Microtubules restrict F-actin to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

Immune synapse formation is a key step for lymphocyte activation. In B lymphocytes, the immune synapse controls the production of high-affinity antibodies, thereby defining the efficiency of humoral immune responses. While the key roles played by both the actin and microtubule cytoskeletons in the formation and function of the immune synapse have become increasingly clear, how the different events involved in synapse formation are coordinated in space and time by actin-microtubule interactions is not understood. Using a microfluidic pairing device, we studied with unprecedented resolution the dynamics of the various events leading to immune synapse formation and maintenance. Our results identify two groups of events, local and global dominated, respectively, by actin and microtubules dynamics. They further highlight an unexpected role for microtubules and the GEF-H1-RhoA axis in restricting F-actin polymerization at the immune synapse to define the cell polarity axis, allowing the formation and maintenance of a unique competent immune synapse.

Phenotyping Polarization Dynamics Of Immune Cells Using A Lipid Droplet - Cell Pairing Microfluidic Platform

The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intra-cellular components, necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied. Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how the ligand concentration, the surface fluidity and the substrate rigidity impact this polarization, we show that its onset depends on the antigen concentration at the synapse, and that the substrate rigidity controls both its onset and its kinetics. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse

Efficient immune responses require Ca2+ fluxes across ORAI1 channels during engagement of T cell receptors (TCR) at the immune synapse (IS) between T cells and antigen presenting cells. Here, we show that ZDHHC20-mediated S-acylation of the ORAI1 channel at residue Cys143 promotes TCR recruitment and signaling at the IS. Cys143 mutations reduced ORAI1 currents and store-operated Ca2+ entry in HEK-293 cells and nearly abrogated long-lasting Ca2+ elevations, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement in Jurkat T cells. The acylation-deficient channel remained in cholesterol-poor domains upon enforced ZDHHC20 expression and was recruited less efficiently to the IS along with actin and TCR. Our results establish S-acylation as a critical regulator of ORAI1 channel trafficking and function at the IS and reveal that ORAI1 S-acylation enhances TCR recruitment to the synapse.

A Novel Peptide-MHC Targeted Chimeric Antigen Receptor T Cell Forms a T Cell-like Immune Synapse

Chimeric Antigen Receptor (CAR) T cell therapy is a promising form of adoptive cell therapy that re-engineers patient-derived T cells to express a hybrid receptor specific to a tumour-specific antigen of choice. Many well-characterised tumour antigens are intracellular and therefore not accessible to antibodies at the cell surface. Therefore, the ability to target peptide-MHC tumour targets with antibodies is key for wider applicability of CAR T cell therapy in cancer. One way to evaluate the effectiveness and efficiency of ligating tumour target cells is studying the immune synapse. Here we generated a second-generation CAR to targeting the HLA-A*02:01 restricted H3.3K27M epitope, identified as a possible therapeutic target in ~75% of diffuse midline gliomas, used as a model antigen to study the immune synapse. The pMHCI-specific CAR demonstrated specificity, potent activation, cytokine secretion and cytotoxic function. Furthermore, we characterised killing kinetics using live cell imaging as well as CAR synapse confocal imaging. Here we provide evidence of robust CAR targeting of a model peptide-MHC antigen and that, in contrast to protein-specific CARs, these CARs form a TCR-like immune synapse which facilitates TCR-like killing kinetics.

Leveraging a physiologically based quantitative translational modeling platform for designing bispecific T cell engagers for treatment of multiple myeloma

Bispecific T cell engager (TCE) is an emerging anti-cancer modality which redirects cytotoxic T cells to tumor cells expressing tumor-associated antigen (TAA) thereby forming immune synapses to exerts anti-tumor effects. Considering the protein engineering challenges in designing and optimizing size and pharmacokinetically acceptable TCEs in the context of the complexity of intercellular bridging between T cells and tumor cells, a physiologically relevant and clinically verified computational modeling framework is of crucial importance to guide the process to understand the protein engineering trade offs. In this study, we developed a quantitative, physiologically based computational framework to predict immune synapse formation for a variety of molecular format of TCEs in tumor tissue. Our model incorporated the molecular size dependent biodistribution using the two pore theory, extra-vascularization of T cells and hematologic cancer cells, mechanistic bispecific intercellular binding of TCEs and competitive inhibitory interaction by shed targets. The biodistribution of TCE was verified by positron emission tomography imaging of [89Zr]AMG211 (a carcinoembryonic antigen-targeting TCE) in patients. Parameter sensitivity analyses indicated that immune synapse formation was highly sensitive to TAA expression, degree of target shedding and binding selectivity to tumor cell surface TAA over shed target. Interestingly, the model suggested a “sweet spot” for TCE’s CD3 binding affinity which balanced the trapping of TCE in T cell rich organs. The final model simulations indicated that the number of immune synapses is similar (~50/tumor cell) between two distinct clinical stage B cell maturation antigen (BCMA)-targeting TCEs, PF-06863135 in IgG format and AMG420 in BiTE format, at their respective efficacious dose in multiple myeloma patients, demonstrating the applicability of the developed computational modeling framework to molecular design optimization and clinical benchmarking for TCEs. This framework can be employed to other targets to provide a quantitative means to facilitate the model-informed best in class TCE discovery and development.

Reshaping the tumor microenvironment with oncolytic viruses, positive regulation of the immune synapse, and blockade of the immunosuppressive oncometabolic circuitry

Immune-related therapies have revolutionized the management of cancer. Oncolytic viruses are now considered part of the immunotherapy armamentarium and have shown promise in clinical trials of patients with glioblastoma. These studies have suggested that tumor microenvironment remodeling is required to achieve an effective response in solid tumors. Here, we showed that Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus expressing the T cell activator OX40L, triggered antitumor immune responses. However, Delta-24-RGDOX also elicited paradoxical activation of the cytokine-driven immunosuppressive IDO-kynurenine-AhR circuitry. The IDO-kynurenine-AhR cascade had the dual effects of preventing optimum viral replication and decreasing the virus-initiated antitumor immune response. To enhance virotherapy, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors. This combination therapy increased the frequency of activated CD8+ T cells and decreased the frequencies of immunosuppressive MDSC and Treg populations in animal models of gliomas and melanoma. Functional studies demonstrated that the IDO blockade-dependent activation of immune cells against tumor antigens could be reversed by the oncometabolite kynurenine. The concurrent targeting of effectors and suppressors in the tumor immune landscape significantly prolonged the survival of glioma- and melanoma-bearing mice. Our data identified the striking role of immunosuppressive pathways in the resistance of solid tumors to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas and other cancers treated with virotherapy.

Gp-100 as a Novel Therapeutic Target in Uveal Melanoma

Uveal melanoma is a rare neoplasm with poor prognosis in the metastatic setting. Unlike cutaneous melanoma, treatment with kinase inhibitors or immune checkpoint inhibitors is not effective. Glycoprotein 100 (Gp-100) is a protein highly expressed in melanocytes and melanoma that has recently been effectively targeted by tebentafusp, a first-in-class bispecific protein of the immune-mobilizing monoclonal T cell receptors against cancer (ImmTACs) family. Tebentafusp targets tumor cells that express a peptide of Gp-100 presented by HLA*A0201, creating an immune synapse that kills targeted tumor cells. Recently, a randomized phase III trial reported an overall survival benefit for tebentafusp in patients with untreated metastatic uveal melanoma. The aim of this comprehensive review is to summarize evidence of Gp-100 as a therapeutic target in melanoma, and the preclinical and clinical development of tebentafusp as a novel therapeutic strategy for patients with uveal melanoma.

CD33 BiTE ® Construct Mediated Immunological Synapse Formation and Downstream Signaling in T Cells Is Dependent on Expression of Costimulatory Molecules on Target Cells

BiTE ® (Bispecific T-cell Engager) constructs represent a novel immunotherapeutic strategy that recruits T cells against cancer cells independent of their TCR specificity. Currently, two CD33xCD3 BiTE ® antibody constructs (AMG 330 & AMG 673) are being investigated in phase I dose escalation trials in patients with relapsed/refractory Acute Myeloid Leukemia (AML) with early evidence of acceptable safety and anti-leukemic activity (Ravandi et al., ASH 2020; Subklewe et al., EHA 2020). So far, details of BiTE ® mediated T-cell engagement and information on parameters contributing to their efficacy need more investigation. Therefore, we aimed to characterize the interplay between target and effector cells to deepen our mechanistic understanding of BiTE ® construct mediated T-cell engagement. Previously, we have created a novel in vitro model system with murine Ba/F3 cells expressing human (hu) CD33 ± huCD80 ± huCD86 ± huPD-L1 to study T-cell proliferation and cytotoxicity induced by AMG 330. Using that system, we showed that expression of T-cell co-signaling receptors on target cells modulate AMG 330 induced T-cell activity (Marcinek et al., ASH 2018, EHA 2019). Here, we hypothesize that expression of costimulatory molecules impacts BiTE ® mediated immune synapse formation and consecutive downstream signaling in BiTE ® construct activated T cells. To study whether AMG 330 can induce synapse formation and TCR triggering we used a previously described reconstituted T-cell system, which consists of non-immune (HEK) cells introduced with genes encoding the TCR and other proteins (e.g. CD45) required for the regulation of TCR phosphorylation (James et al., Nature 2012). HEK-T cells were incubated with huCD33 transduced RajiB cells in presence of fluorescently labeled AMG 330 or a control BiTE® (cBiTE) construct to allow cell conjugation. A spinning disc confocal microscope system was used to image cells. To pinpoint the role of T-cell co-signaling receptors in immune synapse formation we incubated differentBa/F3 cell constructs or primary AML (pAML) cells with healthy donor T cells in the presence of AMG 330 and analyzed intensity of LFA-1 expression within the synapse using an Imaging Flow Cytometer. Furthermore, we determined phosphorylation of ZAP70, AKT and ERK in conjugated T cells after various time points by phosphoflow cytometry. We observed that AMG 330, in contrast to cBiTE®, induced TCR triggering reflected by exclusion of CD45 from the RajiB-T-cell-interface. Simultaneously clustering of CD33 occurred in AMG 330 induced cell-cell-interfaces (Fig. 1A/B). The percentage of conjugates formed with huCD33 + Ba/F3 cells was significantly higher in constructs expressing huCD86, compared to those expressing no costimulatory antigens or additional huPD-L1 (Mean % in huCD33 + Ba/F3: 2.8 vs. huCD33 + CD86 +.Ba/F3: 4.2 [p=0.0031] vs. huCD33 + huCD86 + PD-L1 + Ba/F3: 2.8 [p=0.0018]). This was accompanied by LFA-1 accumulation within the T-cell-Ba/F3 cell synapse (Mean of MFI in huCD33 + CD86 +.Ba/F3: 10,933 > huCD33 + huCD86 + PD-L1 + Ba/F3: 7,749 > huCD33 + Ba/F3: 7,028). For downstream signaling in T cells after engagement with Ba/F3 cell constructs in the presence of AMG 330, we observed that kinase phosphorylation was highest after 10 minutes in CD86 co-expressing Ba/F3 cells (Mean % of phosphorylation in T-cell conjugates with huCD33 + vs huCD33 + huCD86 + vs huCD33 + CD86 +.PD-L1 + Ba/F3: pERK 40.9 vs 54.3 [p=0.0064] vs 51.2 %; pAKT: 69.1 vs 81.5 [p=0.0642] vs 75.1 %; pZAP70: 6.9 vs 12.2 [p<0.0001] vs 7.7 % [p<0.0001]) (Fig. 1C). Finally, we evaluated if these finding could also be observed in pAML samples. For that, we determined LFA-1 expression intensity within AMG 330-induced pAML-T-cell synapses. We used CD33 + pAML samples with either high CD86 and no PD-L1 expression or vice versa. Comparing synapse formation between these samples, LFA-1 intensity was 4.6-fold higher in the CD86 + PD-L1 - sample compared to the CD86 - PD-L1 + pAML. Taken together, our data unravel molecular mechanisms of BiTE® construct induced immune synapse formation, highlighting the role of costimulatory molecules in this process. They support the notion that T cell co-signaling receptors like CD86 and PD-L1 modulate T-cell response in an early event manner. Prospective analyses in clinical trials are needed to validate the relevance of checkpoint molecule expression on target cells as a potential predictive biomarker for response. Figure 1 Figure 1. Disclosures Brauchle: Adivo: Current Employment. Lacher: Roche: Research Funding. Kischel: Amgen GmbH Munich: Current Employment. von Bergwelt: Roche: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Theurich: Amgen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Buecklein: Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Miltenyi: Research Funding; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Subklewe: Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

Preclinical Assessment of CDR101 - a BCMAxCD3xPD-L1 Trispecific Antibody with Superior Anti-Tumor Efficacy

BCMAxCD3 targeting therapies have demonstrated anti-myeloma activity, and high minimal residual disease negativity rates can be achieved with this approach in heavily pre-treated patients with relapsed or refractory multiple myeloma (RRMM). Despite these promising clinical results, patients eventually develop resistant disease and relapse. Thus, there is a high need for novel BCMA therapies that can evade the resistance mechanisms and provide more durable responses. Recently, we reported on the promising activity of the Local Activator and T cell Engager (LocATE) technology, a trispecific molecule that targets CD3, BCMA and PD-L1, redirecting T cells to multiple myeloma (MM) cells while selectively counteracting PD-L1/PD-1 induced immunosuppression at the immune synapse (ASH, 2020). Here we present CDR101, an optimized LocATE candidate with potential for clinical development. First, we analyzed the ability of CDR101 to induce PBMC-mediated cytotoxicity in two MM cell-lines expressing BCMA (U-266 and NCI-H929) and compared it to four BCMAxCD3 bispecific formats currently in clinical development (a half-life extended BCMAxCD3 BiTE, a BCMA-TCB, and two different BCMAxCD3 bispecific monoclonal antibodies) alone or in combination with a separate PD-L1 blocking antibody. CDR101 resulted in at least 10-fold increased T cell-mediated target cell lysis compared to control BCMAxCD3 bispecifics. Strikingly, CDR101 also resulted in increased MM cell killing when compared to free, independent combinations of BCMAxCD3 bispecifics and the PD-L1 inhibitor. These results, together with the observation that MM cells upregulate the expression of PD-L1 in response to treatment with BCMAxCD3 bispecifics, suggest that the superior effect of CDR101 could be attributed to preferential and highly selective inhibition of the PD-1/PD-L1 axis at the cellular interaction within the immune synapse. Next, bone marrow aspirates from newly diagnosed and RRMM patients were treated with increasing concentrations of CDR101 or a BCMAxCD3 bispecific control. After 24h of incubation, percentage of viable CD138-positive cells and activation status of autologous T cells were analyzed by FACS. Overall, CDR101 potently induced lysis of primary MM cells independently of the E:T ratio (range of E:T ratio between 1.3:1 and 33:1). CDR101 achieved higher target cell killing in all samples compared to the bispecific control, with at least 2-fold difference in 3 out of 4 samples at the highest concentration tested. Concomitantly, CDR101 induced a dose-dependent increase of the T cell activation marker CD25, corroborating the ability of CDR101 to counteract PD-L1/PD-1 induced immunosuppression. In vivo anti-tumor activity of CDR101 was evaluated using a human MM (NCI-H929) xenograft model in NPG mice. Treatment with four different doses of CDR101 or BCMAxCD3 bispecific control demonstrated that CDR101 induced stronger and more durable responses compared to the bispecific control leading to complete tumor regression in 55 out of 60 mice at the last day of treatment (day 29) with no relapse until the end of the observation time (day 41). Collectively, CDR101 demonstrated that targeting BCMA with simultaneous blockade of PD-L1 leads to improved myeloma cell killing compared to clinically validated therapies. In contrast to high-affinity PD-L1 immune checkpoint inhibitors, CDR101 selectively inhibits PD-L1 at the immune synapse preventing on-target off-tumor effects. This is expected to translate into a decreased incidence of immune related adverse events (irAEs) and better efficacy arguing for a high clinical potential and swift translation into the clinic. Disclosures Vrohlings: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Jungmichel: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Senn: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Howald: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Schleier: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Scheifele: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Wendelspiess: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Richle: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Merten: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Lenherr-Frey: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Leisner: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA. Borras: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Aging-Associated Epigenetic Reprogramming Alters the Germinal Center Reaction and Targets Pathways Related to Lymphomagenesis

Aging is associated with suboptimal germinal center (GC) responses and inferior outcomes of GC-derived lymphomas such as diffuse large B-cell lymphoma (DLBCL). At the molecular level, aging is characterized by epigenetic alterations of DNA CpG methylation and chromatin architecture that ultimately affect cellular functions. The B-cell/T-cell immune synapse during the transitional stages of the GC is governed by a series of epigenetic switches. Frequent mutations in DLBCL directly impact epigenetic regulators and signaling pathways in the immune synapse. Here, we investigated the impact of aging-associated epigenetic alterations in the functionality of the GC response in parallel with biological characteristics of DLBCL in the elderly. We conducted an integrative characterization of epigenomic, transcriptomic and phenotypic changes of B and T cells during the GC reaction in younger (3-4 months) vs. older (21-23 months) mice by immunophenotyping (flow cytometry), DNA methylation sequencing of sorted sub-populations, single cell (SC) RNA-sequencing (coding and BCR/TCR) and SC-multiomics (integration of RNA-sequencing and ATAC-sequencing). We also performed DNA methylation sequencing, RNA-sequencing and NGS in a cohort of 266 DLBCL including 51 pts >75 y.o. In agreement with reported phenotypes in elderly humans (Collier D. et al., Nature, 2021), the GC reaction in older mice was characterized by a significative reduction in IgG3+ (p=0.0001) and increased in IgM+ (p=0.009) B cells (FAS+ CD38-); whereas the non-GC B cell compartment (FAS- CD38+) displayed an increase in age-associated B cells (ABCs, p=0.0001) and reduction in follicular B-cells (p=0.004). Furthermore, older mice displayed an expansion of a FAS+CD38+ population of B cells (p=0.0001). Regarding T cells, we observed a global reduction in CD4+ (p=0.01) but not in CD8+ cells; however, older mice showed an expansion of PD1+/CD4+ and PD1+/CD8+ T cells (p=0.0004 and p=0.0003, respectively). Furthermore, older mice displayed increased TFH (p=0.0001), Tregs (p=0001) and ICOS+ Treg (p=0001) populations. SC-RNA-seq of B and T cells validated these alterations and identified transcriptionally-defined sub-populations including expansion of clonal GZMK CD8+ TOX+ T cells (TAA cells) and activation of "cytokine production" in T cells (FDR=3.01e-27), both phenotypes associated with "inflammaging" (Mogilenko et al., Immunity, 2020). Epigenetic changes in older B-cells showed aberrant hypermethylation of gene promoters and hypomethylation of intergenic regions including enhancers, resulting in changes of chromatin architecture and gene expression. In GC B-cells but not naïve B-cells (NBC), genes whose promoter was aberrantly hypermethylated in older mice were enriched for targets of SUZ12 (FDR= 5.1e -12), EZH2 (FDR= 5.1e -12) and JARID2 (FDR= 6.8e -10), key components of the PRC2 complex. Older mice B-cells also displayed decreased chromatin accessibility in genes functionally enriched for "semaphorin-plexin signaling pathway" (FDR=5.3e -03) which regulates TFH/B-cell immune synapse as well as decreased expression of linker histone H1 isoforms B-E in GCB cells (q=0.0006; q=0.0003; q=0.008, q=4.64E-10, respectively). Notably, the age-associated reprogramming observed in the germinal center reaction globally targets pro-tumorigenic pathways that are comparably altered in elderly DLBCL. We observed that older pts (>75 y.o.) vs. younger pts (55-65 y.o.) had increased hypermethylation of gene promoters and hypomethylation of intergenic regions with deregulation of epigenetic modifiers (including PCR2 members) and immune synapse genes (including BCR signaling). There were no differences in the prevalence of specific mutations between these two populations. However, DLBCL in elderly are more frequently of the ABC subtype (~60%, p<0.05) and presented with an inflammatory tumor microenvironment (41% vs 27% in younger DLBCL, p<0.001, corrected by COO) that is characterized by increased infiltration of macrophages (p=0.001), Tregs (p=0.001) and CD8+ PD1 highcells (p=0.001), phenocopying changes observed in the germinal center of older mice. Thus, age-associated epigenetic reprogramming results in perturbation of pathways regulating the B-cell/T-cell immune synapse during the normal germinal center reaction and may underlie some of the biological characteristics observed in elderly DLBCL patients. Disclosures Lara-Garcia: Johnson and Johnson: Current holder of individual stocks in a privately-held company; Bristol Myers Squibb: Divested equity in a private or publicly-traded company in the past 24 months; Moderna: Divested equity in a private or publicly-traded company in the past 24 months; Pfizer: Divested equity in a private or publicly-traded company in the past 24 months; Regeneron: Divested equity in a private or publicly-traded company in the past 24 months; Merck: Divested equity in a private or publicly-traded company in the past 24 months; Fortress Biotech: Divested equity in a private or publicly-traded company in the past 24 months. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.