Immunological and molecular studies for the development of vaccine therapy for chronic lymphocytic leukemia

Sammanfattning: Chronic lymphocytic leukaemia (CLL) is a malignant lymphoproliferative disorder which typically affects elderly people. It is the commonest leukemia in the Western adults, accounting for 25-30% of all leukemias and for 10% of all hematological neoplasms. Although new modalities such as combination therapy with fludarabine, cyclophosphamide (CTX) and the anti-CD20 antibody rituximab have greatly improved clinical outcome in a fraction of patients, CLL is largely considered incurable and there is a continuous need to develop new treatment strategies. Anti-cancer active specific immunotherapy aims at activating the patient´s immune system to recognize and eliminate the tumor. A number of clinical observations as well as several preclinical studies indicate that CLL is responsive to immune effector functions. In the first part of this thesis, we investigated the ability of a promiscuous HLA class II epitope, hTERT (611 626) (GV1001) to elicit antileukemic immune responses in vitro. We demonstrated that CLL patients with hTERT-expressing leukemic cells have naturally occurring hTERT-specific T cells that proliferate and can be expanded in vitro and used to lyse autologous CLL cells. We therefore identified telomerase as a vaccine candidate in CLL. We then analyzed hTERT mRNA splicing patterns in CLL by a newly designed quantitative PCR assay and showed that the expression of the functional transcript of hTERT (hTERT-FL) is independent from disease phase in IgHV mutated but not in unmutated patients. This finding highlights the necessity of focusing on this transcript when analyzing hTERT expression and encourages further studies to assess whether hTERT-FL could generate novel epitopes that may serve as immunotherapy targets. In the second part of the thesis, we studied safety, immune and clinical effects of vaccination with autologous DC loaded with apoptotic CLL cells (Apo-DC) in CLL patients in a phase I clinical trial. Using a combination of leukapheresis and affinity-based technologies (CliniMACS®) for monocyte enrichment, we were able to produce a sufficient amount of DC vaccine that met accepted and established quality criteria. Sixteen patients were accrued stepwise in three different cohorts receiving Apo-DC alone, Apo-DC + granulocyte-macrophage-colony-stimulating-factor (GM-CSF), or Apo-DC + GM-CSF + low-dose CTX. Vaccination was well tolerated and increased leukemia-specific immunity in 10/15 (66%) of the patients (2/5, 3/5 and 5/5 in the three cohorts, respectively). No significant difference in time-to progression (TTP) between immuneresponders and non-immune responders was observed. An additional patient was immunized repeatedly for a long period of time and achieved a complete response in blood and a nodular partial response in bone marrow. CD4+CD25highFOXP3+ regulatory T-cells (Tregs) measured in one year follow-up period were significantly lower in immuneresponders vs non-responders (p<0.0001). In this study, we demonstrated that vaccination with Apo-DC is a feasible approach that can generate immune responses and potentially clinical responses and that combination with GM-CSF and low-dose CTX functions as an immunological adjuvant in this setting. In conclusion, the studies presented in this thesis suggest that immunotherapy is a promising approach in CLL and promote further investigation to better define the vaccination strategy and combination with immune enhancing/modulating drugs which holds the greatest potential to generate immune responses and clinical benefit in CLL patients.