CD73 Enzymatic Activity Research Articles

Serum CD73 is a prognostic factor in patients with metastatic melanoma and is associated with response to anti-PD-1 therapy

BackgroundInhibitors of immune checkpoint programmed cell death protein 1 (PD-1) receptor on T cells have shown remarkable clinical outcomes in metastatic melanoma. However, most patients are resistant to therapy. Production...

Enzymatic Activity of CD73 Modulates Invasion of Gliomas via Epithelial-Mesenchymal Transition-Like Reprogramming.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumour in adulthood. Despite strong research efforts current treatment options have a limited impact on glioma stem-like cells (GSCs) which contribute to GBM formation, progression and chemoresistance. Invasive growth of GSCs is in part associated with epithelial–mesenchymal-like transition (EMT), a mechanism associated with CD73 in several cancers. Here, we show that CD73 regulates the EMT activator SNAIL1 and further investigate the role of enzymatic and non-enzymatic CD73 activity in GBM progression. Reduction of CD73 protein resulted in significant suppression of GSC viability, proliferation and clonogenicity, whereas CD73 enzymatic activity exhibited negative effects only on GSC invasion involving impaired downstream adenosine (ADO) signalling. Furthermore, application of phosphodiesterase inhibitor pentoxifylline, a potent immunomodulator, effectively inhibited ZEB1 and CD73 expression and significantly decreased viability, clonogenicity, and invasion of GSC in vitro cultures. Given the involvement of adenosine and A3 adenosine receptor in GSC invasion, we investigated the effect of the pharmacological inhibition of A3AR on GSC maintenance. Direct A3AR inhibition promoted apoptotic cell death and impaired the clonogenicity of GSC cultures. Taken together, our data indicate that CD73 is an exciting novel target in GBM therapy. Moreover, pharmacological interference, resulting in disturbed ADO signalling, provides new opportunities to innovate GBM therapy.

Complementary NAD+ replacement strategies fail to functionally protect dystrophin-deficient muscle

BackgroundDuchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs).MethodsUsing a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38.ResultsAdministering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery.ConclusionsIn the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.

The NADase enzyme CD38: an emerging pharmacological target for systemic sclerosis, systemic lupus erythematosus and rheumatoid arthritis.

Here we review recent literature on the emerging role of nicotinamide adenine dinucleotide (NAD) metabolism and its dysfunction via the enzyme CD38 in the pathogenesis of rheumatologic diseases. We evaluate the potential of targeting CD38 to ameliorate NAD-related metabolic imbalance and tissue dysfunction in the treatment of systemic sclerosis (SSc), systemic lupus erythematous (SLE), and rheumatoid arthritis (RA). In this review, we will discuss emerging basic, preclinical, and human data that point to the novel role of CD38 in dysregulated NAD-homeostasis in SSc, SLE, and RA. In particular, recent studies implicate increased activity of CD38, one of the main enzymes in NAD catabolism, in the pathogenesis of persistent systemic fibrosis in SSc, and increased susceptibility of SLE patients to infections. We will also discuss recent studies that demonstrate that a cytotoxic CD38 antibody can promote clearance of plasma cells involved in the generation of RA antibodies. Recent studies identify potential therapeutic approaches for boosting NAD to treat rheumatologic diseases including SSc, RA, and SLE, with particular attention to inhibition of CD38 enzymatic activity as a target. Key future directions in the field include the determination of the cell-type specificity and role of CD38 enzymatic activity versus CD38 structural roles in human diseases, as well as the indicators and potential side effects of CD38-targeted treatments.

Abstract 5179: Isatuximab based combinations induce potent tumor growth inhibition in pre-clinical models of multiple myeloma and acute lymphocytic leukemia

Abstract CD38 is highly expressed in many hematological malignancies such as multiple myeloma (MM) and acute lymphocytic leukemia (ALL) and can be targeted by antibody-based therapy. Isatuximab, an anti-CD38 IgG1 monoclonal antibody has promising clinical activity in relapsed/refractory multiple myeloma patients. It induces tumor cells death via Fc-mediated cytotoxic effects, direct cancer cell killing, and inhibition of CD38 enzymatic activity. We investigated the mechanisms of action (MoA) of isatuximab against MM and ALL in vitro and evaluated its in vivo pharmacological potency in combination with standard therapies for these two hematologic malignancies. Isatuximab preferentially induced robust antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) in both MM and ALL cell lines. Cancer cells with particularly high level of CD38 were also susceptible to apoptosis or CDC induced by isatuximab. In vitro, isatuximab displayed time- and dose-dependent inhibitory effect on CD38 enzymatic activity shown in LP-1 myeloma cells, suggesting that it could alleviate immunosuppressive effects of CD38-mediated adenosine synthesis in the tumor microenvironment. In vivo efficacy studies were conducted in mouse xenograft models. The in vivo efficacy of isatuximab in combination with the immunomodulatory drugs (IMiDs) pomalidomide or lenalidomide that are standard of care in MM was evaluated. Each combination increased the inhibition of tumor growth (T/C) compared with isatuximab or IMiDs treatment alone. Combination of isatuximab with either bortezomib or carfilzomib, two proteasome inhibitors that are widely used in MM, significantly improved their anti-tumor activity versus each agent alone. Isatuximab also improved tumor growth inhibition mediated by alkylating agent melphalan. Similarly, isatuximab enhanced the activity of different chemotherapies in an ALL tumor model. Its combination with vincristine, cytarabine or cyclophosphamide enhanced antitumor activity translating into a higher number of tumor-free survivors (TFS). These results document the unique MoA of isatuximab, primarily through ADCC and ADCP, and its robust antitumor activity as a single agent and in combination with standard of care treatments used in these hematological malignancies. ChemotherapyMM or T-ALL xenograft modelT/C IsatuximabT/C ChemotherapyT/C combinationPomalidomideMOLP-862%54%34%LenalidomideRPMI-822647%76%24%BortezomibNCI-H92928%26%0 %carfilzomibNCI-H92968%44%6%MelphalanLP-167%13%8%VincristineDND4141% no TFS0% no TFS0% 5/6 TFSAra-CDND4141% no TFS0% 3/6 TFS0% 6/6 TFScyclophosphamideDND4110% 4/7 TFS0% 4/7 TFS0% 7/7 TFS Citation Format: Chen Zhu, Celine Nicolazzi, Helgi Van de Velde, Dmitri Wiederschain, Sukhvinder Sidhu, Marielle Chiron. Isatuximab based combinations induce potent tumor growth inhibition in pre-clinical models of multiple myeloma and acute lymphocytic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5179.

Abstract 6056: A next-gen anti-CD73 Antibody with potential to show better efficacy and safety profile than existing approach

Abstract CD73, an ecto-5'-nucleotidase, plays a major role in de-phosphorylation of the extracellular adenosine monophosphate (AMP) to adenosine, which in turn is a potent immunosuppressive metabolite that modulates the immune reaction within the tumor microenvironment (TME). High concentration of adenosine, predominantly signaling through the A2A receptor, suppress innate and adaptive immune cell responses, leading to tumor escape of immune surveillance. Recent studies have shown a significant overexpression of CD73 in solid tumors alongside it's functional upregulation by inflammatory mediators and hypoxia. Thus, inhibition of CD73 enzymatic activity and its surface expression in the TME may improve anti-tumor immune activity. To test this hypothesis, we designed and developed a fully human antibody with unique structure to recognize CD73. This antibody can block the enzymatic activity of CD73 in addition to reducing the surface expression of CD73. Both enzymatic and non-enzymatic dependent functions of CD73 were significantly dampened. The antibody demonstrates a superior anti-tumor activity over reference antibodies in in-vitro and in-vivo models. Our findings indicate that the antibody has promising potential as an effective therapy for PD-1/PD-L1 nonresponsive or refractory patients, as well as a broader patient population that may benefit from a unique mechanism of action of CD73-targeting antibody. Citation Format: Xin Gan, Lei Shi, Qianqian Shan, Fei Chen, Yun He, Yiping Rong, Qiumei Du. A next-gen anti-CD73 Antibody with potential to show better efficacy and safety profile than existing approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6056.

Abstract 6639: SRF617, a potent enzymatic inhibitor of CD39, demonstrates single-agent activity and cooperates with various cancer therapies in both solid tumor and hematologic malignancies

Abstract CD39 (ENTPD1) is a key enzyme responsible for the degradation of extracellular adenosine triphosphate (ATP) and is upregulated in the tumor microenvironment (TME). High levels of extracellular ATP, often generated in the TME as a result of tissue damage and immunogenic cell death, can initiate proinflammatory responses that are blocked by the enzymatic activity of CD39. In addition, extracellular adenosine accumulates in cancerous tissues through the degradation of ATP by CD39 and other ectonucleotidases (eg, CD73), and constitutes an important mechanism of tumor immune escape, induction of angiogenesis, and metastasis. Thus, inhibition of CD39 can convert a suppressive TME to a proinflammatory environment. SRF617 is an investigational fully human anti-CD39 antibody that binds to human CD39 with nanomolar affinity and potently inhibits its enzymatic function. Results of the current studies show that CD39 is predominantly expressed in tumor stroma and on tumor-infiltrating immune cells in samples from patients with cancer. Similar expression patterns are observed in various murine tumor models. In vivo, SRF617 has significant single-agent anti-tumor activity in a variety of cell line-derived xenograft models that express CD39. Cancer therapies, such as immunogenic cell death agents, can increase inflammation and ATP levels in the TME; combination studies of SRF617 with these agents showed improved preclinical efficacy and led to significant improvement in survival. In addition, a mouse-specific anti-CD39 surrogate antibody demonstrated potent binding and enzymatic inhibition of murine CD39 in vitro and significantly decreased tumor growth in a syngeneic murine tumor model. Pharmacodynamic studies demonstrated mechanistic changes in the tumor-infiltrating leukocytes and plasma chemokine levels. Combination treatment of a murine anti-CD39 surrogate and an anti-mouse PD-1 displayed improved activity and an increase in overall survival in the CT-26 mouse model. In addition, administration of SRF617 in combination with other immunotherapies also demonstrated a similar improvement in activity and survival. In summary, these studies demonstrate that SRF617 is a potent inhibitor of CD39 enzymatic activity both in vitro and in vivo. Inhibition of CD39 potentiates the activity of chemotherapy and immunotherapy agents to improve tumor growth inhibition and survival in mice. These findings support future clinical studies of SRF617 as monotherapy and in combination with other therapeutic agents in treating patients with cancer. Citation Format: Sonia G. Das, Austin Dulak, Gege Tan, Marc Johnson, Tauqeer H. Zaidi, Michael C. Warren, Secil Koseoglu, Erik Devereaux, Marisa O. Peluso, Alison M. Paterson, Benjamin H. Lee, Vito J. Palombella, Pamela M. Holland, Andrew C. Lake. SRF617, a potent enzymatic inhibitor of CD39, demonstrates single-agent activity and cooperates with various cancer therapies in both solid tumor and hematologic malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6639.

Abstract 2266: SAR442085, a next generation anti-CD38 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) against multiple myeloma

Abstract CD38 is a cell surface glycoprotein highly expressed in multiple myeloma (MM) cells making it an attractive target for antibody–based therapy. Anti–CD38 treatment represents a major breakthrough in the treatment of relapsing and refractory multiple myeloma (RRMM) patients. However, a number of patients do not respond to anti–CD38 single agent therapy stressing the need for novel approaches. Here we describe SAR442085, a next generation anti–CD38 antibody, with higher affinity for the activating FcγRIIIa and FcγRIIa receptors expressed on effector cells as compared to the approved anti–CD38 antibody daratumumab, resulting in greater effector functions. SAR442085 targets CD38 with high affinity and is able to destroy CD38 expressing tumor cells in vitro through effector function mechanisms (ADCC and antibody dependent cellular phagocytosis, ADCP) as well as by direct apoptosis. SAR442085 exhibits better ADCC activity as compared to daratumumab with more than 8–fold EC50 improvement against RPMI–8226 MM cells. SAR442085 also inhibits CD38 enzymatic activity. Using primary bone marrow aspirates from MM patients, an increased ability of SAR442085 to engage CD16 was demonstrated, resulting in a higher level of NK cell activation and degranulation against primary plasma cells, as compared to daratumumab. In addition, SAR442085 demonstrated potent in vivo single–agent activity against murine tumor cells expressing human CD38, in a transgenic C57BL/6 mouse model humanized for all Fcγ receptors, with 90% mice survival at 10 and 1.25 mg/kg and up to 90 days median survival time. Overall, these data support the current evaluation of this next generation anti–CD38 antibody in phase I clinical trials in patients with relapsed/refractory MM. Citation Format: Angela Virone-Oddos, Sahar Kassem, Béré Diallo, Alexandre Tang, Alain Fournier, Nizar El-Murr, Nadege Carrie-Constantin, Céline Nicolazzi, Isabelle Arnould, Hervé Avet-Loiseau, Sukhvinder S. Sidhu, Ludovic Martinet, Marielle Chiron. SAR442085, a next generation anti-CD38 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) against multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2266.

CD38: T Cell Immuno-Metabolic Modulator.

Activation and subsequent differentiation of T cells following antigenic stimulation are triggered by highly coordinated signaling events that lead to instilling cells with a discrete metabolic and transcriptional feature. Compelling studies indicate that intracellular nicotinamide adenine dinucleotide (NAD+) levels have profound influence on diverse signaling and metabolic pathways of T cells, and hence dictate their functional fate. CD38, a major mammalian NAD+ glycohydrolase (NADase), expresses on T cells following activation and appears to be an essential modulator of intracellular NAD+ levels. The enzymatic activity of CD38 in the process of generating the second messenger cADPR utilizes intracellular NAD+, and thus limits its availability to different NAD+ consuming enzymes (PARP, ART, and sirtuins) inside the cells. The present review discusses how the CD38-NAD+ axis affects T cell activation and differentiation through interfering with their signaling and metabolic processes. We also describe the pivotal role of the CD38-NAD+ axis in influencing the chromatin remodeling and rewiring T cell response. Overall, this review emphasizes the crucial contribution of the CD38−NAD+ axis in altering T cell response in various pathophysiological conditions.

Detection of Human CD38 Using Variable Lymphocyte Receptor (VLR) Tetramers.

CD38 is a multifunctional cell surface receptor expressed on multiple cell lineages of hematopoietic origin with high levels of expression on human plasma cells. Previously, we isolated the monoclonal variable lymphocyte receptor B (VLRB) MM3 antibody from the evolutionarily distant sea lamprey, which recognized the CD38 ectoenzyme exclusively on human plasma cells in a manner that correlated with CD38 enzymatic activity. The plasma cell-specific binding of VLRB MM3 contrasts with the broad pattern of expression of CD38-determined conventional antibodies specific for this antigen. In an effort to facilitate the application of this unique reagent in combination with conventional antibody panels, we explored a strategy to generate VLRB MM3 tetramers. The resulting reagent maintained the threshold-based recognition of CD38. Increased sensitivity achieved with VLRB MM3 tetramers also showed preferential recognition of germinal center centroblasts over centrocytes. VLRB MM3 tetramers thus provided a unique and versatile single-step staining reagent for the detection of human CD38 that is readily incorporated into multi-color flow cytometry panels.

P2X7 receptor activation increases expression of caveolin-1 and formation of macrophage lipid rafts, thereby boosting CD39 activity.

Macrophages are tissue-resident immune cells that are crucial for the initiation and maintenance of immune responses. Purinergic signaling modulates macrophage activity and impacts cellular plasticity. The ATP-activated purinergic receptor P2X7 (also known as P2RX7) has pro-inflammatory properties, which contribute to macrophage activation. P2X7 receptor signaling is, in turn, modulated by ectonucleotidases, such as CD39 (also known as ENTPD1), expressed in caveolae and lipid rafts. Here, we examined P2X7 receptor activity and determined impacts on ectonucleotidase localization and function in macrophages primed with lipopolysaccharide (LPS). First, we verified that ATP boosts CD39 activity and caveolin-1 protein expression in LPS-primed macrophages. Drugs that disrupt cholesterol-enriched domains - such as nystatin and methyl-β-cyclodextrin - decreased CD39 enzymatic activity in all circumstances. We noted that CD39 colocalized with lipid raft markers (flotillin-2 and caveolin-1) in macrophages that had been primed with LPS followed by treatment with ATP. P2X7 receptor inhibition blocked these ATP-mediated increases in caveolin-1 expression and inhibited the colocalization with CD39. Further, we found that STAT3 activation is significantly attenuated caveolin-1-deficient macrophages treated with LPS or LPS+BzATP. Taken together, our data suggest that P2X7 receptor triggers the initiation of lipid raft-dependent mechanisms that upregulates CD39 activity and could contribute to limit macrophage responses restoring homeostasis.

Abstract B058: CD73 siRNA therapy regulates glioblastoma immune microenvironment

Abstract Glioblastoma (GB) is the most common primary brain tumor, characterized by high aggressiveness and poor prognostic. Although all efforts, current therapy is just palliative. Therefore, new biological targets are needed to treat this invasive cancer. In this regard, immune modulation emerges as a possibility for cancer therapeutic. Literature has shown that the purinergic system can regulate peripheral immune cell functions and adenosine (ADO) plays an important role as an immunosuppressive molecule. CD73 enzyme activity is considered the main extracellular ADO source and it is overexpressed in a variety of tumors, including GB. According to CD73 participation in tumor progression, studies from our group have shown that CD73 knockdown impairs in vitro and in vivo GB growth. Here, we determined the potential of CD73siRNA (CD73 small interference RNA) delivery using the nanotechnology strategy to reduce tumor growth and modulate GB immune microenvironment (GME) in a preclinical immunocompetent GB model. For this purpose, an in vivo experiment was performed. C6 glioma cells were cultivated and implanted in the striatum of Wistar rat brains (male, 60 d) by stereotaxic surgery. Following 5 days of GB implant, the treatment with CD73siRNA complexed to nanoemulsion (NE) via nasal route has started and continuous by 15 days (twice a day). Experimental groups were composed as follows: control group (glioma-bearing animals that receive saline via nasal route) and treated group (CD73siRNA-NE; 10 µg/kg). After 20 days of GB implant surgery, animals were euthanized and the GB histopathological characteristics were analyzed by HE staining; apoptosis cell death analysis and GME composition were determined by flow cytometry using annexin V and specific antibodies for macrophage and microglia staining. In the histopathological analysis, tumors presented GB characteristics, as necrosis, edema, and angiogenesis. The treatment decreased tumor volume by 60% and increased the GME apoptotic cell index at 10%. Also, the drug interfered in GME population cells, the microglia CD11b+CD45low cells (from 3.4 ± 1.9 to 0.2 ± 0.1 %) and macrophages CD11b+CD45high cells (from 1.22 ± 0.3 to 0.4 ± 0.1 %) were practically abolished in the GME. Our data suggest that the lower tumoral volume is due to the higher apoptotic rates, indicating immune participation in cancer cell death. Furthermore, the absence of microglia and macrophages tumor-associated may contribute to the decreased tumor volume, proposing an immune system activation ADO modulated over the GB. Citation Format: Gabriela Spies Lenz, Juliana Hofstätter Azambuja, Roselena Silvestri Schuh, Luana Roberta Michels, Nicolly Espindola Gelsleichter, Liziane Raquel Beckenkamp, Gabriela Goncalves Roliano, Frabricio Figueiró, Juliete N. Scholl, Jean Sévigny, Márcia R Wink, Helder Ferreira Teixeira, Elizandra Braganhol. CD73 siRNA therapy regulates glioblastoma immune microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B058. doi:10.1158/1535-7163.TARG-19-B058

Hexabody-CD38, a Novel CD38 Antibody with a Hexamerization Enhancing Mutation, Demonstrates Enhanced Complement-Dependent Cytotoxicity and Shows Potent Anti-Tumor Activity in Preclinical Models of Hematological Malignancies

HexaBody-CD38 (GEN3014) is a novel, hexamerization-enhanced human IgG1 targeting CD38 with superior complement dependent cytotoxicity (CDC) activity, in addition to other effector mechanisms. HexaBody-CD38 carries the E430G mutation and binds a different epitope than the clinically validated CD38 monoclonal antibody daratumumab, which is currently being established as backbone therapy for the treatment of multiple myeloma. Introduction of the E430G mutationfacilitates the natural process of antibody hexamer formation through increased intermolecular Fc-Fc interactions after antigen binding at the cell surface (Diebolder et al., Science 2014; de Jong et al., PLoS Biol 2016). Improved IgG hexamer formation can increase binding of the hexavalent complement component C1q, thereby potentiating or unlocking antibody-mediated complement-dependent cytotoxicity (CDC). Preclinical data demonstrate highly potent CDC-mediated tumor cell kill in vitro in a panel of cell lines derived from hematological malignancies, including multiple myeloma (MM), B cell lymphoma and acute myeloid leukemia (AML). In these cell lines, at the highest dose tested (10 µg/mL), HexaBody-CD38 induced approximately 2-fold more CDC-mediated lysis compared to daratumumab. Of note, in those cell lines that were responsive to daratumumab in CDC assays (>50% tumor cell lysis), CDC activity of HexaBody-CD38 was superior to daratumumab, with IC50 values for HexaBody-CD38 2.4- to 13-fold lower than for daratumumab. Moreover, HexaBody-CD38 unlocked CDC activity in 17 out of 28 tumor cell lines that were not sensitive to daratumumab in CDC assays (<50% tumor cell lysis), including cell lines with lower expression of CD38 or higher expression of the complement inhibitory protein CD59. Importantly, in pilot experiments that are part of an ongoing larger study, HexaBody-CD38 was able to effectively kill MM cells from patients in CDC assays ex vivo, including in one patient that had relapsed from daratumumab (Figure 1). In addition to superior CDC, HexaBody-CD38 was shown to induce comparable antibody-dependent cell mediated cytotoxicity (ADCC) and antibody-dependent cell mediated phagocytosis (ADCP) as daratumumab. HexaBody-CD38 demonstrated more efficient inhibition of CD38 cyclase activity, which has been postulated to contribute to immune suppression in the tumor microenvironment. Importantly, in the presence of monocytes, HexaBody-CD38 treatment resulted in the removal of CD38 from the cell membrane of CD38 expressing cells, including T regulatory cells. This suggests downmodulation of CD38 as another potential mechanism to reduce CD38-generated metabolites and associated immune suppression. Finally, HexaBody-CD38 induced promising anti-tumor activity in vivo in PDX models of diffuse large B cell lymphoma in nude mice. Anti-tumor activity was associated with CD38 expression levels. In conclusion, HexaBody-CD38 is a novel CD38 antibody that shows superior capacity to induce CDC-mediated tumor cell kill compared to daratumumab, including in tumor samples from MM patients. Furthermore, HexaBody-CD38 induces FcγR-mediated effector functions and effectively inhibits CD38 enzymatic activity, either directly or indirectly by removal of CD38 from the cell membrane, thereby potentially contributing to immune activation. Targeting CD38 with HexaBody-CD38 could have therapeutic potential in daratumumab-naïve and -refractory MM patients, as well as in CD38-positive tumors in which daratumumab does not have single agent efficacy, such as DLBCL and AML. The promise of HexaBody-CD38 warrants further clinical investigation in CD38-positive hematological malignancies, including MM, B cell lymphoma and AML. Disclosures De Goeij: Genmab BV: Employment, Other: stock and/or warrants. Janmaat:Genmab BV: Employment, Other: stock and/or warrants. Andringa:Genmab BV: Employment, Other: stock and/or warrants. Kil:Genmab: Employment, Other: stock and/or warrants. Van Kessel:Genmab: Other: stock and/or warrants. Lingnau:Genmab: Employment, Other: stock and/or warrants. Freidig:Genmab BV: Employment, Other: stock and/or warrants. Mutis:Onkimmune: Research Funding; BMS: Research Funding; Janssen Pharmaceuticals: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Aduro: Research Funding. Sasser:Genmab: Employment, Other: stock and/or warrants. Breij:Genmab: Employment, Other: stock and/or warrants. Van De Donk:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Ahmadi:Genmab Inc: Employment, Other: stock and/or warrants. Satijn:Genmab BV: Employment, Other: stock and/or warrants.

Human dental pulp stem cells regulate allogeneic NK cells' function via induction of anti-inflammatory purinergic signalling in activated NK cells.

ObjectivesMesenchymal stem cells (MSCs) could regulate the function of various immune cells. It remains unclear whether MSCs additionally possess immunostimulatory properties. We investigated the impact of human MSCs on the responsiveness of primary natural killer (NK) cells in terms of induction of anti‐inflammatory purinergic signalling.Material and MethodsWe obtained human bone marrow mesenchymal stem cells (BMMSCs) and dental pulp stem cells (DPSCs). NK cells were isolated from peripheral blood of healthy volunteers. Activated NK cells were cultured with MSCs. Proliferation assay, apoptosis analysis, activating or inhibitory receptor expression and degranulation assay were used to explore NK cells’ function. High‐performance liquid chromatography was used to investigate the purinergic signalling in activated NK cells.ResultsBoth DPSCs and BMMSCs could impair proliferation and promote apoptosis of activated NK cells. Also, activated NK cells could cause DPSCs to lyse. Furthermore, the expression of activating NK cells’ receptors was decreased, but inhibitory receptors of NK cells were elevated following co‐cultivation. NK cells acquired CD73 expression, while MSCs could release ATP into the extracellular space where nucleotides were converted into adenosine (ADO) following co‐culture system. Under the existence of exogenous 2‐chloroadenosine (CADO), the cytotoxic capacity of NK cells was remarkably depressed in a concentration‐dependent manner.ConclusionsDPSCs and BMMSCs could depress NK cells’ function by hydrolysing ATP to ADO using CD39 and CD73 enzymatic activity. Our data suggested that DPSCs might represent a new strategy for treating immune‐related diseases by regulating previously unrecognized functions in innate immune responses.

Blockade pf CD73/adenosine axis improves the therapeutic efficacy of docetaxel in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most common and lethal ovarian cancer which threatens women health. Nowadays, the standard treatment is maximal cytoreductive surgical debulking followed by chemotherapy. However, immunosuppressive environment generated by chemotherapy, which can be reversed by immunotherapy, leads to the failure of standard treatment. Therefore, a combination of chemotherapy and immunotherapy will be a promising strategy for EOC patients. For in vitro study, CD73 expression, CD73 activity, and CD8+ T-cell proliferation were measured after docetaxel (DTXL) treatment with or without CD73 antibody. For in vivo study, tumor growth, tumor weight, and the population of various immune cells in the tumor were analyzed in different drug treatment groups. DTXL can increase both the CD73 expression and enzymatic activity in patient-derived epithelial cell and ID8 cell while causing immunosuppressive response which was reversed by anti-CD73 antibody (aCD73). Moreover, tumor growth and lung metastasis in mouse were significantly diminished after DTXL + aCD73 treatment. Blocking CD73/adenosine pathway could reverse the immunosuppression caused by DTXL, and a combination of DTXL with CD73 inhibitor or anti-CD73 antibody would be a more effective and promising therapy for EOC.

Measurement of CD73 enzymatic activity using luminescence-based and colorimetric assays.

CD73 is a membrane-anchored ectoenzyme that degrades extracellular AMP into adenosine, a potent immunosuppressive factor. In physiological conditions, induction of the CD73-adenosine pathway acts as natural feedback mechanism to prevent excessive immune reactions and subsequent tissue damage. In the past few years, the CD73-adenosine pathway has emerged as a major immunosuppressive mechanism by which multiple types of cancer evade anti-tumor immunity. Research from our group and others have established that blocking the CD73-adenosine pathway represents a promising approach to improve cancer immunotherapy. In this context, an increasing number of research laboratories are becoming interested in CD73 biology and in the development/characterization of CD73 inhibitors. Implementation of simple, rapid and HTS-compatible assays to evaluate CD73 enzymatic active is a critical step for any laboratory willing to study the CD73-adenosine pathway. Over the years, we developed, optimized or adapted various methodologies to assess CD73 enzymatic activity using in vitro assays. In this chapter, we describe two different in vitro assays adapted to the measurement of CD73 enzymatic activity. Both assays are simple, robust, HTS-compatible and can be used in a cell-based fashion.

Abstract CT180: Preliminary phase 1 profile of BMS-986179, an anti-CD73 antibody, in combination with nivolumab in patients with advanced solid tumors

Abstract Background: CD73 is an ectonucleotidase that converts adenosine monophosphate to adenosine, a potent immunosuppressive soluble mediator that inhibits the cytotoxic function of CD8+ T cells and natural killer cells while promoting proliferation of immunosuppressive cells. BMS-986179 is a high-affinity antibody that inhibits CD73 enzymatic activity and downregulates its expression on tumor cells. Blockade of CD73 enhanced the antitumor activity of anti-PD-1 in preclinical models (Barnhart BC, et al. Cancer Res. 2016;76(14 suppl) [abstract 1476]). Here we describe preliminary results of the first-in-human phase 1/2a study of BMS-986179 + nivolumab (NIVO) in patients (pts) with advanced solid tumors (NCT02754141). Methods: Pts with ≥ 1 prior therapy were treated in this open-label, dose-escalation and -expansion study. Escalation began with a 2-week monotherapy lead-in, in which pts received BMS-986179 150-1600 mg IV QW followed by BMS-986179 (same dose) IV QW + NIVO 240 mg IV Q2W. Pharmacokinetics (PK), pharmacodynamics (PD), safety, and preliminary antitumor activity were evaluated. PD analyses included immunohistochemistry, enzyme activity assays in tumor biopsies, and evaluation of receptor occupancy and soluble CD73 in peripheral blood. Results: As of the Dec 19, 2017, data cutoff, 59 pts were treated with BMS-986179 ± NIVO during dose escalation. PK of BMS-986179 was nonlinear at lower doses due to target-mediated drug disposition, and exposure increased proportionally from 1200 to 1600 mg. BMS-986179 demonstrated complete and persistent CD73 target engagement in the tumor and periphery at all doses. Treatment-related AEs (TRAEs) were observed in 30 of 52 pts (58%) who received the combination, with no clear dose relationship. Only 8 pts (15%) experienced grade 3 TRAEs, and 1 discontinued treatment due to a TRAE (grade 3 increased ALT). Both the monotherapy lead-in and the combination were well tolerated, with no grade 4 TRAEs and no treatment-related deaths. BMS-986179 efficiently inhibited CD73 enzyme activity in the tumor vasculature and tumor cells at all doses, without dose dependency. Overall, 7 pts with head and neck, pancreatic, prostate, anal, and renal cancer achieved confirmed partial responses and 10 pts had stable disease. Conclusions: BMS-986179 + NIVO was well tolerated, with CD73 target engagement in the tumor and periphery and a safety profile similar to that of NIVO monotherapy. The combination demonstrated preliminary antitumor activity. These data support the ongoing evaluation of this combination in pts with advanced solid tumors. Citation Format: Lillian L. Siu, Howard Burris, Dung T. Le, Antoine Hollebecque, Neeltje Steeghs, Jean-Pierre Delord, John Hilton, Bryan Barnhart, Emanuela Sega, Kinjal Sanghavi, Anke Klippel, Cyrus Hedvat, Ed Hilt, Mark Donovan, Adrianna Gipson, Paul Basciano, Jennifer Postelnek, Yue Zhao, Raymond P. Perez, Richard D. Carvajal. Preliminary phase 1 profile of BMS-986179, an anti-CD73 antibody, in combination with nivolumab in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT180.

Abstract 3812: A best in class anti-CD38 antibody with antitumor and immune-modulatory properties

Abstract Targeting CD38 in multiple myeloma has resulted in outstanding responses. CD38 is widely expressed on myeloma cells and other hematological malignancies. Not much is known about its expression on solid tumors and its role in the immune system. We have analysed a range of solid tumors for CD38 expression and distribution. To optimally target CD38, we have generated a novel antibody that is depleting CD38-high expressing cells, but also has immune modulatory properties. To dissect CD38 expression in solid tumors we exploited mRNA expression libraries, performed immune histochemistry (IHC) on tumor sections, and flow cytometry on patient tumor material. Bioinformatic analysis of the immune cell atlas revealed varying CD38 expression among all cancers analysed, and CD38 expression could be correlated with immune markers, e.g. Foxp3, PD-1/L1. IHC and flow cytometry confirmed CD38 expression across common cancer types, mostly confined to infiltrating lymphocyte and myeloid subsets. Expression on tumor cells was patient dependent. CD38 expression on immune cells was heterogenous and found on NK cells, T cells, suppressive myeloid cells, as well as regulatory T and B cells. Of note, high expression of CD38 was found to be correlated to a subset of exhausted T cells co-expressing PD-1 and other exhaustion markers. To target CD38 in solid tumors, we have screened a panel of CD38-binding antibodies. All antibodies have the potential to deplete CD38 positive tumor cells in vitro and in vivo. Additionally, their ability to influence effector T and NK cell activation has been evaluated. Among a panel of antibodies binding to distinct epitopes of CD38 and exerting unique functional properties, we have identified a fully human antibody, with strong capacity to deplete CD38-high cells in vitro and in vivo by varying killing mechanisms. This antibody was found to increase TCR-mediated signaling and proinflammatory cytokine secretion by human T cells, and further to enhance NK cell activation in vitro. Low dose injection to non-human primates resulted in increased expression of activation markers on both CD4 and CD8 T cells, while no T cell depletion was observed. Other selected antibodies comprise distinct modalities including strong to weak agonistic activity, differential killing properties, modulation of CD38 enzymatic activity, and offer a selection of candidates applicable for different treatment settings. In summary, we found heterogenous expression of CD38 in solid tumors, mostly confined to immune subsets. To target CD38, we present a potent anti-CD38 antibody with depleting effects on CD38-expressing cancer cells, as well as suppressive immune cells, and the capacity to increase the function of immune effector cells. This dual activity might allow to fully exploit the therapeutic potential of targeting CD38, not only in hematological malignancies but also in solid tumors. Citation Format: Nina Eissler, Simone Filosto, Jake Y. Henry, Michael F. Maguire, Kristina Witt, Andreas Lundqvist, Teresa Marafioti, Pascal Merchiers, Kevin Moulder, Beatriz Goyenechea, Haw Lu, Camilla Fairbairn, Sarah Windler, JD Aurellano, Omar Duramad, Dominic Smethurst, Sergio A. Quezada, Anne Goubier. A best in class anti-CD38 antibody with antitumor and immune-modulatory properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3812.

Abstract 2718: Preclinical development of humanized CD39 and CD73 blocking antibodies targeting the ATP/adenosine immune checkpoint pathway for cancer immunotherapy

Abstract Within the tumor microenvironment, adenosine causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. Adenosine accumulation results from the hydrolysis of extracellular immunoactivating ATP and ADP into AMP by the CD39 (ENTPD1) ectonucleotidase. AMP can be further hydrolyzed into immunosuppressive Adenosine (Ado) by CD73 (NT5E), a cell membrane ectonucleotidase of the NTPDase family.The immunosuppressive role of CD39 expressed on both Tregs and tumor cells has been demonstrated in several reports. Conversely, CD73 expression in the tumor environment has been associated with poor disease outcome and/or with a pro-metastatic phenotype. Blockade of CD39 and CD73 may promote anti-tumor immunity directly by accumulating immunostimulating ATP for CD39 and indirectly by reducing adenosine accumulation for both targets.In cancer tissue, using IHC and flow cytometry analyses, we observed that while CD73 is often expressed by tumor cells, CD39 is more frequently up-regulated on tumor infiltrating cells compared to PBMC or adjacent non-tumor tissue. We next describe the discovery and preclinical development of a unique anti-huCD39 blocking antibody and of an anti-human CD73 antibody for cancer immunotherapy. These anti-CD39 and anti-CD73 antibodies specifically bind with high affinity to huCD39 and huCD73 proteins, respectively. They potently inhibit enzyme activity of their respective targets under their soluble and membrane-associated forms, without inducing down-modulation of these enzymes expressed at the cell surface. Innate's antibodies efficiently reverse Ado-mediated T cell suppression in vitro in presence of ATP and both CD39- and CD73-expressing immune cells and additionally exhibit unique features. The anti-CD39 Ab maintains high concentration of ATP in the extracellular compartment that enhances DC activation and subsequent T cell proliferation in vitro. The anti-CD73 blocking Ab exhibits a more potent ability to block soluble and membrane-associated CD73 enzyme activity than benchmark Abs currently in clinical development. Finally, in vivo blockade of ATP/Ado pathway in CD39ko mice resulted in improved anti-tumor efficacy of immunogenic cell death inducer chemotherapy and of immune checkpoint therapies, including PD1 and CTLA4.Taken together, these data support the clinical development of anti-CD39 and anti-CD73 neutralizing Abs for cancer immunotherapy, potentially in combination with chemotherapy or Immune Checkpoint therapy. The humanized anti-huCD39 and anti-huCD73 monoclonal antibodies are currently in preclinical development.The research leading to CD73 results were obtained within the TumAdoR collaborative consortium that received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200. Citation Format: Ivan Perrot, Marc Giraudon Paoli, Séverine Augier, Marilyne Royannez Blemont, Marion Gaudin, Frédéric Bosco, Rachel Courtois, Stephane Delahaye, Diana Jecko, Nicolas Gourdin, Maryline Salin Agu, Cyril Perrier, Paul Ricaut, Aurélie Docquier, Stéphanie Chanteux, Benjamin Rossi, Agnès Représa, Caroline Denis, Romain Remark, Cécile Bonnafous, Laurent Gauthier, Ariane Morel, Nathalie Bonnefoy, Jérémy Bastid, Yannis Morel, Carine Paturel. Preclinical development of humanized CD39 and CD73 blocking antibodies targeting the ATP/adenosine immune checkpoint pathway for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2718.

A novel function of ADP-ribosyl cyclases to regulate the length of the small intestine revealed in CD38/CD157 double knockout mice

Abstract Mammalian ADP-ribosyl cyclase includes two ectoenzymes; GPI-anchored, Bone marrow stromal cell antigen-1 (BST-1)/CD157 and a type II membrane protein, CD38. Although they are expressed in various tissues including the lung, kidney, intestine, brain, spleen and bone marrow, the enzymatic activity of CD38 is dominant at physiological pH. We generated and analyzed CD157 deficient mice (CD157KO), and reported that CD157 is a positive regulator of humoral immunity in 129 x C57BL/6J (B6) mixed background. Recently, various groups reported novel functions of murine CD157 in intestinal regeneration and depressive or anxious behaviors. Thus, CD157 is now appreciated as an entero-neuro-immune regulator. The phenotypes of CD38KO also revealed the multiple functions of CD38 in immunity, metabolism, social behaviors, as well as in the kidney and lung. To further clarify the function of ADP-ribosyl cyclase family, we generated CD38/CD157 doubly deficient mice (CD38/157DKO) by sequential targeting at ES cell level, and compared with CD38KO, CD157KO and wild type in B6 background. A novel phenotype unique to B6.CD157KO was enhanced humoral immune responses to TNP-LPS, indicating the suppressive role of CD157 in TLR signal on marginal zone B cell. Although CD38/157DKO developed apparently normal with no histological changes in the major tissues by HE staining, the length of the small intestine increased by approximately 20% at 4 months old. Among the lymphoid organs, the cell numbers in the mesenteric lymph node increased by 1.5 times without changes in the lineage or subset, probably reflecting elongation of the small intestine. These data suggest that ADP-ribosyl cyclases, CD38 and CD157, have roles in regulating functions of the small intestine.