Aim: This study aimed to improve the efficacy of the CD44v6-targeting antibody UU-40 for molecular radiotherapy through affinity maturation and IgG subclass optimization. M&M: A panel of affinity-matured antibody candidates was generated and characterized as both human IgG4 and IgG1 with LALA mutations. Surface plasmon resonance and LigandTracer analyses identified several candidates with superior affinity and off-rates compared to the parental UU-40. Biodistribution studies in xenograft models using Lutetium-177 (177Lu)-labeled antibodies showed improved tumor retention for selected candidates, particularly UU-A-155. Species cross-reactivity assays confirmed binding to cynomolgus and rabbit v6-peptides, supporting future toxicity studies. Results: The IgG1 LALA format demonstrated reduced binding to Fc gamma receptors, potentially improving the safety profile. UU-A-155 emerged as the lead candidate for clinical translation, showing superior performance in both affinity and tumor retention. Our findings highlight the importance of comprehensive in vitro and in vivo assessments in antibody development, and provides valuable insights into optimizing antibody-based molecular radiotherapy.