-up is required to determine whether they reflect combination or single-agent treatment. These studies highlight the utility of collecting biomarker samples to confirm target engagement, understand downstream effects, identify patient selection biomarkers, and explore ctDNA biomarkers. Citation Format: Tammie C. Yeh, Helen K. Angell, Vicky Rowlands, Emma V. Jones, Vidalba Rocher Ros, Alison Pritchard, Martine P. Roudier, Nathan Standifer, Mike Kuziora, Philip Martin, Kirill Peskov, Rosie Taylor, J. Carl Barrett, Ben Sidders. Exploratory biomarker analyses of tumor and peripheral blood samples from the phase I durvalumab plus gefitinib trial in EGFR-mutated NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B55.

International audience; This is a pivotal, multicenter, open-label study of moxetumomab pasudotox, a recombinant CD22-targeting immunotoxin, in hairy cell leukemia (HCL), a rare B cell malignancy with high CD22 expression. The study enrolled patients with relapsed/ refractory HCL who had ≥2 prior systemic therapies, including ≥1 purine nucleoside analog. Patients received moxetumomab pasudotox 40 µg/kg intravenously on days 1, 3, and 5 every 28 days for ≤6 cycles. Blinded independent central review determined disease response and minimal residual disease (MRD) status. Among 80 patients (79% males; median age, 60.0 years), durable complete response (CR) rate was 30%, CR rate was 41%, and objective response rate (CR and partial response) was 75%; 64 patients (80%) achieved hematologic remission. Among complete responders, 27 (85%) achieved MRD negativity by immunohistochemistry. The most frequent adverse events (AEs) were peripheral edema (39%), nausea (35%), fatigue (34%), and headache (33%). Treatment-related serious AEs of hemolytic uremic syndrome (7.5%) and capillary leak syndrome (5%) were reversible and generally manageable with supportive care and treatment discontinuation (6 patients; 7.5%). Moxetumomab pasudotox treatment achieved a high rate of independently assessed durable response and MRD eradication in heavily pretreated patients with HCL, with acceptable tolerability.

Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on the cell surface and are frequently used to generate pharmacodynamic (PD) biomarker data in nonclinical and clinical studies of biopharmaceuticals. When combined with the pharmacokinetic (PK) profile, RO data can establish PKPD relationships, which are crucial for informing dose decisions. RO is commonly measured by flow cytometry on fresh blood specimens and is subject to numerous technical and logistical challenges. To ensure that reliable and high quality results are generated from RO assays, careful assay design, key reagent characterization, data normalization/reporting, and thorough planning for implementation are of critical importance during development. In this article, the authors share their experiences and perspectives in these areas and discuss challenges and potential solutions when developing and implementing a flow cytometry‐based RO method in support of biopharmaceutical drug development. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10–producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10–dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4 + CD62L − FoxP3 − , suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.

Objective Interleukin 1-beta (IL-1β) is a major inflammatory cytokine. Blockade of the IL-1β pathway is therapeutically efficacious in type 2 diabetes, but the mechanistic effects on the immune system are incompletely understood. Research design We administered an IL-1 receptor antagonist, anakinra, to 7 type 1 diabetes patients in order to investigate the immunologic and metabolic effects of this drug. Mechanistic assays were performed before and after drug administration. Results A novel signature was observed, with reduced serum interleukin 8 (IL-8) levels and reduced CD11b integrin expression on monocytes associated with increased CXCR1 expression. Conclusions This set of linked phenotypes suggests that blockade of the IL-1β pathway results in the reduced ability of mononuclear cells to traffic to sites of inflammation. Mechanistic studies from large scale trials using IL-1 blockade in type 1 diabetes should focus on changes in monocyte trafficking and the IL-8 pathway.

Introduction: AZD6738 is a potent inhibitor of the ATR protein kinase that is being tested in patients with solid malignancies. In Phase I dose escalation studies, AZD6738 was dosed as monotherapy (continuous schedule from 40 - 480 mg per day), or in combination (intermittent schedule from 80 - 320 mg per day) with olaparib, durvalumab, carboplatin, or radiation. In those studies, thrombocytopenia was the main dose limiting toxicity and changes in peripheral monocytes and proliferating T-cells were seen with AZD6738, with up to 80% decrease on-drug and return to baseline upon cessation of dosing. This effect on monocytes and proliferating T cells was not observed with either single agent olaparib or durvalumab. Based on these observations, we developed two PKPD models linking AZD6738 dose, AZD6738 plasma exposure, and differential blood cell counts to support dose and schedule selection. Methods and Results: The first model characterized the quantitative relationship between the plasma exposure of AZD6738 monotherapy, and the difference between monocyte decrease (on-target activity) and platelet decrease (on-target toxicity). This model was a sigmoid function and predicted biological activity starting at exposures of 2.5 ug/mL (aligned with the in vitro IC90 in LoVo cells, and obtained in ≥ 50% of patients with 80 mg per day) increasing in a concentration dependent manner to 10 ug/mL (obtained in almost all patients dosed at 480 mg per day), after which negligible monocyte response over platelet decrease was observed. Those data were consistent with the preclinical observations which showed tumour regression after at least 3-day concomitant treatment of olaparib and AZD6738 at exposure maintaining IC90 [1]. The second model was a PK-safety model describing the temporal relationship between circulating platelets and exposure of AZD6738 administered in combination with olaparib dosed at 300 mg twice daily continuously. This model allowed generation of various platelet/ time profiles in the most sensitive patient group (10% of the patients) for different AZD6738 doses and schedules. The simulations indicated that, for doses considered biologically relevant by the first model, a period of 21-days free of AZD6738 is needed to give full recovery of potential thrombocytopenia in a 28-day cycle. Conclusion: Using monocyte and proliferative T-cell decreases as biological activity indicators and platelet decrease as a safety indicator, we quantified the exposure/activity/safety relationship for AZD6738. The recommended Phase II dose of AZD6738 (in combination with olaparib) was then driven by maintaining maximally active exposure consistent with manageable safety. Reference: [1]. 2017 AACR Abstract 2494. Acknowledgements: T.A. Yap (The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, London, UK) and J.C. Soria (Institut Gustave Roussy, Paris, France) Citation Format: Alienor Berges, S. Y. Amy Cheung, Andrew J. Pierce, Emma Dean, Brunella Felicetti, Nathan Standifer, Simon Smith, James Yates, Alan Lau, Christine Stephens, Matthew Krebs, Kevin Harrington, Simon J. Hollingsworth. PK-Biomarker-Safety modelling aids choice of recommended Phase II dose and schedule for AZD6738 (ATR inhibitor) [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 CT118.

7004Background: A pivotal multicenter, single-arm study evaluated moxetumomab pasudotox, a first-in-class recombinant immunotoxin, in patients (pts) with relapsed/refractory hairy cell leukemia (HC...

7061Background: Moxe is a recombinant immunotoxin that specifically binds to CD22 on the B cell surface and internalizes leading to cell death. The PK, PD, and immunogenicity of moxe were evaluated...

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+CCR7−CD45RA−effector memory T cells (TEMcells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (TCM) cells. In TEMcells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvβ2, SAP97, ZIP, p56lck, and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific TEMcells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

Type 1 diabetes results from the autoimmune destruction of insulin-producing pancreatic beta-cells by cytotoxic T-lymphocytes (CTLs). In humans, few beta-cell epitopes have been reported, thereby limiting the study of beta-cell-specific CTLs in type 1 diabetes. To identify additional epitopes, HLA class I peptide affinity algorithms were used to identify a panel of peptides derived from the beta-cell proteins islet amyloid polypeptide (IAPP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulin, insulinoma-associated antigen 2 (IA-2), and phogrin that were predicted to bind HLA-A*0201. Peripheral blood mononuclear cells from 24 HLA-A*0201 recent-onset type 1 diabetic patients and 11 nondiabetic control subjects were evaluated for gamma-interferon secretion in response to peptide stimulation in enzyme-linked immunospot assays. We identified peptides IAPP9-17, IGRP215-223, IGRP152-160, islet IA-2(172-180), and IA-2(482-490) as novel HLA-A*0201-restricted T-cell epitopes in type 1 diabetic patients. Interestingly, we observed a strong inverse correlation between the binding affinity of beta-cell peptides to HLA-A*0201 and CTL responses against those peptides in recent-onset type 1 diabetic patients. In addition, we found that self-reactive CTLs with specificity for an insulin peptide are frequently present in healthy individuals. These data suggest that many beta-cell epitopes are recognized by CTLs in recent-onset type 1 diabetic patients. These epitopes may be important in the pathogenesis of type 1 diabetes.

e14063 Background: AZD6738 is a potent, selective inhibitor of the ataxia telangiectasia and Rad3-related (ATR) serine/threonine-specific protein kinase with good selectivity against other phosphatidylinositol 3-kinase-related kinase (PIKK) family members. To support dose/schedule guidance through mathematical modelling of clinical pharmacokinetic/pharmacodynamic relationships, we sought to determine quantitative pharmacodynamic effects of AZD6738 in peripheral blood with respect to drug exposure. Methods: We assessed peripheral blood cell subpopulations in clinical studies of AZD6738 used either as a monotherapy, a combination with the PARP-1 inhibitor olaparib and in combination with the PD-L1 inhibitor durvalumab through both blood analyser and flow cytometry characterization. Peripheral pharmacokinetics of AZD6738 were also assessed. Results: Levels of peripheral monocytes were suppressed in a dose-dependent manner for the duration of AZD6738 dosing resulting in a decrease of up to 80% within the first 7 consecutive days of AZD6738 treatment; these results were consistent across studies. Upon cessation of an AZD6738 dosing interval monocyte levels return to baseline values within 14 days; in combination with durvalumab there is a trend for the rebound in monocyte counts to surpass baseline levels by up to 20%. Monocyte suppression is specific to AZD6738 and not found with either single agent olaparib or single agent durvalumab, possibly attributable to defective base excision repair in monocytes and lack of PARP-1. Monocytes were found to be the most sensitive cell type to AZD6738 treatment compared with neutrophils, platelets, haemoglobin or lymphocytes. Conclusions: This drug effect of AZD6738 is consistent with natural clearance of peripheral monocytes under suppression of monocyte precursor proliferation in the bone marrow, suggestive of a change in the equilibrium between production and elimination of peripheral monocytes. Used as a surrogate marker of target inhibition, a mathematical model of the AZD6738/monocyte dose-exposure-response relationship is described and has been used to inform AZD6738 Phase 2 dose and schedule.

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

OBJECTIVE To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients. RESEARCH DESIGN AND METHODS We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays. RESULTS Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells. CONCLUSIONS We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.

The activation threshold for antigen-specific T cell responses is dependent on the avidity of the trimolecular interaction between TCR, antigen, and MHC. We compared CD4+ T cell avidities for the diabetes-associated autoantigen glutamic acid decarboxylase 555-567 (GAD 555) among serial samples from autoantibody-positive subjects at high risk of progression to type 1 diabetes (T1D). T cells from three at-risk subjects demonstrated significant avidity increases (p

Summary Alloreactivity is one of the most serious problems in organ transplantation. It has been hypothesized that pre-existing alloreactive T cells are actually cross-reacting cells that have been primed by the autologous major histocompatibility complex (MHC) and a specific peptide. CD8+ cytotoxic T lymphocytes that are alloreactive and recognize a virus-peptide that is presented by the autologous MHC have been reported. Here we demonstrate a cross-reactivity that exists between DQ0602 restricted, herpes simplex type 2 VP16 40–50 specific CD4+ T-cell clones, which can be alloreactive to DQ0601. Though most of the DQ0602 restricted T-cell clones we isolated from two different donors were not alloreactive, weakly cross-reacting T-cell clones could be isolated from both donors. Two strongly cross-reacting T-cell clones with high affinity interaction of their T-cell receptor (TCR) with both DQ0602/VP16 40–50 and DQ0601 could be isolated from one donor. DNA sequencing of the a fragment of the Vβ gene used in their TCR confirmed that these two T cells indeed are two independent clones. These clones are cytotoxic and produce cytokines of a T helper 2-like pattern. Possible implications in a DR-matched transplantation setting are discussed.