CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin,in vitro

Abstract

The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30−/CD30L+ mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30+ EVs.

Role of ADAM10 as a CD30 Sheddase in Classical Hodgkin Lymphoma

“…Isolated EVs from cHL cells degrade the artificial ADAM10-selective substrate PEPMCA001 and CD30, the latter resulting in a CD30 reduction to 71% of the inhibited control after 18 h of incubation. These data indicate that CD30 is slowly cleaved on isolated EVs (14). In situ, there might be another kinetic of CD30 cleavage because natural inhibitors and additional enzymes might influence CD30 shedding.…”

“…Inversely, eosinophils, which are typical bystander cells in cHL, bind CD30 + EVs and the coapplication of the ADC BV causes cell damage also in CD30 − eosinophilic cells. Here, the effect depends on the presence of BV and CD30 + EVs (Figure 1) (14). By contrast, the coincubation of the same CD30 concentration of sCD30 was almost ineffective.…”

“…Because the vesicle types have an overlapping diameter, functionality and marker proteins, the International Society of Extracellular Vesicles (ISEV) suggests to collectively refer to them as extracellular vesicles (EVs) (12). Electron microscopy, nanoparticle tracking analysis (NTA), and mass spectrometry revealed that the EVs from Hodgkin cells have an EV-typical diameter between 40 and 800 nm (mean around 140 nm) and they express CD30 and ADAM10 (13,14). They also show a strong exposure of the lipid phosphatidylserine (PS) on the outer membrane leaflet (13).…”

“…Both uncleaved EV-associated CD30 and sCD30 are found in the cell supernatant of cHL cells, but only EV-associated CD30 functionally influences CD30L + bystander cells to release IL-8 (13). In vitro, only a minority of released CD30 is EVassociated, ranging between 1.7 and 3.7% in the supernatants (SN) of L428 and L1236 cells, respectively (14). In situ, the tumor microenvironment might modify this ratio.…”

“…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).…”