The tumor microenvironment is characterized by of high levels of extracellular nucleotides that are metabolized through the dynamic and sequential action of cell surface enzymes (ectoenzymes). These ectoenzymes operate according to their spatial arrangement, as part of (1) continuous (molecules on the same cell) or (2) discontinuous (molecules on different cells) pathways, the latter being facilitated by restricted cellular microenvironment. The outcome of this catabolic activity is an increase in the local concentration of adenosine, a nucleoside involved in the control of inflammation and immune responses. The aim of the work presented here was to demonstrate that a previously unexplored enzymatic pathway may be an alternate route to produce extracellular adenosine. Our data show that this new axis is driven by the nucleotide-metabolizing ectoenzymes CD38 (an NAD+ nucleosidase), the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1, also known as CD203a or PC-1) and the 5′ ectonucleotidase (5′-NT) CD73, while bypassing the canonical catabolic pathway mediated by the nucleoside tri- and diphosphohydrolase (NTPDase) CD39. To determine the relative contributions of these cell surface enzymes to the production of adenosine, we exploited a human T-cell model allowing for the modular expression of the individual components of this alternative pathway upon activation and transfection. The biochemical analysis of the products of these ectoenzymes by high-performance liquid chromatography (HPLC) fully substantiated our working hypothesis. This newly characterized pathway may facilitate the emergence of an adaptive immune response in selected cellular contexts. Considering the role for extracellular adenosine in the regulation of inflammation and immunogenicity, this pathway could constitute a novel strategy of tumor evasion, implying that these enzymes may represent ideal targets for antibody-mediated therapy.

Nicotinamide adenine dinucleotide (NAD+) is an essential co-enzyme reported to operate both intra- and extracellularly. In the extracellular space, NAD+ can elicit signals by binding purinergic P2 receptors or it can serve as the substrate for a chain of ectoenzymes. As a substrate, it is converted to adenosine (ADO) and then taken up by the cells, where it is transformed and reincorporated into the intracellular nucleotide pool. Nucleotide-nucleoside conversion is regulated by membrane-bound ectoenzymes. CD38, the main mammalian enzyme that hydrolyzes NAD+, belongs to the ectoenzymatic network generating intracellular Ca2+-active metabolites. Within this general framework, the extracellular conversion of NAD+ can vary significantly according to the tissue environment or pathological conditions. Accumulating evidence suggests that tumor cells exploit such a network for migrating and homing to protected areas and, even more importantly, for evading the immune response. We report on the experience of this lab to exploit human multiple myeloma (MM), a neoplastic expansion of plasma cells, as a model to investigate these issues. MM cells express high levels of surface CD38 and grow in an environment prevalently represented by closed niches hosted in the bone marrow (BM). An original approach of this study derives from the recent use of the clinical availability of therapeutic anti-CD38 monoclonal antibodies (mAbs) in perturbing tumor viability and enzymatic functions in conditions mimicking what happens in vivo.

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is increased in inflammatory bowel disease (IBD) patients, and its serum levels correlate with a worse prognosis. In the present manuscript, we show that eNAMPT serum levels are increased in IBD patients that fail to respond to anti-TNFα therapy (infliximab or adalimumab) and that its levels drop in patients that are responsive to these therapies, with values comparable with healthy subjects. Furthermore, eNAMPT administration in dinitrobenzene sulfonic acid (DNBS)-treated mice exacerbates the symptoms of colitis, suggesting a causative role of this protein in IBD. To determine the druggability of this cytokine, we developed a novel monoclonal antibody (C269) that neutralizes in vitro the cytokine-like action of eNAMPT and that reduces its serum levels in rodents. Of note, this newly generated antibody is able to significantly reduce acute and chronic colitis in both DNBS-and dextran sulfate sodium (DSS)-induced colitis. Importantly, C269 ameliorates the symptoms by reducing pro-inflammatory cytokines. Specifically, in the lamina propria, a reduced number of inflammatory monocytes, neutrophils, Th1, and cytotoxic T lymphocytes are found upon C269 treatment. Our data demonstrate that eNAMPT participates in IBD and, more importantly, that eNAMPT-neutralizing antibodies are endowed with a therapeutic potential in IBD. Key messages& What are the new findings? & Higher serum eNAMPT levels in IBD patients might decrease response to anti-TNF therapy. & The cytokine-like activity of eNAMPT may be neutralized with a monoclonal antibody. & Neutralization of eNAMPT ameliorates acute and chronic experimental colitis. & Neutralization of eNAMPT limits the expression of IBD inflammatory signature. & Neutralization of eNAMPT impairs immune cell infiltration in lamina propria.

Surface functionalization with antitransferrin receptor (TfR) mAbs has been suggested as the strategy to enhance the transfer of nanoparticles (NPs) across the blood-brain barrier (BBB) and to carry nonpermeant drugs from the blood into the brain. However, the efficiency of BBB crossing is currently too poor to be used in vivo. In the present investigation, we compared 6 different murine mAbs specific for different epitopes of the human TfR to identify the best performing one for the functionalization of NPs. For this purpose, we compared the ability of mAbs to cross an in vitro BBB model made of human brain capillary endothelial cells (hCMEC/D3). Liposomes functionalized with the best performing mAb (MYBE/4C1) were uptaken, crossed the BBB in vitro, and facilitated the BBB in vitro passage of doxorubicin, an anticancer drug, 3.9 folds more than liposomes functionalized with a nonspecific IgG, as assessed by confocal microscopy, radiochemical techniques, and fluorescence, and did not modify the cell monolayer structural or functional properties. These results show that MYBE/4C1 antihuman TfR mAb is a powerful resource for the enhancement of BBB crossing of NPs and is therefore potentially useful in the treatment of neurologic diseases and disorders including brain carcinomas.

Daratumumab (DARA) is an anti-CD38 human mAb in phase III clinical trials in myeloma patients. DARA binding induces killing of tumor cells via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis. This work reports on the results obtained while dissecting the events following in vivo CD38 ligation by DARA. Treatment of myeloma cells with DARA + anti-human IgG at 37 °C influenced myeloma cytoskeleton, with redistribution of the CD38 molecules and a formation of distinct polar aggregates visualized by confocal microscopy. DARA effects are different from those observed using 8 different murine anti-human CD38 mAbs, which tended to internalize. The findings observed after DARA ligation were confirmed by exposing myeloma cells to DARA immobilized on CHO cells modified to express 4 distinct human FcRs. CHO cells adhere to plastic and mimics the events that take place in the myeloma niche. First, the interaction between Dara and the different FcRs was determined according to a biosensor-based approach on an IAsys Plus equipment (Affinity Sensors). Results show that DARA possesses the highest avidity for CD64, due to the higher kinetic stability of the complex. Conversely, the differences in the FcR-DARA recognition phase (kass) had only minor effects on final stability of the complex. The results also indicated that NK cells and monocytes are the blood populations with higher kdiss for DARA. The effects observed at 37 °C on myeloma cells in the presence of immobilized DARA were amplified when compared to those with soluble mAb. Results indicate that DARA induces CD38 target to aggregate, polarize and to release microvesicles (MV) from extrusions of the myeloma membrane. MV in culture supernatants and in bone marrow plasma of myeloma patients were characterized for concentration (particles/ml) and size by means of Malvern NanoSight NS300 equipment. DARA treatment was followed by high amounts of MV of different sizes released from myeloma cells. Same experiments repeated using DARA + anti-human IgG labeled with Alexa 488 analyzed by Malvern equipment highlighted the presence of DARA on the surface of MV. The induction of MV may be relevant for in vivo therapy: MV are outward buds of the membrane, which host molecules clustered in microdomains. The presence of CD38 has been confirmed. MV phenotype was analyzed looking for the ectoenzymes that join CD38 in the regulation of adenosine in the myeloma niche. MV phenotype included not only the presence of the expected CD38, but also of CD203a/PC-1, CD39 and CD73, the ectoenzymatic pathway leading to ADO production. A first conclusion is that DARA treatment is followed in vivo by a marked release of MV at the tumor site. The fate of the MV bearing DARA on their surface is multiple: on one side, MV may interact locally with different cells and populations of the niche. Another possibility is that MV are released into the blood stream and interact with cell populations therein. The lipid bilayer of MV consents passive movements even through tissues. MV appear as minicellular signals delivering instructions at a distance from their place of origin. Further, the ectoenzymes analyzed are also involved in cell migration or in interaction with countereceptors (e.g., CD31) expressed by endothelial cells. This issue, was investigated by testing FITC-conjugated DARA on a Laboratory-established human myeloma line. MV-DARA-FITC were then exposed to PBMC preparations obtained from normal donors. The results from a cytofluorimetric analysis highlighted the tendency of the labeled MV to cluster around CD16+ (NK cells) and CD14+ subsets (monocytes). At the moment, it is only possible to conclude that MV are associated with cell membranes, a binding likely mediated by FcRs. Not known yet whether MV interact with other cell types (e.g., macrophages, dendritic cells or lymphocytes). DARA shows a high affinity to FcRs of immune cell types (NK cells, monocytes, B cells). Given clinical data that indicate a robust increase in T cell counts, activation and clonality following DARA treatment should be expected. MV containing DARA and portions of myeloma cell membranes could help drive antigen presentation and T cell response in some patients. This is being investigated further. Disclosures Mark: Bioinvent International: Consultancy, Research Funding. Giuliani:Janssen Pharmaceutica: Research Funding; Celgene Italy: Research Funding. Sasser:Janssen Pharmaceuticals: Employment. Malavasi:Janssen: Honoraria, Research Funding.