ADP-ribosyl Cyclase Activity Of CD38 Research Articles

Abstract 5468: SAR650984, a humanized anti-CD38 antibody potently modulates intracellular and extracellular nucleotide levels of cancer cells

Abstract CD38 is a type II transmembrane glycoprotein with both ectozyme activity and receptor function that has been implicated in cancer cell adhesion, signal transduction and calcium signaling. CD38 is highly expressed at the surface of many hematological cancer cells. SAR650984 is a humanized IgG1 antibody targeting CD38 in early clinical development to treat patients with CD38+ hematological malignancies. Several potential mechanisms of action of SAR650984 have been identified including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct apoptosis induction (ASH 2008 Abstract #2756). In addition, SAR650984 displays potent inhibition of recombinant CD38 ADP-ribosyl cyclase activity in a biochemical assay (AACR 2013, Abstract #4735). To further explore the impact of anti-CD38 antibodies on CD38 ecto-enzymatic activities in cancer cells, we have developed a robust LC-MS-based cellular CD38 enzymatic assay that monitors the depletion of CD38 substrates nicotinamide adenosine dinucleotide (NAD) or nicotinamide mononucleotide (NMN), and the production of cyclic adenosine diphosphoribose (cADPR) and adenosine diphosphoribose (ADPR) from CD38 cyclase and CD38 NAD glycohydrolyase activities, respectively. Preliminary results demonstrate that in response to the treatment of SAR650984, CD38 cyclase activity in multiple myeloma and lymphoma cancer cells was significantly inhibited by more than 90% as shown by a rapid and dose-dependent decrease in the rate of both substrate NAD/NMN depletion as well as extracellular cADPR production. Interestingly, a significant reduction of intracellular cADPR (over 60%) was also observed after SAR650984 treatment. On the other hand, extracellular ADPR level was only moderately affected by SAR650984 treatment with a reduction about 20% which could be hypothetically attributed to the relatively different potency of SAR650984 toward CD38 cyclase and hydrolase activity, or to its potential binding preference to different CD38 oligomers. Emerging data suggest that nucleotides such as NAD and cADPR are important regulators of cell growth, survival and homeostasis. The altered levels of intracellular and extracellular nucleotides resulting from CD38 inhibition by SAR65084 in tumor microenvironment therefore could contribute to the anti-tumor efficacy of the antibody in addition to the previously identified mechanisms. Collectively, these results provide new insights toward understanding the multiple antitumor mechanisms of SAR650984. Additional studies to further understand these observations are currently ongoing. Citation Format: Bailin Zhang, Guang Yang, Shujia Dai, Tim He, Daniel Simard, Zhili Song, Stuart Licht, Francisco Adrian, Hong Cheng, Ti Cai. SAR650984, a humanized anti-CD38 antibody potently modulates intracellular and extracellular nucleotide levels of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2014-5468

SAR650984, a novel humanized CD38-targeting antibody, demonstrates potent antitumor activity in models of multiple myeloma and other CD38+ hematologic malignancies.

The CD38 cell surface antigen is expressed in diverse hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. Here, we assessed the antitumor activity of the anti-CD38 antibody SAR650984. Activity of SAR650984 was examined on lymphoma, leukemia and multiple myeloma cell lines, primary multiple myeloma samples, and multiple myeloma xenograft models in immunodeficient mice. We identified a humanized anti-CD38 antibody with strong proapoptotic activity independent of cross-linking agents, and potent effector functions including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP), equivalent in vitro to rituximab in CD20+ and CD38+ models. This unique antibody, termed SAR650984, inhibited the ADP-ribosyl cyclase activity of CD38, likely through an allosteric antagonism as suggested by 3D structure analysis of the complex. In vivo, SAR650984 was active in diverse NHL, ALL, and multiple myeloma CD38+ tumor xenograft models. SAR650984 demonstrated single-agent activity comparable with rituximab or cyclophosphamide in Daudi or SU-DHL-8 lymphoma xenograft models with induction of the proapoptotic marker cleaved capase-7. In addition, SAR650984 had more potent antitumor activity than bortezomib in NCI-H929 and Molp-8 multiple myeloma xenograft studies. Consistent with its mode of action, SAR650984 demonstrated potent proapoptotic activity against CD38+ human primary multiple myeloma cells. These results validate CD38 as a therapeutic target and support the current evaluation of this unique CD38-targeting functional antibody in phase I clinical trials in patients with CD38+ B-cell malignancies.

Abstract 4735: SAR650984: Characterization of a potent phase I humanized anti-CD38 antibody for the treatment of multiple myeloma and other hematologic malignancies.

Abstract SAR650984, a humanized antibody targeting the type II transmembrane glycoprotein CD38 is currently in phase I dose escalation in patients with selected CD38+ hematological malignancies. CD38 is an ectoenzyme which catalyzes the formation of nucleotide metabolites involved in calcium signalling. Nearly 90% of MM patients express CD38 and this expression correlates with poor disease prognosis. The robust multiple mechanisms of action of SAR650984 include antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis induction, leading to in vivo efficacy in different tumor models, as reported previously (AACR 2010 #714; AACR 2009 #859, #2048, #2797; ASH 2008 #2756). Here, we report additional studies aimed to further elucidate the mechanism of action of SAR650984. These studies include solving the crystal structure of the SAR650984/CD38 complex, measuring enzymatic inhibition, testing in vitro activity against human primary MM cells, studying the mechanism of apoptosis induction in vivo and lastly assessing in vivo activity in combination with bortezomib (Velcade®). Fab fragments derived from SAR650984 were co-crystallized with soluble human CD38 to a 1.5 Å resolution. Loop H3 of the paratope (residues Asp100, Tyr101, Tyr102, Gly103, Ser104 and Asn105) protrudes out of the SAR650984 Fab structure and is particularly critical for the complex formation. Significant conformational changes are observed in CD38 upon binding of SAR650984 while the configuration of the key residues that are involved in the ADP-ribosyl cyclase activity of CD38 is conserved as well as the access to the catalytic site. SAR650984 shows potent inhibition of the catalytic activity of CD38 (86 % at 10 nM), Taken together this would suggest that SAR650984 is not a direct orthosteric antagonist but more likely an allosteric antagonist of CD38. Consistent with the in vitro activity observed against cell lines, SAR650984 also demonstrated potent pro-apoptotic activity against a panel of CD38+ human primary MM cells. In SCID mice bearing SU-DHL-8 lymphoma, SAR650984 induced apoptosis of tumor cells, evidenced by a robust and sustained induction of cleaved caspase 7. In combination with bortezomib, a standard of care for MM treatment, SAR650984 demonstrated synergy in NCI-H929 MM xenografts in SCID mice. Together with previous reports the humanized anti-CD38 antibody, SAR650984, has demonstrated potent anti-tumor activities including ADCC, CDC and induction of apoptosis which translated into potent in vivo anti-tumor efficacy. These data support the ongoing clinical development of SAR650984 as a promising therapeutic antibody for various CD38+ hematologic malignancies. Citation Format: Marie-Cécile Wetzel, Céline Nicolazzi, François Vallée, Jutta Deckert, Charles Dumontet, Adriana Plesa, Aimo Kannt, Vincent Mikol, Marielle Chiron, Loïc Vincent, Veronique Blanc. SAR650984: Characterization of a potent phase I humanized anti-CD38 antibody for the treatment of multiple myeloma and other hematologic malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4735. doi:10.1158/1538-7445.AM2013-4735

Regulation of CD38 Expression in Human Airway Smooth Muscle Cells

The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose, a Ca(2+)-mobilizing agent. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinases and NF-κB and AP-1. The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and contributes to airway hyperresponsiveness and airway remodeling. We hypothesized that PI3Ks mediate CD38 expression and are involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin) or class I-selective (GDC0941) or isoform-selective PI3K inhibitors (p110α-PIK-75 and p110β-TGX-221) with or without TNF-α. HASM cells were transfected with a catalytically active form of PI3K or phosphatase and tensin homolog (PTEN) or nontargeting or p110 isoform-targeting siRNAs before TNF-α exposure. CD38 expression and activation of Akt, NF-κB, and AP-1 were determined. LY294002 and wortmannin inhibited TNF-α-induced Akt activation, whereas only LY294002 inhibited CD38 expression. P110 expression caused Akt activation and basal and TNF-α-induced CD38 expression, whereas PTEN expression attenuated Akt activation and CD38 expression. Expression levels of p110 isoforms α, β, and δ were comparable in nonasthmatic and asthmatic HASM cells. Silencing of p110α or -δ, but not p110β, resulted in comparable attenuation of TNF-α-induced CD38 expression in asthmatic and nonasthmatic cells. NF-κB and AP-1 activation were unaltered by the PI3K inhibitors. In HASM cells, regulation of CD38 expression occurs by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation, and PI3K signaling may not be involved in the differential elevation of CD38 in asthmatic HASM cells.

Differential induction of CD38 expression by TNF-α in asthmatic airway smooth muscle cells

The ADP-ribosyl cyclase activity of CD38, a membrane protein expressed in human airway smooth muscle (ASM) cells, generates cyclic ADP-ribose (cADPR), a Ca²(+)-mobilizing agent. cADPR-mediated Ca²(+) responses to agonists are augmented in human ASM cells by TNF-α. CD38-deficient mice fail to develop airway hyperresponsiveness following intranasal TNF-α or IL-13 challenge, suggesting a role in asthma. The role of CD38 in human asthma remains unknown. We hypothesized that CD38 expression will be elevated in ASM cells from asthmatic donors (ASMA cells). CD38 mRNA and ADP-ribosyl cyclase activity were measured in cells maintained in growth-arrested conditions and exposed to vehicle or TNF-α (10-40 ng/ml). TNF-α-induced induction of CD38 expression was greater in ASMA than in ASM cells from nonasthmatic donors (ASMNA). In four of the six donors, basal and TNF-α-induced ERK and p38 MAPK activation were higher in ASMA than ASMNA cells. JNK MAPK activation was lower in ASMA than ASMNA cells. Nuclear NF-κB (p50 subunit) and phosphorylated c-Jun were comparable in cells from both groups, although nuclear c-Fos (part of the AP-1 complex) levels were lower in ASMA than ASMNA cells. NF-κB or AP-1 binding to their consensus sequences was comparable in ASMNA and ASMA cells, as are the decay kinetics of CD38 mRNA. The findings suggest that the differential induction of CD38 by TNF-α in ASMA cells is due to increased transcriptional regulation involving ERK and p38 MAPK activation and is independent of changes in NF-κB or AP-1 activation. The findings suggest a potential role for CD38 in the pathophysiology of asthma.

Novel peptide inhibitor of ecto-ADP-ribosyl cyclase of bone marrow stromal cell antigen-1 (BST-1/CD157)

cADP-ribose, which is produced by ecto-ADP-ribosyl cyclase of CD38 or bone marrow stromal cell antigen-1 (BST-1), is a key regulator of Ca2+-induced calcium release in an Ins(1,4,5)P3-independent manner. In the present study, we have identified a specific peptide inhibitor for ADP-ribosyl cyclase of BST-1. A 15-mer random peptide phage library was screened with a soluble form of BST-1 expressed in baculovirus-infected insect cells. After biopanning, two potent sequences reactive towards BST-1 were isolated. The two synthetic peptides corresponding to the identified sequences were shown to antagonize each other's ability to inhibit the binding of the two isolated phage to BST-1, suggesting that they bind at a common binding site on BST-1. One of the peptides (SNP-1) was shown to inhibit ADP-ribosyl cyclase activity of BST-1 in an uncompetitive manner with a Ki value of 180+/-40 nM (n=3). SNP-1 also inhibited cyclic ADP-ribose hydrolase activity of BST-1 dose-dependently. Selected phage did not cross-react with a soluble form of CD38. SNP-1 did not show any inhibitory effect on ADP-ribosyl cyclase activity of CD38, and therefore this peptide inhibitor will be useful to serve as a starting tool for understanding the roles of these intriguing ecto-enzymes. This is the first report of a specific ADP-ribosyl cyclase inhibitor.

Ectocellular CD38-catalyzed synthesis and intracellular Ca 2+-signalling activity of cyclic ADP-ribose in T-lymphocytes are not functionally related

Cyclic ADP-ribose (cADPR) is a natural metabolite of β-NAD + with a potent Ca 2+-mobilizing activity in different cell types, including T-lymphocytes. We investigated (i) whether stimulation of T-lymphocytes with different agonists affects the intracellular concentration of cADPR, and (ii) whether the lymphocyte antigen CD38, through its ectocellular ADP-ribosyl cyclase and cADPR-hydrolase enzymatic activities, can account for the regulation of the intracellular levels of cADPR and the Ca 2+-mobilizing effects of this nucleotide in Jurkat and HPB.ALL T-lymphocytes. The anti-CD3 antibody OKT3, the sphingolipid sphingosine and lysophosphatidic acid induced an increase in intracellular cADPR with concomitant increases in the intracellular Ca 2+ concentration ([Ca 2+] i). In contrast, activation of an ectocellular ADP-ribosyl cyclase by preincubation of cells with β-NAD + led to a dose-dependent increase in cADPR, but no changes in [Ca 2+] i were observed. However, extensive washing of the cells following preincubation with NAD + demonstrated that the increases in cADPR were not intracellular but due to cell surface-associated nucleotide. Accordingly, measurements of ADP-ribosyl cyclase activity in intact T-cells showed ectocellular synthesis of cADPR, but no evidence was obtained for a shift of this activity into the cells which could account for intracellular accumulation of cADPR. Taken together, the results indicate no direct involvement of the ADP-ribosyl cyclase activity of CD38 on the regulation of the cADPR-mediated intracellular Ca 2+-signalling in T-lymphocytes.

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P </= 0.05). Insulin secretion from pancreatic islets by glucose is significantly inhibited by the addition of the NIDDM sera with anti-CD38 antibodies (P </= 0.04-0.0001), and the inhibition of insulin secretion is abolished by the addition of recombinant CD38 (P </= 0.02). The increase of cADPR levels in pancreatic islets by glucose was also inhibited by the addition of the sera (P </= 0.05). These results strongly suggest that the presence of anti-CD38 autoantibodies in NIDDM patients can be one of the major causes of impaired glucose-induced insulin secretion in NIDDM.

Cytoskeleton-dependent inhibition of the ADP-ribosyl cyclase activity of CD38 in thrombin-stimulated platelets

Stimulation of human platelets with thrombin caused a 42% inhibition of the ADP-ribosyl cyclase activity of membrane CD38. This effect was mediated by the activation of the platelet thrombin receptor rather than by proteolysis of CD38, and was not due to a different distribution of the synthesised nucleotide or to a reduced accessibility of CD38 to the substrate. The inhibitory effect of thrombin required actin polymerisation and was not observed when interaction of CD38 with the cytoskeleton was prevented by cytochalasin D. Finally, we analysed whether cADPR could play a role as a Ca 2+-mobilising agent in human platelets. Using saponin-permeabilised cells, we found that unlike IP 3, cADPR did not induce any release of Ca 2+ from intracellular stores. These results indicate that the enzymatic activity of membrane CD38 can be modulated by platelet activation, and that the function of this glycoprotein is probably not related to Ca 2+ mobilisation.