DuoHexaBody-CD37 induces direct cytotoxic signaling in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a common aggressive form of non-Hodgkin lymphoma. Tetraspanin CD37 is highly expressed on mature B cells and being studied as a therapeutic target for NHL, including DLBCL. DuoHexaBody-CD37 is a biparatopic antibody with an E430G hexamerization-enhancing mutation targeting two non-overlapping CD37 epitopes shown to promote complement-dependent cytotoxicity. However, the impact of DuoHexaBody-CD37 on direct cytotoxic signaling has not yet been studied. Here, we demonstrate that DuoHexaBody-CD37 induces direct cytotoxicity in DLBCL-derived tumor cell lines independent of the subtype. DuoHexaBody-CD37 induced significant CD37 clustering and was retained at the cell surface in contrast to rituximab, which was internalized. Unbiased screening identified the modulation of 26 (phospho)proteins upon DuoHexaBody-CD37 treatment of primary B cells or DLBCL cells. Whereas DLBCL cells predominantly upregulated p-SHP1(Y564) upon DuoHexaBody-CD37 treatment, primary B cells showed significantly increased p-AKT(S473) and MAPK signaling which is linked to cell survival. Studies using CD37-mutants identified the N-terminus to be involved in DuoHexaBody-CD37-induced signaling. Finally, DuoHexaBody-CD37 treatment inhibited cytokine pro-survival signaling in DLBCL cells. These findings provide novel insights into the signaling functions of CD37 upon DuoHexaBody-CD37 treatment, and open up opportunities for developing CD37-targeted immunotherapy in combination with small molecule inhibitors to maximize tumor cell death.