Faculty Bibliography

OBJECTIVE:Apoptotic cell receptors contribute to the induction of tolerance by modulating dendritic cell function following the uptake of apoptotic cells or microparticles. Dendritic cells that have bound or ingested apoptotic cells produce only low amounts of pro-inflammatory cytokines and fail to prime effector T cell responses. Specifically, ligation of the apoptotic cell receptor CR3 (CD11 b/CD18) on human monocyte-derived dendritic cells (moDC) down-modates proinflammatory cytokine secretion, but the consequences for human Th17 cell homeostasis and effector responses remain unknown. Here, we aimed to establish whether CD11b-ligated moDC modulate Th17 cell effector reponses to assess their potential for future use in moDC-based suppressive immunotherapy. METHODS:We generated a bead-based surrogate system to target CD11b on monocyte-derived human dendritic cells and examined the effects of CD11b ligation on Th17-skewing cytokine secretion, priming, expansion and functional plasticity in DC/T cell co-culture systems at the poly- and monoclonal level. RESULTS:T cell compartment was efficiently constricted by targeting the CD11b receptor on moDC. This tolerogenic capacity was primarily dependent on cytokine skewing. Furthermore, ligation of CD11b on healthy homozygous carriers of the rs11143679 (ITGAM) variant - a strong genetic susceptibility marker for human systemic lupus erythematosus - also down-modulated the secretion of Th17-skewing cytokines. CONCLUSION/CONCLUSIONS:Overall, our findings underline the potential of targeted CD11b ligation on human dendritic cells for the engineering of suppressive immunotherapy for Th17-related autoimmune disorders.

BACKGROUND:Cancer-testis antigen NY-ESO-1 is a highly immunogenic melanoma antigen which has been incorporated into adjuvant vaccine clinical trials. Three such early-phase trials were conducted at our center among patients with high-risk resected melanoma. We herein report on the pooled long-term survival outcomes of these patients in comparison to historical controls. METHODS:All melanoma patients treated at NYU Langone Health under any of three prospective adjuvant NY-ESO-1 vaccine trials were retrospectively pooled into a single cohort. All such patients with stage III melanoma were subsequently compared to historical control patients identified via a prospective institutional database with protocol-driven follow-up. Survival times were calculated using the Kaplan-Meier method, and Cox proportional hazard models were employed to identify significant prognostic factors and control for confounding variables. RESULTS:A total of 91 patients were treated with an NY-ESO-1 vaccine for the treatment of high-risk resected melanoma. Of this group, 67 patients were stage III and were selected for comparative analysis with 123 historical control patients with resected stage III melanoma who received no adjuvant therapy. Among the pooled vaccine cohort (median follow-up 61 months), the estimated median recurrence-free survival was 45 months, while the median overall survival was not yet reached. In the control cohort of 123 patients (median follow-up 30 months), the estimated median recurrence-free and overall survival were 22 and 58 months, respectively. Within the retrospective stage III cohort, NY-ESO-1 vaccine was associated with decreased risk of recurrence (HR = 0.56, p < 0.01) and death (HR = 0.51, p = 0.01). Upon controlling for sub-stage, the adjuvant NY-ESO-1 clinical trial cohort continued to exhibit decreased risk of recurrence (HR = 0.45, p < 0.01) and death (HR = 0.40, p < 0.01). CONCLUSIONS:In this small retrospective cohort of resected stage III melanoma patients, adjuvant NY-ESO-1 vaccine immunotherapy was associated with longer recurrence-free and overall survival relative to historical controls. These data support the continued investigation of adjuvant NY-ESO-1 based immunotherapy regimens in melanoma.

Background Immunotherapy has demonstrated promising antitumor activity in various advanced cancers. Combined tumor targeting from multiple drugs with unique mechanisms may provide further improved outcomes. Tremelimumab (TRE) is a CTLA-4 antibody and durvalumab (DUR) blocks PD-L1. Poly-ICLC is a toll-like receptor 3 agonist. Intratumoral (intra-T) injection of poly-ICLC directly alters the tumor microenvironment (TME), and by creating an in situ vaccination, may trigger a clinically effective systemic anti-tumor response when also combined with DUR and TRE. Methods This is an ongoing Phase 1/2, open-label, multicenter study (NCT02643303). The study evaluates the use of intra-T administration of TRE and IV DUR + poly-ICLC (intra-T and intramuscular [IM]) to determine the safety, preliminary efficacy and immune activity of this regimen in patients with advanced, measurable, biopsyaccessible tumors: head and neck squamous cell carcinoma, breast cancer, sarcoma, merkel cell carcinoma, cutaneous T cell lymphoma, melanoma, genitourinary cancer, and other solid tumors. Phase 1 determines the recommended combination dosing (RCD) for the regimen with dose de-escalation based on dose limiting toxicities (DLTs) and standard 3 + 3 rules. Starting doses are: DUR, 1500 mg IV; TRE, 75 mg IV; TRE, 10 mg intra-T; poly-ICLC, 1 mg intra-T/IM. Phase 1 starts with Cohort 1A (DUR + poly-ICLC). Upon demonstration of tolerability, enrollment proceeds with Cohort 1B (DUR + IV TRE + poly-ICLC) and Cohort 1C (DUR + intra-T TRE + poly-ICLC). The RCD is the highest dose at which < 2/6 patients have DLTs. In Phase 2, up to 66 evaluable patients are treated using the RCD regimen, with enrollment of 6 patients per tumor type initially, and enrollment of 6 additional patients per 3 tumor types contingent upon at least 1 response among the initial 6 patients. Study endpoints are RCD and safety, objective response rate, progression-free survival, and overall survival. Exploratory endpoints are biological activity, including effects on the TME and immunological responses

Background: Immunotherapy has demonstrated promising antitumor activity in various advanced cancers. Combined tumor targeting from multiple drugs with unique mechanisms may provide further improved outcomes. Tremelimumab (TRE) is a CTLA-4 antibody and durvalumab (DUR) blocks PD-L1. Poly-ICLC is a toll-like receptor 3 agonist. Intratumoral (intra-T) injection of poly-ICLC directly alters the tumor microenvironment (TME), and by creating an in situ vaccination, may trigger a clinically effective systemic anti-tumor response when also combined with DUR and TRE. Methods: This is an ongoing Phase 1/2, open-label, multicenter study (NCT02643303). The study evaluates the use of intra-T administration of TRE and IV DUR + poly-ICLC (intra-T and intramuscular [IM]) to determine the safety, preliminary efficacy and immune activity of this regimen in patients with advanced, measurable, biopsy-accessible tumors: head and neck squamous cell carcinoma, breast cancer, sarcoma, merkel cell carcinoma, cutaneous T-cell lymphoma, melanoma, genitourinary cancer, and other solid tumors. Phase 1 determines the recommended combination dosing (RCD) for the regimen with dose de-escalation based on dose limiting toxicities (DLTs) and standard 3 + 3 rules. Starting doses are: DUR, 1500 mg IV; TRE, 75 mg IV; TRE, 10 mg intra-T; poly-ICLC, 1 mg intra-T/IM. Phase 1 starts with Cohort 1A (DUR + poly-ICLC). Upon demonstration of tolerability, enrollment proceeds with Cohort 1B (DUR + IV TRE + poly-ICLC) and Cohort 1C (DUR + intra-T TRE + poly-ICLC). The RCD is the highest dose at which < 2/6 patients have DLTs. In Phase 2, up to 66 evaluable patients are treated using the RCD regimen, with enrollment of 6 patients per tumor type initially, and enrollment of 6 additional patients per 3 tumor types contingent upon at least 1 response among the initial 6 patients. Study endpoints are RCD and safety, objective response rate, progression-free survival, and overall survival. Exploratory endpoints are biological activity, including effects on the TME and immunological responses. Enrollment opened on 28 Dec 2016

BACKGROUND:Melanoma is a heterogeneous malignancy. We set out to identify the molecular underpinnings of high-risk melanomas, those that are likely to progress rapidly, metastasize, and result in poor outcomes. METHODS:We examined transcriptome changes from benign states to early-, intermediate-, and late-stage tumors using a set of 78 treatment-naive melanocytic tumors consisting of primary melanomas of the skin and benign melanocytic lesions. We utilized a next-generation sequencing platform that enabled a comprehensive analysis of protein-coding and -noncoding RNA transcripts. RESULTS:Gene expression changes unequivocally discriminated between benign and malignant states, and a dual epigenetic and immune signature emerged defining this transition. To our knowledge, we discovered previously unrecognized melanoma subtypes. A high-risk primary melanoma subset was distinguished by a 122-epigenetic gene signature ("epigenetic" cluster) and TP53 family gene deregulation (TP53, TP63, and TP73). This subtype associated with poor overall survival and showed enrichment of cell cycle genes. Noncoding repetitive element transcripts (LINEs, SINEs, and ERVs) that can result in immunostimulatory signals recapitulating a state of "viral mimicry" were significantly repressed. The high-risk subtype and its poor predictive characteristics were validated in several independent cohorts. Additionally, primary melanomas distinguished by specific immune signatures ("immune" clusters) were identified. CONCLUSION/CONCLUSIONS:The TP53 family of genes and genes regulating the epigenetic machinery demonstrate strong prognostic and biological relevance during progression of early disease. Gene expression profiling of protein-coding and -noncoding RNA transcripts may be a better predictor for disease course in melanoma. This study outlines the transcriptional interplay of the cancer cell's epigenome with the immune milieu with potential for future therapeutic targeting. FUNDING/BACKGROUND:National Institutes of Health (CA154683, CA158557, CA177940, CA087497-13), Tisch Cancer Institute, Melanoma Research Foundation, the Dow Family Charitable Foundation, and the Icahn School of Medicine at Mount Sinai.

Background: Somatic mut in DDR genes have been associated with increased sensitivity to cisplatin in UC. Higher mut load has correlated with response to immune checkpoint blockade. We hypothesized that DDR mut result in higher mut load and DDR mut UC may be particularly sensitive to both chemotherapy and immune checkpoint blockade. Methods: Three cohorts were utilized: (1) TCGA UC cohort (n = 389), (2) Mount Sinai (MS) cohort (n = 67) of UC (cystectomy) specimens subjected to targeted exome sequencing for 341 genes (MSK-IMPACT), and (3) Phase 2 trial of gemcitabine, cisplatin, plus ipilimumab (GCI) in metastatic UC from which 28/36 enrolled patients (pts) had specimens suitable for whole exome sequencing. DDR mut were defined as somatic alterations in one of 52 genes. Deleterious (del) mut were defined as nonsense, frameshift, splice site, or hotspot point mut. Results: The mut load using all genes in the TCGA cohort, and restricted to the 341 IMPACT genes, were highly correlated (r = 0.81, p < 0.001). Associations between del DDR mut and mut load are shown (Table). In the MS cohort, CD8+cells/mm2 by IHC were higher in tumors with del DDR mut versus no DDR mut (p = 0.04). In the GCI cohort, the sensitivity, specificity, positive predictive value, and negative predictive value of a del DDR mut for objective response to treatment was 40.9% (95% CI 20.7-63.7%), 86.7% (95% CI 42.1-99.4%), 90% (55.5-99.8%), and 31.6% (95% CI 12.6-56.6%), respectively. Median progression-free survival in the GCI cohort was 308 days (95% CI 270-NR) in del DDR mut and 196 days (95% CI 185-372) in others (p = 0.24). Notably, 2/9 pts with del DDR mutations, and with the highest mut loads, achieved complete responses after GCI and are alive without evidence of disease at 2.1+ and 1.8+ years. Conclusions: DDR mut are associated with higher mut load in UC, a high likelihood of response to GCI, and may identify a subset of pts achieving durable disease control. GC plus PD-1/PD-L1 blockade should be explored in DDR mut UC

The nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome, a multiprotein complex, is an essential intracellular mediator of antiviral immunity. In murine dendritic cells, this complex responds to a wide array of signals, including ion efflux and influenza A virus infection, to activate caspase-1-mediated proteolysis of IL-1beta and IL-18 into biologically active cytokines. However, the presence and function of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells, in response to various triggers, including viral infection, has not been defined clearly. Here, we delineate the contribution of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome to the secretion of IL-1beta, IL-18, and IL-1alpha by human dendritic cells (monocyte-derived and primary conventional dendritic cells). Activation of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells by various synthetic activators resulted in the secretion of bioactive IL-1beta, IL-18, and IL-1alpha and induction of pyroptotic cell death. Cellular IL-1beta release depended on potassium efflux and the activity of proteins nucleotide-binding oligomerization domain-like receptor protein 3 and caspase-1. Likewise, influenza A virus infection of dendritic cells resulted in priming and activation of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome and secretion of IL-1beta and IL-18 in an M2- and nucleotide-binding oligomerization domain-like receptor protein 3-dependent manner. The magnitude of priming by influenza A virus varied among different strains and inversely corresponded to type I IFN production. To our knowledge, this is the first report describing the existence and function of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells and the ability of influenza A virus to prime and activate this pathway in human dendritic cells, with important implications for antiviral immunity and pathogenesis.

Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.

The efficacy of PD-1/PD-L1 targeted therapies in addition to anti-CTLA-4 solidifies immunotherapy as a modality to add to the anticancer arsenal. Despite raising the bar of clinical efficacy, immunologically targeted agents raise new challenges to conventional drug development paradigms by highlighting the limited relevance of assessing standard pharmacokinetics (PK) and pharmacodynamics (PD). Specifically, systemic and intratumoral immune effects have not consistently correlated with standard relationships between systemic dose, toxicity, and efficacy for cytotoxic therapies. Hence, PK and PD paradigms remain inadequate to guide the selection of doses and schedules, both starting and recommended Phase 2 for immunotherapies. The promise of harnessing the immune response against cancer must also be considered in light of unique and potentially serious toxicities. Refining immune endpoints to better inform clinical trial design represents a high priority challenge. The Cancer Immunotherapy Trials Network investigators review the immunodynamic effects of specific classes of immunotherapeutic agents to focus immune assessment modalities and sites, both systemic and importantly intratumoral, which are critical to the success of the rapidly growing field of immuno-oncology.

OBJECTIVE: Plasma soluble CD40 ligand (sCD40L) is increased during HIV-1 infection, but it is unknown whether it circulates in monomeric or multimeric forms, and whether the circulating forms have differential effects on myeloid dendritic cell function and adaptive regulation. DESIGN: sCD40L forms were measured in plasma samples from HIV-infected donors. The effects of sCD40L forms on dendritic cell function were measured in vitro. METHODS: To delineate which forms of sCD40L are present in plasma from HIV-infected donors, immunoblots were performed following enrichment of plasma for medium and low-abundance proteins. Dendritic cells from seronegative donors were exposed to multiple forms of sCD40L prior to Toll-like receptor stimulation and dendritic cell function and adaptive regulation was assessed in vitro. RESULTS: Monomeric and multimeric forms of sCD40L were identified in plasma from antiretroviral therapy-treated HIV-infected donors. Although monomeric and multimeric forms of sCD40L had differential effects on dendritic cell activation when given alone, both strongly suppressed secretion of the Th1 skewing cytokine, interleukin-12, upon subsequent Toll-like receptor stimulation. Furthermore, dendritic cells exposed to both monomeric and multimeric sCD40L induced regulatory T-cell formation and T-cell anergy. CONCLUSION: Elevated sCD40L during HIV infection impairs dendritic cell function, contributing to innate and adaptive immune dysfunction. Antiretroviral adjunctive therapies that decrease sCD40L may provide immune modulatory benefits.