Specific ADAM10 inhibitors localize in exosome-like vesicles released by Hodgkin lymphoma and stromal cells and prevent sheddase activity carried to bystander cells

Abstract

Shedding of ADAM10 substrates, like TNFα, MICA or CD30, is reported to affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. Soluble forms of these molecules and ADAM10 can be carried and spread in the microenvironment by exosomes released by tumor cells. We reported new ADAM10 inhibitors able to prevent MICA shedding in Hodgkin lymphoma (HL), leading to recognition of HL cells by cytotoxic lymphocytes.In this paper, we show that the mature bioactive form of ADAM10 is released in exosome-like vesicles (ExoV) by HL cells and lymph node mesenchymal stromal cells (MSC). We demonstrate that ADAM10 inhibitors are released in ExoV by MSC or HL cells, endocytosed by bystander cells and localized in the endolysosomal compartment in HL MSC. ExoV released by HL cells can enhance MICA shedding by MSC, while ExoV from MSC induce TNFα or CD30 shedding by HL cells. Of note, ADAM10 sheddase activity carried by ExoV is prevented with the ADAM10 inhibitors LT4 and CAM29, pretreating either the ExoV-producing or the ExoV-receiving cells. In particular, both inhibitors reduce CD30 shedding maintaining the anti-tumor effects of the ADC Brentuximab-Vedotin or the anti-CD30 Iratumumab on HL cells.Thus, spreading of ADAM10 activity due to ExoV can result in the release of cytokines, like TNFα, a lymphoma growth factor, or soluble molecules, like sMICA or sCD30, that potentially interfere with host immune surveillance or immunotherapy. ADAM10 blockers can interfere with this process, allowing the development of anti-lymphoma immune response and/or efficient ADC-based or human antibody-based immunotherapy.

Role of ADAM10 as a CD30 Sheddase in Classical Hodgkin Lymphoma

“…However, BB-3644 inhibits different metalloproteinases and this might have been a reason why a clinical phase I dose-escalation study with BB-3644 showed dose-limiting musculoskeletal toxicities and was discontinued (28). More selective ADAM10 inhibitors, such as the synthetic LT4 and INCD8765, the ADAM10 antibody 8C7, and an exocite-binding artificial substrate might provide a better alternative with less side effects (Figure 1) (21,29,30).…”

“…In situ, there might be another kinetic of CD30 cleavage because natural inhibitors and additional enzymes might influence CD30 shedding. Thus, EV-associated ADAM10 from other cells might participate in CD30 cleavage (21). Nevertheless, CD30 is not completely cleaved when EVs harbor in the circulation, because in the blood of cHL patients, a low percentage of CD30 is EV-associated (14).…”

Synthesis and in vitro Evaluation of ADAM10 and ADAM17 Highly Selective Bioimaging Probes

“…They were used in a recent study to show that ADAM10 mature form is released in exosome‐like vesicles (ExoV) by HL cells and lymph node mesenchymal stromal cells. Using these probes it has been also demonstrated that ADAM10 inhibitors can be released in ExoV by tumor cells and can be endocytosed by bystander cells thus enhancing the antibody‐drug conjugate Brentuximab‐Vedotin antitumor effects on Hodgkin lymphoma cells …”

“…Photoprobe 3 maintained the same activity profile of its unlabeled analogues 1 and 2,while fluorescent probes 6a and 6b showed aslight decrease in potency toward ADAM10 with respectt ot he parent compounds 4 and 5.A ll the probes were enough potent and selective for their targets and were successfully used for ADAMss taining on differentl ymphoma cell lines.F inally,a gold-labeled derivativeo f4,c ompound 6c,w as developed as an additional bioimagingp robe to trace the intracellular route specifically followed by ADAM10. [34] To this aim small GNPs (6 nm) wereu sed to avoid interference with ADAM10 physiological internalization and nonspecific endocytosis of bigger GNP, [34] though GNP around4 0-60 nm are best for efficient drug delivery,w hich is the topic of future work.…”

“…Using thesep robesi th as been also demonstrated that ADAM10i nhibitors can be released in ExoV by tumor cells and can be endocytosedb yb ystander cells thus enhancing the antibody-drug conjugate Brentuximab-Vedotin antitumor effects on Hodgkin lymphoma cells. [34] Experimental Section Chemistry Melting points were determined on aK ofler hotstage apparatus and are uncorrected. 1 Ha nd 13 CNMR spectra were recorded on a Bruker Avance III HD 400 MHz spectrometer.C hemical shifts (d)a re reported in parts per million and referenced according to deuterated solvent.…”