cancers Article Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia Raquel Delgado 1,2 , Karoline Kielbassa 1,3,4,5 , Johanna ter Burg 1 , Christian Klein 6 , Christine Trumpfheller 6 , Koen de Heer 7 , Arnon P. Kater 3,4,5,8 and Eric Eldering 1,3,4,5, *   Citation: Delgado, R.; Kielbassa, K.; ter Burg, J.; Klein, C.; Trumpfheller, C.; de Heer, K.; Kater, A.P.; Eldering, E. Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia. Cancers 2021, 13, 3084. https://doi.org/ 10.3390/cancers13123084 Academic Editor: Daruka Mahadevan Received: 7 June 2021 Accepted: 14 June 2021 Published: 21 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Experimental Immunology, Amsterdam University Medical Center, 1105 AZ Amsterdam, The Netherlands; R.Delgado@amsterdamumc.nl (R.D.); k.kielbassa@amsterdamumc.nl (K.K.); hannekterburg@hotmail.com (J.t.B.) 2 Chronic Diseases Research Center, NOVA Medical School, 1150-082 Lisbon, Portugal 3 Cancer Center Amsterdam, Amsterdam University, 1081 HV Amsterdam, The Netherlands; A.P.Kater@amsterdamumc.nl 4 Amsterdam Institute for Infection and Immunity, 1081 HV Amsterdam, The Netherlands 5 LYMMCARE (Lymphoma and Myeloma Center Amsterdam), 1105 AZ Amsterdam, The Netherlands 6 Cancer Immunotherapy Discovery, Roche Innovation Centre Zurich, 8952 Schlieren, Switzerland; christian.klein.ck1@roche.com (C.K.); christine.trumpfheller@roche.com (C.T.) 7 Department of Hematology, Flevoziekenhuis, 1315 RA Almere, The Netherlands; k.deheer@amsterdamumc.nl 8 Department of Hematology, Amsterdam University Medical Center, 1105 AZ Amsterdam, The Netherlands * Correspondence: e.eldering@amsterdamumc.nl; Tel.: +31-205667018 Simple Summary: Previous observations have shown that CD40 activation of CLL cells via coculture with CD40L-expressing fibroblasts increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. We studied the activity of the fully human-agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile and sensitization for cell death by aCD20 mAbs. We found that the pro-survival effect of selicrelumab is minimal, while cell death by combined selicrelumab plus anti-CD20 antibody treatment is maintained. Thus, further investigation of applying selicrelumab combined with anti-CD20 mAbs in a therapeutic setting might be considered. Abstract: Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro. Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following CD40 stimulation was quantified by flow cytometry and ELISA. The anti- apoptotic profile of CLL induced by stimulation was evaluated by the expression of BCL-2 proteins with Western blot, and resistance to venetoclax with flow cytometry. Cell death induced by the combination of selicrelumab and aCD20 mAbs was quantified by flow cytometry. Results: CLL cells treated with selicrelumab upregulated co-stimulatory molecules such as CD86, TNF-α and death receptor CD95/Fas. In contrast to the CD40 ligand-transfected NIH3T3 cells, induction of resistance to venetoclax by selicrelumab was very moderate. Importantly, selicrelumab stimulation positively sensitized CLL cells to CD20-induced cell death, comparable to CD40 ligand-transfected NIH3T3 cells. Conclusions: Taken together, these novel insights into selicrelumab-stimulatory effects in CLL may be considered for developing new therapeutic strategies, particularly in combination with obinutuzumab. Cancers 2021, 13, 3084. https://doi.org/10.3390/cancers13123084 https://www.mdpi.com/journal/cancers