615577f361e0e646601a5d66c76b5ca9 336381.pdf c7190b53889b97b98055df30fc6902f6ee072967 336381.pdf 847d8d600aa757733bc80f08e5dd9883192c9d84fa89c3713cf31e93b2a5c75e 336381.pdf Title: Non-Competitive Binding of Isatuximab and Daratumumab to CD38: Implications for Targeted Therapy in Multiple Myeloma Subject: Background/Objectives: CD38-targeting monoclonal antibodies isatuximab and daratumumab have revolutionized multiple myeloma (MM) treatment, but a deeper understanding of their distinct mechanisms is crucial for therapeutic optimization. Methods: We used flow cytometry to assess isatuximab and daratumumab binding competition in MM cell lines and patient-derived bone marrow cells. The dynamics of CD38 expression were evaluated at different time points before and after antibody-mediated removal. The effects of IMiDs (pomalidomide, lenalidomide) on CD38 expression and isatuximab-induced apoptosis, either alone or in combination with IMiDs, were also examined. Moreover, MM cell migration was assessed through CXCR4-mediated assays, and cell adhesion was evaluated via CD49d-dependent assays. Results: Isatuximab and daratumumab did not compete for CD38 binding, confirming distinct epitope recognition. Following depletion with either antibody, CD38 expression on the MM cell surface began to recover within 2 h, suggesting a dynamic regulation of CD38 availability. While daratumumab lacked direct apoptosis, isatuximab induced significant direct cell death. Pomalidomide enhanced isatuximab-induced apoptosis by increasing CD38 expression, whereas lenalidomide had no significant effect. Additionally, both antibodies effectively inhibited MM cell migration and significantly reduced cell adhesion. Conclusions: Their non-competitive binding and shared impact on cell dynamics suggest opportunities for optimizing treatment strategies through combinatorial or sequential approaches in MM therapy. Keywords: multiple myeloma; monoclonal antibodies; CD38; cytotoxicity Author: Rubén Osuna-Gómez, Jordi López-Pardo, Maria Mulet, Josep Nomdedéu, Elisabet Cantó, Rosa Ortin, Ángela Sánchez Cayuela, Ma Àngels Ortiz, Albert Guinart-Cuadra and Silvia Vidal Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Sep 30 12:38:51 2025 CEST ModDate: Tue Sep 30 12:41:26 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 11064588 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- PGPDYS+VnURWPalladioL-Bold Type 1 Custom yes yes yes 127 0 LOCLZE+VnURWPalladioL Type 1 Custom yes yes yes 128 0 GTCLSJ+URWPalladioL-Roma Type 1 Custom yes yes yes 129 0 FCCXRS+URWPalladioL-Ital Type 1 Custom yes yes yes 130 0 MWMQFV+URWPalladioL-Bold Type 1 Custom yes yes yes 131 0 JDWIZJ+EURM10 Type 1 Builtin yes yes yes 184 0 VYVZGZ+CMSY10 Type 1 Builtin yes yes yes 215 0 CLEDGE+PalatinoLinotype TrueType WinAnsi yes yes no 234 0 CLEELO+PalatinoLinotype TrueType MacRoman yes yes no 400 0 CLEDGC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 401 0 CLEDGE+PalatinoLinotype TrueType WinAnsi yes yes no 402 0 CLEDGE+PalatinoLinotype TrueType WinAnsi yes yes no 504 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-10-07 04:15:28 CEST RepresentationInformation: 336381.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-10-06 18:50:16 CEST Size: 11064588 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 818 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Cell Lines, Reagents and BM Samples Item: Title: Assessment of Daratumumab and Isatuximab Binding Item: Title: CD38 Recovery Assay Item: Title: Viability Assay Item: Title: Migration and Adhesion Assays Item: Title: Statistics Item: Title: Results Children: Item: Title: Isatuximab and Daratumumab Do Not Compete for Binding to CD38 on MM Cell Lines Item: Title: Isatuximab and Daratumumab Do Not Compete for Binding of CD38 on BM Cells from MM Patients Item: Title: Pomalidomide Increases CD38 Levels and Enhance Isatuximab Apoptosis in MM Cell Lines Item: Title: CD38 Expression Rapidly Recovers on MM Cells Following Antibody Removal Item: Title: Isatuximab Interferes with In Vitro Migration and Cell Adhesion of MM Cells Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Non-Competitive Binding of Isatuximab and Daratumumab to CD38: Implications for Targeted Therapy in Multiple Myeloma Author: Rubén Osuna-Gómez, Jordi López-Pardo, Maria Mulet, Josep Nomdedéu, Elisabet Cantó, Rosa Ortin, Ángela Sánchez Cayuela, Ma Àngels Ortiz, Albert Guinart-Cuadra and Silvia Vidal Subject: Background/Objectives: CD38-targeting monoclonal antibodies isatuximab and daratumumab have revolutionized multiple myeloma (MM) treatment, but a deeper understanding of their distinct mechanisms is crucial for therapeutic optimization. Methods: We used flow cytometry to assess isatuximab and daratumumab binding competition in MM cell lines and patient-derived bone marrow cells. The dynamics of CD38 expression were evaluated at different time points before and after antibody-mediated removal. The effects of IMiDs (pomalidomide, lenalidomide) on CD38 expression and isatuximab-induced apoptosis, either alone or in combination with IMiDs, were also examined. Moreover, MM cell migration was assessed through CXCR4-mediated assays, and cell adhesion was evaluated via CD49d-dependent assays. Results: Isatuximab and daratumumab did not compete for CD38 binding, confirming distinct epitope recognition. Following depletion with either antibody, CD38 expression on the MM cell surface began to recover within 2 h, suggesting a dynamic regulation of CD38 availability. While daratumumab lacked direct apoptosis, isatuximab induced significant direct cell death. Pomalidomide enhanced isatuximab-induced apoptosis by increasing CD38 expression, whereas lenalidomide had no significant effect. Additionally, both antibodies effectively inhibited MM cell migration and significantly reduced cell adhesion. Conclusions: Their non-competitive binding and shared impact on cell dynamics suggest opportunities for optimizing treatment strategies through combinatorial or sequential approaches in MM therapy. 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