Tetrocarcin-A as immunostimulatory tool in B-CLL

Chronic B lymphocytic leukemia (B-CLL) represents a lymphoproliferative disease with clonal expansion of immunoincompetent B lymphocytes. Clinically, the disease is characterized by a distinct immune deficiency and by the direct consequences of B-cell accumulation, i.e. splenomegaly, lymphadenopathy, bone marrow infiltration and progressive lymphocytosis. Beside dysregulation of apoptotic pathways at the cellular level of the neoplastic clone, profound disturbances in the immune system leading to severe infections, autoimmune diseases and to the frequent occurrences of secondary solid tumors are found in B-CLL. A pathologic T-cell distribution together with an impairment of T-cell functions were thought to be primarily involved in the immune deficiency and the increased frequencies of infection episodes occurring in these patients. T-cell deficiencies of B-CLL patients are additionally aggravated by the most efficient cytotoxic agents currently available for treatment of B-CLL such as fludarabine phosphate or 2-CdA, thereby counteracting attempts for reconstitution of active immune surveillance. Thus, the degree of aggressive therapy feasible under in vivo conditions remains limited and new drugs with more selective targeting of the neoplastic clone and/or with immunostimulatory activities are urgently required. Following the concept of vaccination against infectious diseases, the induction of a long-lasting immunological memory directed toward the tumor following cytoreductive therapy is now discussed as a new option in tumor therapy. In the first part of our project, we will elucidate whether Tetrocarcin-A (TC-A), a novel antitumor agent, beside killing B-CLL tumor cells, might have local immunostimulatory effects in vivo, and whether these features are specific for TC-A and absent in other cytotoxic agents already available for the therapy of B-CLL patients. In detail, the role of heat shock proteins induced in tumor cells dying following treatment with TC-A in the activation of dendritic cells, monocytes and natural killer cells, will be analyzed. In the second part, it will be evaluated whether TC-A-mediated ex vivo generation of apoptotic bodies or cell lysates from dying tumor cells can be used for a vaccination strategy. These studies will be completed with experiments in a hu-PBL-SCID xenograft mouse model where the primary and secondary immune response generated by vaccination with DC will be assessed.

Following the concept of vaccination against infectious diseases, the induction of a long-lasting immunological memory directed to tumor cells is now discussed as a new option in tumor therapy. This approach requires cytoreductive therapies which decrease tumor load and thus relieve tumor-mediated immune suppression but do not harm immune effector cell such as T cells. The T cell population can then be activated and expanded for a long-term control of therapy-resistant tumor cells in order to decrease the risk of disease relapse. In this project we evaluated whether cytoreduction using the microbial agent Tetrocarcin-A fulfills the criteria of a cytoreductive agent which could be used in such an approach. Our previous work suggested this since TC-A effectively induced apoptosis preferentially in tumor cells as compared to T cells and cell death of tumor cells was associated with a significant upregulation of heat shock protein (HSP) levels. These proteins have been shown to increase activity of the immune system by acting of dendritic cells (DC), monocytes and NK cells. We could not corroborate previous findings of an immunostimulatory activity of HSPs in DC since maturation markers, costimulatory molecules and T cell stimulatory of DC did not change, neither following treatment with HSP-containing supernatants (SN) from dying tumor cells, purified HSP fractions from SN or recombinant HSP. We therefore increased the palette of cytoreduction agents analyzed by including vincristine, fludarabine and rituximab. Vincristine was highly selective in inducing apoptosis in B-CLL and, though not increasing HSP levels increased migration and tumor lytic activity of monocytes but completely blocked the proliferative activity of T cells. In contrast, Fludarabine depleted also T cells very effectively but the surviving ones were unexpectedly more proliferating in response to allogeneic DC than their untreated counterparts. We then focused on the characterization of the molecular mechanisms of this interesting observation, having ni mind that fludarabine represents the current standard treatment in B-CLL, both in monotherapy as well as in combinatory regimens. We combined our in vitro studies with the analysis of blood samples from B-CLL patients treated with fludarabine- containing regimens in vivo which confirmed our in vitro results, however, still the exact mechanisms of this drug interaction with DC and T cell functions are topic of ongoing work in our laboratory. In conclusion, we did not observe local immunostimulatory effects of HSP produced by tumor cells undergoing TC-A-induced cell death but detected ones in a setting in which HSP was not induced. Of clinical importance we observed hyperproliferative activity of T cells in fludarabine-containing regimens which might on the one hand contribute to the relatively frequent occurrence of autoimmune phenomena in fludarabine treated patients. Its role for increasing the long-term remission-free survival of patients by helping in the establishment of immunological memory against tumor cells remains to be determined.