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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.<\/jats:p>","DOI":"10.7554\/elife.106425","type":"journal-article","created":{"date-parts":[[2025,4,22]],"date-time":"2025-04-22T10:56:47Z","timestamp":1745319407000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.106425","source":"Crossref","is-referenced-by-count":0,"title":["DuoHexaBody-CD37 induces direct cytotoxic signaling in diffuse large B-cell lymphoma"],"prefix":"10.7554","volume":"14","author":[{"given":"Simar","family":"Pal Singh","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05wg1m734","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medical BioSciences, Radboud Institute for Medical Innovation, Radboud University Medical Center","place":["Nijmegen, 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