P30-OHKUBO

CD30⁺ EVs Enable the Targeting of CD30^- DLBCL Cells by the CD30 Antibody-Drug Conjugate Brentuximab Vedotin

Brentuximab Vedotin (BV), an anti-CD30 antibody-drug conjugate, shows efficacy in CD30-negative diffuse large B-cell lymphoma (DLBCL), but the mechanism is unclear. Lobastova et al. investigated whether CD30⁺ extracellular vesicles (EVs) enable BV binding and uptake in CD30-negative tumor cells.

To study CD30⁺ EVs mediating BV toxicity in non-Hodgkin B cell leukemia, they assessed three target cell lines for endogenous CD30, EV binding, and CD30 enrichment. Flow cytometry showed Karpas 422 and DoHH-2 lacked CD30, while P30-OH/KUBO was positive (Fig. 1A, left). All lines bound fluorescent EVs from L540 and THP-1 cells (Fig. 1A, middle). Only L540-derived EVs increased CD30 on CD30^- cell surfaces (Fig. 1A, right). CD30 enrichment was measured by comparing CD30 MFI in EV-treated vs. untreated cells. Karpas 422 showed better CD30 surface enrichment than DoHH-2 after 2 h; in CD30⁺ P30-OH/KUBO, EVs contributed little to total CD30. Membrane-labeled EVs gave stronger signals than anti-CD30 staining. Only a small fraction of target cells bound CD30⁺ EVs (Fig. 1B), likely due to EV heterogeneity. CD30 internalization may also differ by cell type. We then studied anti-CD30 internalization. After co-incubating cells with EVs and fluorescent anti-CD30 (using SGN30 or Ki-3 due to BV labeling issues), confocal microscopy revealed CD30⁺ EVs promoted antibody uptake in Karpas 422 and DoHH-2, but not with THP-1 EVs (Fig. 1C). Intracellular signals exceeded surface signals, partly explaining low surface CD30 in prior assays. Imaging flow cytometry quantified SGN30-FITC uptake, yielding internalization scores of 3.1 (Karpas 422, N=2156) and 2.1 (P30-OH/KUBO, N=1086) with L540 EVs, confirming CD30⁺ EVs enable antibody uptake (Fig. 1D).