ALBERT

Description: Tissue-derived adenosine, acting via the adenosine A2A receptor (A2AR), is emerging as an important negative regulator of T-cell function. In this report, we demonstrate that A2AR stimulation not only inhibits the generation of adaptive effector T cells but also promotes the induction of adaptive regulatory T cells. In vitro, antigen recognition in the setting of A2AR engagement induces T-cell anergy, even in the presence of costimulation. T cells initially stimulated in the presence of an A2AR agonist fail to proliferate and produce interleukin-2 and interferon (IFN)-γ when rechallenged in the absence of A2AR stimulation. Likewise, in an in vivo model of autoimmunity, tissue-derived adenosine promotes anergy and abrogates tissue destruction. Indeed, A2AR stimulation inhibits interleukin-6 expression while enhancing the production of transforming growth factor-β. Accordingly, treating mice with A2AR agonists not only inhibits Th1 and Th17 effector cell generation but also promotes the generation of Foxp3+ and LAG-3+ regulatory T cells. In this regard, A2AR agonists fail to prevent autoimmunity by LAG-3−/− clonotypic T cells, implicating an important role for LAG-3 in adenosine-mediated peripheral tolerance. Overall, our findings demonstrate that extracellular adenosine stimulates the A2AR to promote long-term T-cell anergy and the generation of adaptive regulatory T cells.

Description: Adoptive cellular immunotherapy (ACT) using chimeric-antigen receptor (CAR)-modified T cells has shown unprecedented initial response rates in patients with advanced B cell malignancies and is emerging as a powerful tool for cancer treatment. However, there remains room for improvement in initial response rates, and relapse occurs in a significant fraction of those patients that do respond. Reasons for failure to respond are being elucidated. At least two mechanism of relapse - lack of persistence of infused CAR-T cells and the loss of antigen-expression by tumor cells - are well documented. We have developed a novel platform for ACT called Marrow-infiltrating Lymphocytes (MILs). MILs are a population of polyclonal T cells expanded from the bone marrow (BM) with distinct properties that set them apart from T cells expanded from peripheral blood lymphocytes (PBLs). MILs are enriched with memory T cells and contain tumor antigen-specific T cells that are typically not detected in PBLs. MILs are being developed for several tumor types, including both hematological and solid tumors. Distinguishing features of MILs compared to PBLs include their memory phenotype, inherent tumor antigen-specificity, and ability to persist long-term. As such, we hypothesized that MILs would provide a robust platform for CAR-T cell therapy with the potential to boost initial response rates and reduce the risk of relapse. Herein, data is presented demonstrating superior anti-tumor activity of CAR-modified MILs (CAR-MILs) compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). We designed a 2nd generation 4-1BB-CD3z CD38 CAR using an scFv derived from Daratumumab. The CD38 CAR was linked to GFP by a T2a cleavage peptide so that GFP could be used as a marker of CAR expression. The CD38 CAR was expressed in a lentiviral construct and demonstrated equal transduction efficiencies and CAR expression levels in MILs and PBLs. The CAR modified MILs demonstrated superior antigen specific killing compared to PBLs against the 8226 myeloma (MM) cell line but not its isogenic CD38 knocked out line. More importantly, CAR-MILs retained their inherent tumor antigen-specificity and capacity to respond through their endogenous tumor antigen-specific T cell receptors - a property that PBL-CARs did not appear to possess. Tumor antigen-specific T cells were quantified by co-culturing MILs with autologous antigen-presenting cells pulsed with allogeneic multiple myeloma cell line lysates as a source of antigen and measuring IFNγ-production as a read-out. An equal or greater number of tumor antigen-specific IFNγ-producing T cells were measured in CD38 CAR-MILs compared to matched unmodified MILs. Finally, we compared the in vitro cytolytic potential of CD38 CAR-MILs to matched CD38 CAR-PBLs. Co-culture killing assays were performed at decreasing CAR-T effector: target (E:T) ratios. At high E:T ratios, CAR-MILs and CAR-PBLs lysed comparable percentages of CD38+ targets with similar kinetics. However, at lower E:T ratios, CAR-MILs killed a greater percentage of CD38+ targets with faster kinetics compared to CAR-PBLs. In conclusion, we have demonstrated 1) the feasibility of producing CAR-modified MILs, 2) that CAR-MILs retain their inherent tumor antigen-specificity and functional capacity - something that was lacking with CAR-PBLs, and 3) that CAR-MILs kill more efficiently in vitro than matched CAR-PBLs. These data suggest that CAR-MILs may provide both better antigen specific killing but more importantly by targeting the endogenous antigenic repertoire, they could also prevent or minimize the risk of relapse via antigen escape variants and thus increase the overall efficacy of adoptive CAR T cell therapy. Disclosures Lutz: WIndMIL Therapeutics: Employment, Patents & Royalties. Rudraraju:WIndMIL Therapeutics: Employment. DeOliveira:WIndMIL Therapeutics: Employment. Jana:WIndMIL Therapeutics: Employment. Weiss:WIndMIL Therapeutics: Employment. Borrello:WIndMIL Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Noonan:WIndMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Description: Background The type of T cell used in generating chimeric antigen receptor (CAR) T cells is an important choice. Evidence suggests that T cells that are early in the effector/memory differentiation pathway with more stemness and greater potential to persist are better than more differentiated T cells with less stemness that are more readily exhausted and have less potential to persist. Marrow-infiltrating Lymphocytes (MILsTM) is a novel form of adoptive T cell therapy composed of patient-autologous, polyclonal CD4 and CD8 T cells that are activated and expanded from the bone marrow. Genetically unmodified MILsTM have demonstrated antitumor activity in patients with multiple myeloma and are being developed for several other tumor types, including non-small cell lung cancer and other solid tumors. Distinguishing features of bone marrow T cells used to produce MILsTM include their memory phenotype, inherent tumor antigen-specificity, higher CD8:CD4 ratio and ability to persist long-term when compared to peripheral blood lymphocytes (PBLs) which is the T cell source used to produce currently approved CAR-T therapies. Based on these differences, we hypothesize that MILsTM provide a more robust and better fit platform for CAR-T therapy compared to PBLs. Using a CD38-specific, 4-1BB/CD3z-signaling CAR as an initial model, we have demonstrated the feasibility of producing CAR-modified MILsTM (CAR-MILsTM) and showed that CAR-MILsTM demonstrate superior killing in vitro compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). Herein, we build on our previous data and add a second BCMA-specific CAR model. We use the two multiple myeloma model systems to compare cytolytic potential, functionality, and expression of phenotypic markers of memory, stemness and exhaustion between patient-matched CAR-MILsTM and CAR-PBLs. Methods Matched pairs of CAR-MILsTM and CAR-PBLs were produced from the bone marrow and blood of multiple myeloma patients. Two different in vitro cytotoxicity assays, the RTCA xCelligence real-time impedance and FACS assays, were used to evaluate antigen-specific killing of target tumor cells. Functionality of CD4 and CD8 CAR-T cells, at the single-cell level, was evaluated by measuring the secretion of 32 cytokines and chemokines following in vitro antigen-specific stimulation using IsoPlexis IsoCode chips and analyzed using IsoPeak. Expression of markers of T cell memory (CD45RO & CCR7/CD62L), stemness (CD27) and exhaustion (PD1 & TIM3) on CAR-MILsTM and CAR-PBLs prior to and following antigen-specific stimulation was evaluated by flow-cytometry (FACS). Results CAR-MILsTM demonstrated superior killing of tumor target cells in vitro, regardless of the antigen specificity of the CAR, when compared to matched CAR-PBLs and this superiority persisted even upon repeated antigen encounter - a factor that may be critical in guaranteeing better anti-tumor efficacy and persistence. CAR-MILsTM demonstrated increased polyfunctionality (secretion of 2+ cytokines per cell) and an increased polyfunctional strength index (PSI) following antigen-stimulation compared to CAR-PBL in both CD4 and CD8 T cells. The enhanced PSI in CAR-MILsTM was predominately mediated by effector, stimulatory and chemoattractive proteins associated with antitumor activity including Granzyme B, IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and enhanced secretion of these same proteins was reported to be associated with improved clinical responses in patients with Non-Hodgkin lymphoma treated with CD19-specific CAR-T therapy. Expression of memory markers on CD4 and CD8 T cells were similar in CAR-MILsTM and CAR-PBLs both prior to and following antigen-stimulation. Although expression of CD27, PD1 and TIM3 were similar at baseline, CAR-MILs maintained higher levels of CD27 and lower levels of PD1 and TIM3 compared to CAR-PBLs following antigen-stimulation in both CD4 and CD8 T cells. Conclusions Collectively, our data suggest that CAR-MILsTM have several advantages over CAR-PBLs, including increased cytolytic potential, enhanced polyfunctionality, increased stemness and less exhaustion. Based on these differences and the inherent antitumor properties of MILsTM, we speculate that CAR-MILsTM would be more potent and effective than currently approved CAR-T products derived from PBLs. Disclosures Lutz: WindMIL Therapeutics: Employment, Equity Ownership. Jana:WindMIL Therapeutics: Employment, Equity Ownership. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. DeOliveira:WindMIL Therapeutics: Employment, Equity Ownership. Zhou:Isoplexis: Employment, Equity Ownership. Mackay:Isoplexis: Employment, Equity Ownership. Borrello:Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX.

Description: Avalanche hazard evaluation relies in part on representative snowpack stability observations. Thus, understanding the spatial patterns of snowpack instabilities and their environmental determinants is crucial. This case study integrates intensive field observations with spatial modeling to identify associations between incoming radiation, surface hoar development and its subsequent shear strength across an inclined forest opening. We examined a buried surface hoar layer in southwest Montana, USA, over five sampling days, collecting 824 SnowMicroPen resistance profiles and performing 352 shear frame tests. Spatial models of incoming long- and shortwave radiation were generated for the surface hoar formation period using modeled hemispheric sky visibility, physically based parameters and the Bird Clear Sky Radiation Model in a Geographic Information System. Before burial, the surface hoar persisted despite moderate winds and relatively high air temperatures. The buried surface hoar layer thickness varied between 3 and 21 mm within a distance of 30 m. Modeled incoming radiation explained spatial variations in layer thickness and shear strength. In areas exposed to large amounts of radiation, the surface hoar layer was strong and thin, while areas with limited incoming radiation (due to high sky visibility and shading) possessed a thicker surface hoar layer that sheared more easily. This demonstrates the usefulness of microclimate modeling for slope-scale avalanche hazard evaluation. We also identify that over the 3 week sample period, strengthening occurred without thinning of the surface hoar layer.

Description: Accumulation is a key parameter governing the mass balance of the Greenland ice sheet. Several studies have documented the spatial variability of accumulation over wide spatial scales, primarily using point data, remote sensing or modeling. Direct measurements of spatially extensive, detailed profiles of accumulation in Greenland, however, are rare. We used 400 MHz ground-penetrating radar along the 1009 km route of the Greenland Inland Traverse from Thule to Summit during April and May of 2011, to image continuous internal reflecting horizons. We dated these horizons using ice-core chemistry at each end of the traverse. Using density profiles measured along the traverse, we determined the depth to the horizons and the corresponding water-equivalent accumulation rates. The measured accumulation rates vary from ~0.1 m w.e. a–1 in the interior to ~0.7 m w.e. a–1 near the coast, and correspond broadly with existing published model results, though there are some excursions. Comparison of our recent accumulation rates with those collected along a similar route in the 1950s shows a ~10% increase in accumulation rates over the past 52 years along most of the traverse route. This implies that the increased water vapor capacity of warmer air is increasing accumulation in the interior of Greenland.

Description: A series of NASA airborne lidars have been used in support of satellite laser altimetry missions. These airborne laser altimeters have been deployed for satellite instrument development, for spaceborne data validation, and to bridge the data gap between satellite missions. We used data from ground-based Global Positioning System (GPS) surveys of an 11 km long track near Summit Station, Greenland, to assess the surface–elevation bias and measurement precision of three airborne laser altimeters including the Airborne Topographic Mapper (ATM), the Land, Vegetation, and Ice Sensor (LVIS), and the Multiple Altimeter Beam Experimental Lidar (MABEL). Ground-based GPS data from the monthly ground-based traverses, which commenced in 2006, allowed for the assessment of nine airborne lidar surveys associated with ATM and LVIS between 2007 and 2016. Surface–elevation biases for these altimeters – over the flat, ice-sheet interior – are less than 0.12 m, while assessments of measurement precision are 0.09 m or better. Ground-based GPS positions determined both with and without differential post-processing techniques provided internally consistent solutions. Results from the analyses of ground-based and airborne data provide validation strategy guidance for the Ice, Cloud, and land Elevation Satellite 2 (ICESat-2) elevation and elevation-change data products.

Description: Surface melt on a glacier can perturb the glaciochemical record beyond the natural variability. While the centre of the Greenland ice sheet is usually devoid of surface melt, many high-Arctic and alpine ice cores document frequent summertime melt events. Current hypotheses interpreting melt-affected ice-core chemistry rely on preferential elution of certain major ions. However, the precise nature of chemistry alteration is unknown because it is difficult to distinguish natural variability from melt effects in a perennially melt-affected site. We use eight trace-element snow chemistry records recovered from Summit, Greenland, to study spatial variability and melt effects on insoluble trace chemistry and physical stratigraphy due to artificially introduced meltwater. Differences between non-melt and melt-affected chemistry were significantly greater than the spatial variability in chemistry represented by nearest-neighbour pairs. Melt-perturbed trace elements, particularly rare earth elements, retained their seasonal stratigraphies, suggesting that trace elements may serve as robust chemical indicators for annual layers even in melt-affected study areas. Results suggest trace-element transport via meltwater percolation will deposit eluted material down-pit in refrozen areas below the nearest-surface chemistry peak. In our experiments, snow chemistry analyses are more sensitive to melt perturbations than density changes or unprocessed near-infrared digital imagery.

Description: A series of NASA airborne lidars have been used in support of satellite laser altimetry missions. These airbornelaser altimeters have been deployed for satellite instrument development, for spaceborne data validation, and to bridge the data gap between satellite missions. We used data from ground-based Global Positioning System (GPS) surveys of an 11 km long track near Summit Station, Greenland, to assess the surface elevation bias and measurement precision of three airborne laser altimeters including the Airborne Topographic Mapper (ATM), the Land, Vegetation, and Ice Sensor (LVIS), and the Multiple Altimeter Beam Experimental Lidar (MABEL). Ground-based GPS data from the monthly ground-based traverses, which commenced in 2006, allowed for the assessment of nine airborne lidar surveys associated with ATM and LVIS between 2007 and 2016. Surface elevation biases for these altimeters over the flat, ice-sheet interior are less than 0.12 m, while assessments of measurement precision are 0.09 m or better. Ground-based GPS positions determined both with and without differential post-processing techniques provided internally consistent solutions. Results from the analyses of ground-based and airborne data provide validation strategy guidance for the Ice, Cloud, and land Elevation Satellite 2 (ICESat-2) elevation and elevation-change data products.