A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg(2+) ion hexacoordinated at the metal ion-dependent adhesion site (MIDAS) ...in the integrin A domain. This interaction stabilizes the A domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking mAb 107 binds MIDAS of integrin CD11b/CD18 A domain (CD11bA), but in contrast, it favors the inhibitory Ca(2+) ion over the Mg(2+) ion at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of the Ca(2+) ion at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca(2+) ion. Binding of the Fab fragment of mAb 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that the denticity of the ligand Asp/Glu can modify the divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca(2+) ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists.
This paper reviews some of the results and the speculations presented at the Torino CD38 Meeting in June, 2006 and focused on CD38 and CD157 seen as a family of molecules acting as surface receptors ...of immune cells. This partisan view was adopted in the attempt to combine the enzymatic functions with what the immunologists consider key functions in different cell models. At the moment, it is unclear whether the two functions are correlated, indifferent, or independent. Here we present conclusions inferred exclusively on human cell models, namely T and B lymphocytes, dendritic cells, and granulocytes. As an extra analytical tool, we try to follow in the history of life when the enzymatic and receptorial functions were generated, mixing ontogeny, membrane localization, and cell anchorage.
Ectoenzymes are a family of cell surface molecules whose catalytic domain lies in the extracellular region. A subset of this family, nucleotide-metabolizing ectoenzymes, are key components in the ...regulation of the extracellular balance between nucleotides (e.g. NAD+ or ATP) and nucleosides (e.g. adenosine). Their substrates and products are signalling molecules that act by binding to specific receptors, triggering signals that regulate a variety of functions, ranging from the migration of immune cells, to synaptic transmission in the brain, to hormone/receptor interactions in the glands. Almost two decades of accumulated data indicate that these regulatory processes significantly affect the endocrine system, a tightly controlled information signal complex with clear evidence of fine regulation. Functional models discussed in this review include insulin secretion, bone modelling and the association between hormones and behaviour. The emerging pattern is one of a system operating as a scale-free network that hinges around hubs of key molecules, such as NAD+ or ATP. The underlying natural link between nucleotides, ectoenzymes and the endocrine system is far from being clearly demonstrated. However, the body of evidence supporting the existence of such connection is growing exponentially. This review will try to read the available evidence in a hypothesis-oriented perspective, starting from the description of NAD+ and of ecto- and endoenzymes involved in its metabolism.
CD38, a surface receptor that controls signals in immunocompetent cells, is densely expressed by cells of multiple myeloma (MM). The immune system of MM patients appears as functionally impaired, ...with qualitative and quantitative defects in T cell immune responses. This work answers the issue whether CD38 plays a role in the impairment of T lymphocyte response. To this aim, we analyzed the signals implemented by monoclonal antibodies (mAb) ligation in peripheral blood mononuclear cells (PBMC) obtained from MM patients and compared to benign monoclonal gammopathy of undetermined significance (MGUS). PBMC from MM both failed to proliferate and secrete IFNγ induced by CD38 ligation while it retained the ability to respond to TCR/CD3. The impaired CD38-dependent proliferative response likely reflects an arrest in the progression of cell cycle, as indicated by the reduced expression of PCNA. CD38 signaling showed an enhanced ability to induce IL-6 secretion. PBMC from MM patients displays a deregulated response possibly due to defects of CD38 activation pathways and CD38 may be functionally involved in the progression of this pathology via the secretion of high levels of IL-6 that protects neoplastic cells from apoptosis.
Abstract
Background CD38 is a pleiotropic cell surface glycoprotein with receptorial and enzymatic functions. The molecule is generally expressed at low levels by different hematological and solid ...tissues: plasma cells score the highest surface levels of CD38 among mature lymphoid cells. CD38 has become the target of therapeutic antibodies in multiple myeloma (MM). Daratumumab (Dara) has been approved as efficient monotherapy or in combination with other anti-myeloma agents. The results obtained are good in patients refractory to standard myeloma therapies. Dara mediates clinical effects through multiple mechanisms. These include complement- and antibody-dependent cell cytotoxicity, antibody-dependent phagocytosis, programmed cell death and modulation of enzymatic activities. Promising are Dara immunotherapeutic functions in virtue of its ability to induce cytotoxicity exploiting both arms of innate and adaptive immune responses. Recent studies suggest that Dara plays immunomodulatory roles in addition to its direct effects on cytotoxicity.
Results CD38 engagement by Dara on MM cells is followed by a selective polar aggregation of the target molecule in myeloma membranes, with subsequent release of microvesicles (MV) of 100-1,000 nm into the extracellular space. We validated the hypothesis that MV released by MM in the bone marrow (BM) niche may express functional ectoenzymes (CD38, CD39, CD73, and CD203a), potentially capable of metabolizing both ATP and NAD+ and to produce adenosine (ADO). Results indicate that MV obtained after Dara treatment tend to cluster around (and to be internalized in) NK cells, monocytes and MDSC, cells all expressing IgG Fc Receptors (FcR). NK cells, which apparently disappear in patients during Dara treatment: because of this, they were selected for testing MV-mediated effects. Comparative analysis of the genes modulated after exposing NK cells to the MV/Dara complex were followed by functional in vitro experiments. Both sets of results confirmed reduced proliferative ability and enhanced NK cell-mediated killing of MM cells. A further support to the immune modulatory roles exerted by Dara comes from the observation that MV surface represent a clustering of the expected CD38/Dara complex, flanked by a set of ectoenzymes involved in the generation of ADO. Moreover, MV express high amount of CD55 and CD59 molecules, receptors which impair the ability of the complement to exert in situ a cytolytic activity. Another set of observations indicate that PD-L1 tend to accumulate in the surface of MV after Dara treatment, anticipating a role in the modulation of immune checkpoint pathways (PD-1/PD-L1).
Conclusions Results of our observations indicate that MV obtained from MM cells treated with Dara may be a particulate system to influence the BM niche and the successive immune responses elicited by different mechanisms and cell effectors.
Citation Format: Barbara Castella, Angelo C. Faini, Yuliya Yakymiv, Fabio Morandi, Alessandra Larocca, Stefania Oliva, Alberto L. Horenstein, Massimo Massaia, Fabio Malavasi. Induction of structural and functional effects of myeloma cells after daratumumab treatment 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 2122.
Human CD38 is a target of different therapeutic antibodies and the results from in vivo applications are favorable. However, the use of CD38 targeting may be improved due to insights still being ...gleaned from basic research, also covering its paralogue CD157. Myeloma was adopted to confirm in vivo our in vitro models of CD38, which is a receptor and an ectoenzyme. Our hypothesis is that ectoenzymes involved in NAD+ metabolism are part of the myeloma escape strategy. Extracellular nucleotides (ATP, NAD+), high in tumor environments, also serve as intracellular signal transducers, with their degradation products modulating the communications between myeloma and normal cells. Hypoxia helps reprogram the local metabolism. In the extracellular milieu, nucleotides bind purinergic type P2 receptors or are metabolized by ectoenzymes and converted to adenosine (ADO), an immunosuppressor that may also bind different P1 receptors. The resulting anergic status helps the tumor evade the host immune response by promoting Tregs and MDSC, and depressing NK and T effectors. Another question is whether therapeutic antibodies interfere with the survival mechanisms of myeloma. The unprecedented fact that anti-CD38 antibody therapy targets not only a molecule expressed by the tumor but also by effectors and inhibitory cells is a further source of complication. The direct action of the antibodies on the tumor is being investigated in depth, but little is known about their presentation to the target cells. The steps of Fc domain presentation of the antibodies to the target molecule dissected in vitro using CHO cells expressing 4 distinct human FcRs. Exposure of CD38 to Daratumumab (DARA) bound to CHO/FcRs causes at 37 °C a selective aggregation to one pole of the myeloma cells. Such aggregations (rich in CD38 and bound DARA) are released as microvesicles (MVs) into the culture medium or in vivo into biological fluids. These MVs differ from those spontaneously released simply in the presence of the antibody on their surface. In conclusion, MVs are equipped with an enzymatic network potentially capable of metabolizing both ATP and NAD+ to produce ADO. Secondly, they may fuse with neighboring cells and leave the myeloma niche, eventually reaching the blood. The therapeutic IgG on their surface acts as a link which favors the capture by FcRs+ cells. This model was confirmed by tracking the migrated MVs which accumulate around NK cells (>30%) and monocytes (>90%). The same MVs then enter the cytoplasm of NK, monocytes and MDSCs, even if the functional effects induced by their internalization remain to be defined. Preliminary results conducted on purified NK cells exposed in vitro to MV from myeloma membranes treated with DARA indicate that they contain gene products of potential relevance. Indeed, a set of genes involved in the immune response and in the regulation of cell death is up-modulated. Instead, another set of genes related to mitosis and cell cycle is down-modulated. Under evaluation is what happens when MVs are taken up by FcR+-dendritic cells, which may reveal possible vaccinal effects. The transfer of the results of basic research on CD38 to therapy is just beginning: exploration of its multiple functions are expected to provide new, valuable insights.
Malavasi:Tusk Therapeutics: Research Funding; Janssen: Consultancy, Honoraria, Research Funding.
Breast ductal carcinoma
in situ
is an intraductal proliferation of malignant epithelial cells that diffuse within the ductal system without stromal invasion. Our finding that a subset of these tumors ...express CD31/platelet endothelial cell adhesion molecule-1 suggests that breast cancer represents an informative model for studying the involvement of the molecule in the morphogenesis, differentiation, and diffusion of this disease. Transfection of CD31 in MDA-MB-231 cells caused reduction in growth, loss of CD44, and acquisition of a ductal morphology. The same effects were maintained
in vivo
, in which CD31
+ tumors grew with
in situ
-like aspects, papillary differentiation, and a secretory phenotype. CD44 was down-modulated, with the CD31
+ cells blocked in the G
1 phase. The morphology was highly similar to what was observed in some human CD31
+ ductal carcinomas
in situ
. MDA-MB-231 mock cells grew in solid sheets, lacking stromal material, and displaying high levels of CD44 and proliferation. CD31
+ cells acquired motility characteristics in
in vitro
assays, a finding confirmed
in vivo by the diffusion of human tumor cells throughout the normal ducts residual in the murine mammary gland. In conclusion, CD31 expression reverts the undifferentiated morphology and aggressive behavior of MDA-MB-231 cells, indicating its active role in the morphogenesis of breast ductal
in situ
carcinomas.
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