Targeted therapies and immunotherapy in chronic lymphocytic leukemia and Richter syndrome: preclinical and translational studies

Thesis title: Targeted therapies and immunotherapy in chronic lymphocytic leukemia and Richter syndrome: preclinical and translational studies Background Chronic lymphocytic leukemia (CLL) is a disease characterized by the progressive accumulation of mature monoclonal B lymphocytes, driven by genetic alterations and sustained by dynamic interactions with the tumor microenvironment. Despite major advances introduced by targeted therapies, including Bruton’s tyrosine kinase (BTK) and BCL-2 inhibitors, CLL remains largely incurable, and immune dysfunction represents a key driver of disease persistence and therapeutic resistance. Patients with CLL frequently exhibit T-cell exhaustion, impaired cytotoxic activity, and defective immune surveillance, thereby limiting the efficacy of immunotherapeutic strategies. Richter transformation (RT), the evolution of CLL into an aggressive lymphoma, further underscores the unmet clinical need for innovative therapeutic approaches. This thesis aimed to investigate how targeted therapies and immunotherapy can reshape immune responses in CLL and RT, and to identify strategies capable of enhancing antitumor immunity and therapeutic efficacy through preclinical and translational studies. Methods A multidisciplinary approach integrating preclinical models, longitudinal patient analyses and translational immunological studies was employed. Peripheral blood samples from CLL patients receiving targeted agents were analyzed using multiparametric flow cytometry, functional assays and molecular profiling to assess immune cell phenotype and function. In vivo experimental models were used to evaluate immune checkpoint modulation and cytokine-based immunotherapy combinations. In addition, CAR T cells were generated from patients treated with targeted therapies to investigate how treatment influences T-cell fitness, differentiation status, functional activity and the CAR T-cell manufacturing process. Clinical trial data and real-world patient cohorts were incorporated to correlate immunological findings with therapeutic responses. Results CLL was confirmed to be associated with profound immune dysregulation, characterized by exhausted T-cell phenotypes and impaired effector functions. Targeted therapies demonstrated significant immunomodulatory effects beyond their direct antitumor activity, partially restoring immune competence and reshaping T-cell and natural killer cell compartments. Combination of immunotherapeutic strategies enhanced immune activation and improved antitumor responses in preclinical settings. Importantly, prolonged exposure to targeted agents influenced T-cell differentiation dynamics, identifying a potential optimal window for lymphocyte apheresis for CAR T-cell manufacturing. CAR T-cell products generated during treatment with targeted therapies showed improved memory phenotypes and preserved cytotoxicity; however, manufacturing processes could re-induce exhaustion features. In RT models and clinical settings, combined targeted and immune-based approaches showed encouraging activity despite the aggressive biology of the disease. Conclusions This work demonstrates that immune modulation is central to improving therapeutic outcomes in CLL and RT. Targeted therapies function not only as antitumor agents but also as regulators of immune fitness, creating opportunities to enhance immunotherapy efficacy. Optimizing treatment sequencing, combination strategies and cellular therapy manufacturing may help overcome immune dysfunction and resistance mechanisms. These findings support a translational framework integrating targeted therapy with immunotherapy to achieve more durable responses and advance personalized treatment strategies for CLL and its aggressive variants