Chlorambucil Conjugation Enhances the Potency of Rituximab: Synthesis and Evaluation of the Novel [177Lu]Lu-Labeled Rituximab-Chlorambucil Conjugate toward Therapy of Non-Hodgkin’s Lymphoma

Abstract

In this study, a novel antibody-drug conjugate (ADC) consisting of Rituximab and Chlorambucil (Rituximab-CMB) was synthesized. The average number of drug molecules attached per Rituximab molecule was determined using MALDI-TOF mass spectrometry, revealing a range of 4–6 drug molecules per antibody. To further improve the therapeutic potential of the ADC, it was radiolabeled with the therapeutic radionuclide ¹⁷⁷Lu via a DOTA chelator, achieving a final radiochemical purity of over 95%. In vitro assays demonstrated that the Rituximab-CMB conjugate had greater cytotoxicity compared to that of both unconjugated Rituximab and Chlorambucil alone. Moreover, [¹⁷⁷Lu]Lu-labeled-Rituximab-CMB (15.67 MBq/mg) exhibited higher radiotoxicity (37.08 ± 1.40% cell death) compared to [¹⁷⁷Lu]Lu-labeled-Rituximab (83.99 MBq/mg) (25.25 ± 0.8% cell death) when administered at similar radioactivity doses. Ex vivo experiments indicated that coinjecting cold Rituximab with the radiolabeled formulations significantly improved tumor accumulation and reduced nontarget organ uptake. SPECT-CT imaging results supported these findings, further confirming the enhanced tumor-targeting and biodistribution of the radiolabeled ADC.