Canonical and non-canonical adenosinergic pathways.

Adenosine (ADO) is an immunosuppressive molecule with multiple functions in different human organs. ADO is released through the concerted action of surface molecules endowed with enzymatic functions, that belong to two different adenosinergic pathways. The canonical pathway is started by CD39, that converts ATP to AMP. On the other hand, the non-canonical pathway metabolizes NAD+ to ADPR, through the action of CD38. The latter byproduct is then converted to AMP by CD203a/PC-1. Both pathways converge to CD73, that fully degrades AMP to the final product ADO. In this Review we take into account the most relevant finding regarding the expression of ectoenzymes belonging to both adenosinergic pathways in different cell types, including regulatory cell subsets and neoplastic cells. Moreover, we summarize the role of these molecules in different physiological and pathological settings. Finally, we discuss potential therapeutic application of specific inhibitors of ectoenzymes and/or ADO receptors.

Functional insights into nucleotide-metabolizing ectoenzymes expressed by bone marrow-resident cells in patients with multiple myeloma.

Human myeloma cells grow in a hypoxic acidic niche in the bone marrow. Cross talk among cellular components of this closed niche generates extracellular adenosine, which promotes tumor cell survival. This is achieved through the binding of adenosine to purinergic receptors into complexes that function as an autocrine/paracrine signal factor with immune regulatory activities that i) down-regulate the functions of most immune effector cells and ii) enhance the activity of cells that suppress anti-tumor immune responses, thus facilitating the escape of malignant myeloma cells from immune surveillance. Here we review recent findings confirming that the dominant phenotype for survival of tumor cells is that where the malignant cells have been metabolically reprogrammed for the generation of lactic acidosis in the bone marrow niche. Adenosine triphosphate and nicotinamide-adenine dinucleotide extruded from tumor cells, along with cyclic adenosine monophosphate, are the main intracellular energetic/messenger molecules that serve as leading substrates in the extracellular space for membrane-bound ectonucleotidases metabolizing purine nucleotides to signaling adenosine. Within this mechanistic framework, the adenosinergic substrate conversion can vary significantly according to the metabolic environment. Indeed, the neoplastic expansion of plasma cells exploits both enzymatic networks and hypoxic acidic conditions for migrating and homing to a protected niche and for evading the immune response. The expression of multiple specific adenosine receptors in the niche completes the profile of a complex regulatory framework whose signals modify multiple myeloma and host immune responses.

Microvesicles released from multiple myeloma cells are equipped with ectoenzymes belonging to canonical and non-canonical adenosinergic pathways and produce adenosine from ATP and NAD.

Multiple myeloma (MM) derives from malignant transformation of plasma cells (PC), which accumulate in the bone marrow (BM), where microenvironment supports tumor growth and inhibits anti-tumor immune responses. Adenosine (ADO), an immunosuppressive molecule, is produced within MM patients' BM by adenosinergic ectoenzymes, starting from ATP (CD39/CD73) or NAD+ [CD38/CD203a(PC-1)/CD73]. These ectoenzymes form a discontinuous network expressed by different BM cells. We investigated the expression and function of ectoenzymes on microvesicles (MVs) isolated from BM plasma samples of patients with MM, using asymptomatic forms of monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) as controls. The percentage of MVs expressing ectoenzymes at high levels was higher when derived from MM patients than controls. BM CD138+ PC from MM patients expressed high levels of all ectoenzymes. Paired MVs samples confirmed a higher percentage of MVs with high ectoenzymes expression in MM patients than controls. Pooled MVs from MM patients or controls were tested for ADO production. The catabolism of ATP, NAD+, ADPR and AMP to ADO was higher in MVs from MM patients than in those from controls. In conclusion, our results confirmed the hypothesis that MVs in MM niche are main contributor of ADO production. The ability of MVs to reach biological fluids strongly support the view that MVs may assume diagnostic and pathogenetic roles.

The Role of Extracellular Adenosine Generation in the Development of Autoimmune Diseases.

Adenosine (ADO) is an immunosuppressive molecule, which suppresses the immune responses by interacting with specific receptors expressed by immune effector cells. ADO is produced from ATP through the enzymatic activities of CD39 and CD73. Alternatively, ADO can be generated starting from NAD+, which is metabolized by the concerted action of CD38, CD203a/PC-1, and CD73. The role of ADO in immunity has been characterized in the last years in physiology and in pathological settings. This review examines a panel of reports focused on the functions of ADO in the context of human autoimmune/inflammatory diseases and the selected animal models. The final aim is to consider the role of adenosinergic ectoenzymes and ADO receptors as novel therapeutic targets for selected diseases.

CD38 increases CXCL12-mediated signals and homing of chronic lymphocytic leukemia cells.

Homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth, a critical step in disease progression, is principally coordinated by the CXCL12/CXCR4 axis. A cohort of 62 CLL patients was divided into migrating and nonmigrating subsets according to chemotaxis toward CXCL12. Migrating patients phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) proteins more than nonmigrating patients (P<0.0002). CD38 expression was the parameter most strongly associated with heightened CXCL12 signaling (P<0.0001), confirmed by independent statistical approaches. Consistent with this observation, CD38(-) CLL cells in samples with bimodal CD38 expression responded less to CXCL12 than the intact clone (P=0.003). Furthermore, lentivirus-induced de novo expression of CD38 was paralleled by increased responses to CXCL12, as compared with cells infected with a control virus. CD38 ligation with agonistic monoclonal antibodies (mAbs) enhanced CXCL12 signaling, whereas blocking anti-CD38 mAbs inhibited chemokine effects in vitro. This is attributed to physical proximity on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by (i) coimmunoprecipitation and (ii) confocal microscopy experiments. Blocking anti-CD38 mAbs significantly compromised homing of CLL cells from blood to lymphoid organs in a mouse model. These results indicate that CD38 synergizes with the CXCR4 pathway and support the working hypothesis that migration is a central step in disease progression.

CD38 as a target of IB4 mAb carrying saporin-S6: design of an immunotoxin for ex vivo depletion of hematological CD38+ neoplasia.

An anti-CD38 mAb (IB4) coupled to saporin-S6, a type 1 ribosome-inactivating protein (RIP), was designed for ex vivo or loco-regional therapeutical applications in myeloma and lymphoma. The ability of this immunotoxin to eliminate CD38+ cells was studied in vitro on selected CD38+ human cell lines (Raji, HBL6, L540 and CEM) and on CD38+ neoplastic cells from a Non Hodgkin Lymphoma (NHL) patient. HBL6, Raji and L540 cells resulted very sensitive to the IB4/saporin-S6 conjugate, concentrations as low as 100 pM of the immunotoxin completely inhibited protein synthesis. CD38+ neoplastic cells from the NHL patient were completely eliminated after treatment with immunotoxin at 10 nM concentration. CFU-c rescue by bone marrow precursors was maintained after exposure to the immunotoxin. These results indicate that IB4/saporin-S6 is endowed with strong and specific cytotoxic effects on selected CD38+ tumor cells lineages. Consequently, it is reasonable to propose a clinical use of the IB4/saporin-S6 for ex vivo purging of unwanted cells (e.g. depletion of contaminating neoplastic cells in aphereses obtained from G-CSF-treated patients) or for loco-regional therapies of CD38+ tumors.

Monoclonal antibodies and therapy of human cancers.

This survey is an overview of the applications of murine, humanized and recombinant monoclonal antibodies for in vivo diagnostic and therapeutic applications. Monoclonal antibodies (mAb) have been applied to the diagnosis and therapy of an array of human diseases. The initial failures of early clinical trials have been overcome through the production of a new generation of mAb which features reduced immunogenicity and improved targeting abilities. The early models of mAb therapy were focused on enhancing the cytolytic mechanisms against the tumor cells. More recently, successful mAb-based therapies were targeted to molecules involved in the regulation of growth of cancer cells. This has highlighted the relevance of understanding receptor-mediated signaling events, and may provide new opportunities for anti-tumor antibody targeting. Despite all the difficulties, clinical data is outlining an increasingly significant role for antibody-mediated cancer therapy as a versatile and powerful instrument in cancer treatment. One reasonable expectation is that treatment at an earlier stage in the disease process or in minimal residual disease may be more advantageous.

Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient.

Human monoclonal antibodies (HuMAb) specific for a 14-kDa perchloric acid-soluble protein (defined as UK114) were produced by somatic fusion of the human-mouse myeloma K6H6/B5 with Epstein-Barr virus-transformed peripheral B lymphocytes from a cancer patient previously treated with UK101 preparations, containing the UK114 protein. Three IgM-secreting clones were selected on the criteria of specificity for the purified UK114 protein immobilized onto plastic and adapted to grow in a serum-free medium. The reactivity of these antibodies showed a broad distribution pattern restricted to fresh tumor tissues and tumor cell lines, mainly of the adenocarcinoma type. None of the normal cells, nonmalignant cell lines, and normal tissues surrounding the neoplastic lesions were reactive. The immunochemical analysis of the target antigens showed that the HuMAb recognize a molecule of 220 kDa selectively expressed by the surface of tumor cells, as well as a cytoplasmic 14-kDa protein. The 220-kDa antigen was different from other tumor-associated antigens with similar molecular mass and, so far, unique. In the presence of human complement, two of three HuMAb are cytotoxic for tumor cells expressing the 220-kDa surface antigen. The tumor specificity and the lytic ability attributed to these HuMAb are promising features for the exploration of future clinical applications.

CD38 binding to human myeloid cells is mediated by mouse and human CD31.

Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukocyte trafficking. In the present study, we found that the soluble form of the CD38 antigen (sCD38) bears a binding domain of low affinity for a cellular receptor on U937 cells. Cross-linking and peptide-mapping studies confirmed the physical association and the identification of the U937 receptor as a 130 kDa protein. The binding of sCD38 to the receptor was differentially inhibited by several monoclonal antibodies against the CD31 cell-adhesion molecule. Thus the interaction was analysed through direct association of soluble and membrane CD38 with soluble recombinant murine CD31 with three N-terminal and with all six extracellular Ig domains. Cross-linking experiments on U937 intact cells, and ligand blot assays of the immunoprecipitated CD38 molecule, indicated that (i) the recognized epitope is determined by the tertiary structure of the molecule, and that (ii) the binding domain involved resides in the ectocellular portion of the CD31 molecule, more precisely in the first three N-terminal domains. A comparative functional activity between murine and human CD31 was also explored. The data presented suggest that (i) human CD31 bears a highly functional similarity with its murine counterpart, as it is a receptor in myeloid cells with more than one ligand (the alphavbeta3 integrin and the CD38 molecule), and that (ii) the activity of sCD38 as decoy molecule for CD31 may play an important role in cell-cell interactions in physiological and pathological conditions.

c-kit is expressed in soft tissue sarcoma of neuroectodermic origin and its ligand prevents apoptosis of neoplastic cells.

During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Consequently, accumulating evidence suggests that the c-kit/SCF system plays a crucial role in all of these processes and in tumors which derive from them. Especially in neuroblastoma (infant tumors of neuroectoderm crest derivation such as melanocytes) it would appear that an autocrine loop exists between c-kit and SCF, and that the functional block of the c-kit receptors with monoclonal antibodies (MoAbs) results in a significant decrease in cellular proliferation. We studied the expression and role of c-kit and SCF in cell lines of soft tissue sarcoma of neuroectodermic origin, such as Ewing's sarcoma (ES) and peripheral neuro-ectodermal tumors (PNET). Using flow cytometry with MoAb CD117 PE, c-kit expression was highlighted in all six of the cell lines examined. This receptor was specifically and functionally activated by SCF, as shown by the binding experiments and the intracellular phosphotyrosine and immunoprecipitation studies that were performed. Using reverse transcriptase polymerase chain reaction analysis, five of the six cellular lines expressed the mRNA of SCF. In the medium measured by using an enzyme- linked immunosorbent assay, low concentrations of SCF were found: only the TC32 cellular line produced significantly higher levels (32 pg) than control. In serum-free culture the addition of SCF reduced the percentage of apoptotic cells from 25% to 90% in five out of the six cellular lines. This observation was confirmed by (1) the functional block of c-kit with MoAb: after 7 days of culture more than 30% of the cells were apoptotic (range 31.5% to 100%) in five out of six cell lines and there was also a decrease in the percentage of cells in phase S, and (2) c-kit antisense oligonucleotides: in the cellular lines treated with oligonucleotides (in relation to the untreated lines) there was a notable reduction (P < .001) both in the absolute number of cells and the 3H-thymidine uptake. These results indicate that ES and PNET express c-kit and its ligand SCF and that SCF is capable of protecting the tumor cells against apoptosis. Furthermore, the reverse transcriptase-polymerase chain reaction performed on the biopsies revealed the presence of mRNA both of SCF and c-kit in practically all of the samples studied. Our in vitro data lead us to assume that SCF may also inhibit tumor cell apoptosis in vivo.

Identification and characterization of an active soluble form of human CD38 in normal and pathological fluids.

Human CD38 is a transmembrane glycoprotein involved in lymphocyte activation and adhesion to endothelium. The ectocellular domain of the molecule possesses properties of a bifunctional enzyme catalyzing both the synthesis from NAD+ and the hydrolysis of the calcium-releasing metabolite cyclic ADP-ribose (cADPR). Surface expression of CD38 (mCD38) is rapidly and almost completely down-modulated upon ligation by specific mAb in cells from different lineages. The data presented here also show that, in addition to the existence of a mCD38, a soluble form of CD38 (sCD38) is detectable in the cell culture supernatant of allo-activated T lymphocytes and of several tumor cell lines. sCD38 is also present in vivo and is assayable in normal (fetal serum and amniotic fluid) and pathological (serum and ascites from patients with multiple myeloma, and serum from patients with AIDS) biological fluids. Immunoaffinity chromatography, SDS-PAGE and Western blot analyses with mAb and polyclonal antibodies, along with metabolic labeling, yield a body of data concerning the structure of sCD38, which displays a M(r) of 39 kDa. Native sCD38 maintains the ability to inhibit the binding activity of different anti-CD38 mAb and still catalyzes the synthesis and the hydrolysis of cADPR at the same ratio observed with mCD38. Furthermore, cross-linking experiments indicate that the purified soluble molecule binds a 120 kDa molecule expressed by monocytoid cells and identified as a candidate ligand for human mCD38.

Human CD38 ligand. A 120-KDA protein predominantly expressed on endothelial cells.

Human CD38, a pleiotropic molecule with ADP-ribosyl cyclase activity, regulates activation and growth of several cell types. Its in vivo function is incompletely determined, mainly due to the lack of evidence concerning the existence of a single or multiple ligands. We recently observed that CD38 rules a selectin-type adhesion between lymphoid cells and HUVECs. A panel of murine mAbs raised against HUVEC included one (Moon-1) constantly blocking the CD38-mediated adhesion of several cell lines to HUVEC. Tissue distribution studies and an extended immunohistochemical analysis on the majority of normal human tissues revealed that the Moon-1 molecule displays a unique pattern of expression, being present at high levels on resting and activated vascular endothelium, on the majority of monocytes, platelets, NK cells, and to a lesser extent on T, B, and myeloid cells. The Moon-1 structure of an apparent molecular mass of 120 kDa proved to be a ligand for human CD38, as inferred by the direct binding observed when using a chimeric mouse CD8 alpha-human CD38 (mCD8 alpha-hCD38) molecule as a probe in Western blot experiments. Furthermore, Ab-induced modulation experiments highlighted an association between the Moon-1 molecule and human CD38 on the surface of cell lines coexpressing the two structures, which also share a common regulation system of surface expression. Finally, direct ligation of Moon-1 on T cell lines caused a relevant increase in the cytoplasmic concentration of calcium ([Ca2+]i).

99mTc direct labeling of anti-CEA monoclonal antibodies: quality control and preclinical studies.

The anti-carcinoembryonic B2C114 monoclonal antibody was radiolabeled with 99mTc by a direct method and quality control tested in vitro by instant thin layer chromatography, gel column scanning and cellulose acetate electrophoresis and assessed in vivo for radioimmunodetection on a murine spontaneous mammary carcinoma. The optimal results of percent 99mTc bound to protein were obtained at a dithiothreitol:antibody molar ratio ranging from 800:1 to 1000:1 and at a methylene diphosphonate:stannous fluoride weight ratio of 4.3:1. Although cysteine removed up to 18% of the label during the first 4 h, the stability of the tracer appeared to be excellent in human serum at 37 degrees C and when challenged with DTPA. 99mTc-labeled B2C114 demonstrated good and specific in vivo tumor targeting.

Direct labeling of monoclonal antibodies with 99mTc and radioimmunodetection of a murine mammary carcinoma with 99mTc-B2C114.

Two anti-CEA antibodies, B2C114 and IORCEA1, were radiolabeled with 99mTc by two direct methods (mercaptoethanol and ascorbic acid reduction), and the radio-immunoimaging properties of B2C114 were assessed in mice bearing an M3-reactive tumor. The labeling efficiency was greater than 90% as measured by ITLC in saline, methylethylketone and with serum albumin impregnated sheets using ethanol: water: NH4OH (2:5:1). The label was stable to challenge with excess DTPA, and in the case of ascorbic acid reduction, serum analysis showed that 10-15% of the radioactivity was lost during incubation. In vitro studies demonstrated that the radiolabeled antibodies retained their immunoreactivity. Biodistribution studies in normal Balb/c mice showed that the pattern of uptake was quite similar for both antibodies. Biodistribution of the 99mTc-B2C114 and image studies in the animal model showed that the tumor was clearly visualized and that B2C114 labeled with 99mTc is a possible candidate for human radioimmunodetection of CEA-expressing tumors.

Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer.

An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.

Marcación del anticuerpo monoclonal B2C114 con 111In usando un quelante funcional / 111In labelling of B2C114 monoclonal antibody with a bifunctional chelating agent

El anticuerpo monoclonal (AcMo) B2C114, dirigido contra el antígeno carcinoembrionario (CEA) fue conjugado con el anhídrido bicíclico del DTPA (CA-DTPA) usando diferentes relaciones molares CA-DTPA: AcMo desde 1:1 hasta 30:1. Se determinó para cada caso la eficiencia de acoplamiento y el número de moléculas de DTPA por molécula de AcMo. Se realizó la marcación con 111In para todas las relaciones molares CA-DTPA: AcMo y se determinó la pureza radioquímica por cromatografía instantánea en placa delgada (ITLC Gelman SG) y cromatografía en gel (Sephadex G-25). La biodistribución del AcMo marcado en ratones normales Balb/c y en portadores de tumor reactivo (M3), a diferentes horas post-inyección, mostró una acumulación creciente en el tumor al cabo de 72 h, con captación en hígado y riñón. Se observó también que al aumentar la relación molar CA-DTPA se incrementó el porcentaje de radiactividad asociada al riñón, lo cual indicaría una mayor inestabilidad del radiofármaco