ALBERT

Description: Endogenous cannabinoids (endocannabinoids) are small molecules biosynthesized from membrane glycerophospholipid. Anandamide (AEA) is an endogenous intestinal cannabinoid that controls appetite and energy balance by engagement of the enteric nervous system through cannabinoid receptors. Here, we uncover a role for AEA and its receptor, cannabinoid receptor 2 (CB2), in the regulation of immune tolerance in the gut and the pancreas. This work demonstrates a major immunological role for an endocannabinoid. The pungent molecule capsaicin (CP) has a similar effect as AEA; however, CP acts by engagement of the vanilloid receptor TRPV1, causing local production of AEA, which acts through CB2. We show that the engagement of the cannabinoid/vanilloid receptors augments the number and immune suppressive function of the regulatory CX3CR1hi macrophages (Mϕ), which express the highest levels of such receptors among the gut immune cells. Additionally, TRPV1−/− or CB2−/− mice have fewer CX3CR1hi Mϕ in the gut. Treatment of mice with CP also leads to differentiation of a regulatory subset of CD4+ cells, the Tr1 cells, in an IL-27–dependent manner in vitro and in vivo. In a functional demonstration, tolerance elicited by engagement of TRPV1 can be transferred to naïve nonobese diabetic (NOD) mice [model of type 1 diabetes (T1D)] by transfer of CD4+ T cells. Further, oral administration of AEA to NOD mice provides protection from T1D. Our study unveils a role for the endocannabinoid system in maintaining immune homeostasis in the gut/pancreas and reveals a conversation between the nervous and immune systems using distinct receptors.

Description: Immune checkpoint therapy (ICT) has transformed cancer treatment in recent years; however, treatment response is not uniform across tumor types. The tumor immune microenvironment plays a critical role in determining response to ICT; therefore, understanding the differential immune infiltration between ICT-sensitive and ICT-resistant tumor types will help to develop effective treatment strategies. We performed a comprehensive analysis of the immune tumor microenvironment of an ICT-sensitive tumor (melanoma, n = 44) and an ICT-resistant tumor (pancreatic cancer, n = 67). We found that a pancreatic tumor has minimal to moderate infiltration of CD3, CD4, and CD8 T cells; however, the immune infiltrates are predominantly present in the stromal area of the tumor and are excluded from tumoral area compared with melanoma, where the immune infiltrates are primarily present in the tumoral area. Metastatic pancreatic ductal adenocarcinomas (PDACs) had a lower infiltration of total T cells compared with resectable primary PDACs, suggesting that metastatic PDACs have poor immunogenicity. Further, a significantly higher number of CD68+ macrophages and VISTA+ cells (also known as V-domain immunoglobulin suppressor of T cell activation) were found in the pancreatic stromal area compared with melanoma. We identified VISTA as a potent inhibitory checkpoint that is predominantly expressed on CD68+ macrophages on PDACs. These data suggest that VISTA may be a relevant immunotherapy target for effective treatment of patients with pancreatic cancer.

Description: Background: Outcomes of patients (pts) with Richter transformation (RT) remain dismal with a median survival of less than 1 year with chemoimmunotherapy (CIT). Dysfunction of T cells, NK cells and other immune subsets is common in pts with CLL. Checkpoint blockade is an emerging treatment approach for pts with RT (Ding et al. Blood 2017; Younes et al. ASH 2017). Methods: We designed an investigator-initiated phase II clinical trial combining nivolumab (anti-PD1 monoclonal antibody) with ibrutinib in pts with R/R CLL or RT (NCT02420912). We report data on the RT cohort. Nivolumab was given 3 mg/kg IV every 2 weeks, starting cycle 1 day 1 for a total of 24 cycles. Ibrutinib was given 420 mg once daily starting cycle 2 day 1 (Ibrutinib could be added during cycle 1, in case of worsening disease) and continued until disease progression or unacceptable toxicities. Each cycle was 4 weeks. Eligibility criteria included age ≥18 years, adequate organ function (total bilirubin ≤1.5 x ULN, ALT and AST ≤3 x ULN, creatinine ≤1.5 x ULN). Pts were included if they had received at least one therapy for CLL or RT (pts with del(17p) could be treatment-naïve). Response assessments were done by PET scan and bone marrow after cycle 1, cycle 3, cycle 6, cycle 9, and cycle 12, cycle 18, and cycle 24. Results: A total of 23 pts with RT have been enrolled. The median age was 65 years (range, 49-88); 10 women, 13 men. The median number of prior therapies for CLL/RT was 3 (range, 0-10); one pt with previously untreated CLL who developed RT with del(17p) was enrolled. Prior therapies included CIT (n=18), ibrutinib (n=11), acalabrutinib (n=1), P13K inhibitor (n=4), venetoclax (n=3), allo-SCT (n=2). A total of 10 pts (43%) responded (complete metabolic response, n=8; partial metabolic response, n=2). The median duration of response (censored for allo-SCT) for the responding pts (n=10) is 9.3 months (Figure 1). Two pts previously treated with ibrutinib responded. A total of 4 pts underwent a subsequent allo-SCT after achieving a response to therapy. Four additional pts underwent allo-SCT after receiving a subsequent salvage therapy. The median overall survival for the entire group (n=23) is 13.8 months (Figure 1). One pt had G3 transaminitis and one pt had grade 4 lipase/amylase elevation. One pt developed grade 2 pneumonitis, and one pt had grade 2 uveitis. Correlative studies, including flow-cytometry and immunohistochemistry for PD1 and PDL1 are ongoing. Conclusions: The combination of nivolumab and ibrutinib has clinical activity in pts with RT with a 43% response rate. Disclosures Jain: Pfizer: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Astra Zeneca: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Verastem: Research Funding; Servier: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Servier: Research Funding; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cellectis: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding; Abbvie: Research Funding; Adaptive Biotechnologioes: Research Funding; Genentech: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Adaptive Biotechnologioes: Research Funding; Cellectis: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Celgene: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Research Funding; Abbvie: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thompson:AbbVie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kadia:Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; BMS: Research Funding; Novartis: Consultancy; BMS: Research Funding; Celgene: Research Funding; Amgen: Consultancy, Research Funding; Celgene: Research Funding; Abbvie: Consultancy; Takeda: Consultancy; Jazz: Consultancy, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Abbvie: Consultancy; Novartis: Consultancy. Pemmaraju:novartis: Research Funding; samus: Research Funding; stemline: Consultancy, Honoraria, Research Funding; daiichi sankyo: Research Funding; abbvie: Research Funding; celgene: Consultancy, Honoraria; cellectis: Research Funding; plexxikon: Research Funding; Affymetrix: Research Funding; SagerStrong Foundation: Research Funding. Khoury:Stemline Therapeutics: Research Funding. O'Brien:Aptose Biosciences Inc.: Consultancy; Vaniam Group LLC: Consultancy; Pfizer: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Regeneron: Research Funding; Alexion: Consultancy; Gilead: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Kite Pharma: Research Funding; Abbvie: Consultancy; Celgene: Consultancy; Astellas: Consultancy; Pharmacyclics: Consultancy, Research Funding; Acerta: Research Funding; Amgen: Consultancy; Janssen: Consultancy; Sunesis: Consultancy, Research Funding. Wierda:Genentech: Research Funding; AbbVie, Inc: Research Funding.

Description: Background: The combination of an anthracycline and cytarabine has remained a standard of care induction regimen for newly diagnosed AML pts for more than 40 years. PD-1 positive CD8+ T-cells are increased in bone marrow (BM) of AML pts and blocking PD-1/PD-L1 pathways enhances anti-leukemia effect of cytotoxic chemotherapy by increasing CD8+ T-cells in murine models. Addition of Nivo to IA induction may prolong event-free, relapse-free and overall survival (EFS, RFS, OS; respectively). We report on updated feasibility and efficacy data. Methods: Pts aged 18-65 yrs with newly diagnosed AML (≥20% blasts by WHO criteria) and high risk MDS (≥10% blasts) were eligible if they had adequate performance status (ECOG ≤2) and organ function. Treatment included 1-2 induction cycles of (A) 1.5 g/m2 over 24 hours (days 1-4) and (I) 12 mg/m2 (days 1-3). Nivo 3 mg/kg was started on day 24±2 and continued every 2 weeks for up to a yr. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi), up to 5 consolidation cycles of attenuated dose I+A was given. Eligible pts received allogeneic stem cell transplant (alloSCT) at any time during or after consolidation. Results: 3 AML pts were treated at a run-in phase with Nivo 1 mg/kg without drug-related toxicity. Subsequently, 41 pts were treated as above. Median (med) age was 54 yrs (range, 26-66) with 42 AML (32 de novo, 7 secondary, 3 therapy-related) and 2 high risk MDS. 19 pts had adverse ELN genetic risk with 8 TP53 mutations (Fig 1A). All pts were evaluable for response: 34 (77%) achieved CR/CRi (28 CR, 6 CRi) and 18/34 (53%) had undetectable MRD by flow cytometry (FC) following induction. Med number of cycles to response was 1 (range 1-2) and med number of total cycles received was 2 (range, 1-6). Counting maintenance schedule, med number of Nivo doses was 2 (range, 0-25) and 15 pts (34%) received ≥4 doses; 4 pts did not receive Nivo: insurance issues (n=2), early death (n=1) and rapid disease progression (n=1). The 4- and 8-week mortality was 5%. At med follow-up of 17.25 mos (range 0.5-30.4), med OS was 18.54 mos (range, 0.5-30.4) and med RFS for the 34 CR/CRi pts was 18.5 mos (range 1.7-25.6); med EFS was not reached (range 0.5-13.7). When compared to a contemporary cohort of pts treated with I+A alone, there is a trend of improvement of OS at a short follow-up duration (med 18.54 vs 13.2 mos; p=0.2). Six pts had grade 3/4 immune-related toxicities with rash (n=2), colitis (n=2), transaminitis and pancreatitis (n=1; each). Grade 3/4 cholecystitis was possibly attributed to Nivo in 1 pt. 18 (41%) pts proceeded to alloSCT. Donor source was matched related in 3, matched unrelated in 12 and haplo-identical in 3 pts. Conditioning regimen was Fludarabine+busulfan-based in 12, and fludarabine+melphalan in 6 pts. 13 (72%) pts developed graft versus host disease (GVHD)(grades I/II in 8, III/IV in 5), which responded to treatment in 8 (Fig 1B). Multicolor FC studies were conducted on baseline (prior to 1st Nivo dose) and on-treatment BM aspirate and peripheral blood to assess T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cells and leukemic blasts, respectively. Baseline BM was evaluated on 19 responders and 5 non-responders. Pts who achieved CR/CRi had a trend to higher frequency of live CD3+ total T cell infiltrate compared to non-responders in baseline BM aspirates (Fig 1C). We evaluated expression of immune markers on T cell subsets: CD4 T effectors [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 Teff co-expressing PD1/TIM3 (p

Description: Background: Inhibitory killer-cell immunoglobulin receptors (KIRs) negatively regulate NK cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy in AML patients in remission upon haploidentical stem cell transplantation (SCT) from KIR-mismatched donors (Ruggeri L et al., Science 2002). Blockade of KIR2DL1, 2 and 3 receptors induced augmented NK-cell mediated lysis of tumor cells (Romagne F et al., Blood 2009). Hypomethylating agents possess anti-leukemia activity and concomitantly alter immune regulation. Blockade of the KIR-receptors by lirilumab may improve response rates and abrogate immune-mediated resistance to hypomethylating agents. Patients and Methods: Pts are eligible if they have AML and failed prior therapy (including prior therapy with a hypomethylating agent), have adequate performance status (ECOG ≤ 2), and organ function. AZA was given at the dosage of 75mg/m2 on days 1-7; lirilumab was given on Day 8 at the dosage of 1 and 3 mg/kg in 2 consecutive cohorts of 6 pts each. Courses were repeated approximately every 4-5 weeks. No dose-limiting toxicities were observed and lirilumab 3mg/kg was established as the recommended phase 2-dose (RP2D) in combination with AZA. 9 additional pts have been treated at the RP2D. Responses were evaluated at the end of 3 courses of therapy. Results: To date, 21 pts (11 de novo, 10 secondary AML), median age 60 years (range, 33 - 89), 48% with adverse cytogenetics, median prior therapies of 3 (range, 1-8), and prior allogeneic SCT in 6 (28%) have been enrolled. All 21 pts had baseline next generation sequencing for 28-genes and frequently identified mutations included TP53 (n=6), TET2 (N=6), ASXL1 (n=5), RUNX1 (n=5), EZH2 (n=3), DNMT3A (N=2), and CEBPA (n=2). 12 pts are ≥ 3 months into therapy and evaluable for response at this time: 1 achieved complete remission (CR), 1 achieved complete remission with insufficient count recovery (CRi), 2 (17%) had ≥50% bone marrow (BM) blast reduction, 2 (17%) had hematologic improvement (HI) 〉 6 months, and 6 (50%) had progressed. Nine pts were too early for response assessment. The 4- and 8-week mortality is 0 and 5%, respectively. The median duration of response, overall survival (OS) and event-free survival for the 12 evaluable pts were 2.5 months (range, 1.1 - 3.0) 4.4 months and 3.2 months, respectively. Grade 3/4 toxicities irrespective of causality were similar to those seen with AZA based therapies in salvage patients included 15 episodes of neutropenic infections, 6 pneumonia, 1 UTI, 1 skin infection, 2 abdominal pain, and 1 mucositis. Immune mediated toxicities were observed in 3 (14%) pts (1 pneumonitis Grade 3, 1 colitis Grade 2, 1 infusion reaction Grade 2), respectively. The immune mediated toxicities responded rapidly to steroids and all 3 pts could be rechallenged safely with lirilumab. Six pts were postSCT and no Grade 3/4 GVHD flares were noted. No pts have come off study due to toxicities and the azacytidine or lirilumab were not discontinued in any pts due to toxicities. Multicolor flow-cytometry studies and Mass-cytometry (CyTOF) studies are being conducted by the Immunotherapy Platform on baseline and on-treatment BM aspirate (end of cycle 1, 2, 4, 8) and peripheral blood to assess NK- and T-cell costimulatory markers. Conclusion: Full doses of AZA and lirilumab were well tolerated in heavily pretreated pts with relapsed AML with poor risk features, including pts with post-allogeneic SCT relapse. The efficacy data are still preliminary. AZA with lirilumab is being investigated in earlier salvage in AML and frontline and salvage settings in myelodysplastic syndrome. Disclosures Daver: Kiromic: Research Funding; Ariad: Research Funding; Sunesis: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; BMS: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

Description: Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by unleashing T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition alone demonstrated limited activity in AML (Berger et al, Clin Cancer Res 2008). Rational combination strategies to enhance the anti-tumor and immunogenic effects of PD1 inhibitors in AML are needed. AZA up-regulates PD-1 and PD-L1 in AML and the up-regulation of these genes has been associated with emergence of resistance to AZA (Yang et al., Leukemia 2013).These could be blocked with the PD-1 inhibitor nivolumab. Methods: Pts were eligible if they had AML and failed prior therapy, had adequate performance status (ECOG ² 2), and organ function. The first six pts received AZA 75mg/m2 Days 1-7 with nivolumab 3mg/kg on Day 1 and 14. Courses were repeated approximately every 4-5 weeks indefinitely. Only one of six pts had a dose limiting toxicity (grade 3 pneumonitis) and this dose was established as RP2D. 45 additional pts have been treated at the RP2D. Responses were evaluated at the end of 3 courses of therapy. Results: 51 pts with a median age of 69 years (range, 45 - 90), secondary AML (55%), poor risk cytogenetics (47%), median number of prior regimens 2 (range, 1-7) have been enrolled. All 51 pts had baseline next generation sequencing and most frequently detected mutations included DNMT3A (n=12), TP53 (n=11), TET2 (N=9), ASXL1 (n=7), and RAS (n=7). 35 pts are evaluable for response: 6 (18%) achieved complete remission (CR)/ complete remission with insufficient recovery of counts (CRi) (3 CR, 3 CRi), 5 (15%) had hematologic improvement (HI), 9 (26%) had ³50% BM blast reduction,3 pts (9%) had stable disease 〉 6 months, and 12 (34%) had progression. 16 pts are early for response assessment (

Description: Background The combination of the αPD-1 (nivolumab) and hypomethylating agent azacytidine demonstrated encouraging response rate and overall survival in relapsed/refractory acute myeloid leukemia (AML) patients, compared to azacytidine alone1 (NCT02397720). However, the percentage of the patient who achieved an IWG 2016 response to such therapy was still limited (overall response rate = 33%), so it is desirable to have early predictive biomarkers to facilitate patient stratification and selection for future trials. A better understanding of T cells (primary targets of αPD-1 therapy) from bone marrow (BM) and peripheral blood (PB) in AML pre-therapy and on-therapy should yield valuable insights on the treatment-induced anti-tumor response. Methods We performed whole transcriptomic profiling (RNA-sequencing, RNA-seq) on T cells from a cohort of AML patients who were enrolled in the clinical trial mentioned above, and treated with azacytidine and nivolumab (Table 1). Sixty-four FACS-sorted patient-derived T cell samples from cryopreserved peripheral blood (PB) or bone marrow (BM) aspirates, which were collected pre-therapy (T0) and after the first round of treatment (end of cycle one, EOC1), were evaluated. By leveraging subset definitions based on scRNA-seq results from T cells of cancer patients, we implemented deconvolution of our bulk T-cell RNA-seq data to obtain the relative abundance of different T-cell subsets (in-silico dissection without physical isolation). Results We performed ex vivo cDNA library preparation on 2,000-100,000 sorted T cells and yielded a minimal of 17 million sequencing reads per BM T-cell library and 2.6 million sequencing reads per PBMC T-cell library. We compared the gene expression profile of peripheral blood T cells from AML patients (CD4: n = 16; CD8: n = 15) and healthy donors (HD, n = 8)2,3 to validate our methodology. The deconvolution results were consistent with previously published flow-cytometry data profiling cancer patients4,5 (Figure 1). In comparison with HDs, circulating CD4 T cells from AML patients consisted of a higher frequency of Treg (AML versus HD: 5.1% versus 0.6%, P