Nestorovich, EkaterinaFulfulan, Amro Abdulrahim2023-08-012023-08-012023-07-12https://hdl.handle.net/20.500.14154/68762Chronic lymphocytic leukemia (CLL) is a hematopoietic disease caused by the accumulation of CD5+/CD19+ mature CLL B cells in blood. Despite the promising results of anti-CD20 monoclonal antibodies and Bruton's Tyrosine Kinase inhibitors (BTKis) in CLL treatment, yet patients relapse and reduce their median progression-free survival. This necessitates exploring novel therapeutic approaches to surpass current CLL treatment limitations. The Fc receptor for IgM (FCMR/ FcμR), a type-1 transmembrane glycoprotein, is essential to the humoral immune response of B cells. Studies have shown that FCMR mRNA is markedly upregulated in CLL B cells compared to their normal counterparts, particularly in cells bearing unfavorable prognostic markers (unmutated-IGHV, CD38). Higher-risk cases also exhibited elevated FCMR mRNA levels, indicating the potential influence of surface FcμR on CLL progression. FcμR binds to the Fc fragment of IgM with high avidity, resulting in immediate clathrin-mediated internalization and lysosomal delivery. This underscores FcμR as a promising biomarker for CLL-targeted therapies. Our investigation centered on Antibody-Drug Conjugates (ADCs), a form of targeted therapy that couples a cytotoxic drug with a monoclonal antibody. We developed novel Dual Variable Domain antibodies (DVD-Abs) that possess an h38C2 IgG1 backbone and anti-FcμR rabbit variable domains, resulting in a chimeric DVD-Ab targeting FcμR. The h38C2 IgG1 Ab has a uniquely reactive lysine residue (Lys99), allowing a site-click and homogenous attachment to cytotoxic drug. Four anti-FcμR DVD-Abs formats were constructed but we focused on DVD-IgG and Fc-silenced DVD-IgGs. For therapeutic applications, the DVD-Abs were homogenously conjugated with cytotoxic agents such as MMAF to create DVD-IgG-β-Lactam-MMAF, and Fc-silenced DVD-IgG-DBCO-PEG4-Valine-Citruline-PAB-MMAF. We hypothesize that the DVD-ADCs will broaden CLL therapeutic approaches by providing long-lasting drug delivery to cancer while minimizing off-target toxicity. DVD-Abs bound strongly to FcμR-overexpressing Mino, NU-DHL-1, and NU-DUL-1 cell lines but not to corresponding FcμR knockout (FcμR-KO) cell lines. Additionally, DVD-Abs demonstrated high specificity towards FcμR on CLL cells compared to donor B cells. The binding of DVD-Abs to FcµR remained unaffected by human IgM or HM7 blockade on Mino and CLL cells as Fc silenced DVD-IgGs (clones: A1 and A4) recognize a certain epitope (ISALEGLL) in the FcµR stalk. Intriguingly, FcμR-presenting cell lines and CLL B cells rapidly internalized the Ab-FcμR complex, effectively directing DVD-IgGs into lysosomal compartments. The binding of ADCs demonstrated potent cytotoxic effects on FcµR-expressing cell lines, but not on FcµR-KO cell lines. This potency also included CLL B cells without impacting the viability of normal cells, highlighting their potential for targeted cancer therapy. Based on our findings, we propose the IgM Fc receptor as a novel therapeutic target and anti-FcμR ADCs as a novel strategy to target CLL in a hope that they will be translated into clinics in the forthcoming.110en-USADCsAntibody-drug conjugatesIgM Fc receptorTOSOFCMRFcuRCLLChronic Lymphocytic LeukemiaFree drugsDCMMMAFMMAEDual Variable DomainDVDAntibodiesDVD-AbsDM1MinoNU-DHL-1NU-DUL-1Thesis