The Immune “Combination Therapy” of Leukemia Using Adoptive Transfer and TGF-β Blockade.

Abstract 3017

The immunosuppressive cytokine Transforming Growth factor-beta (TGF-β), either tumor or T cell-derived, can significantly alter T-dependent immune responses to tumors in mouse models. However, the role of TGF-β neutralization on other cell types of the tumor microenvironment and the ensuing impact on immunotherapies is still unclear. Moreover, pre-clinical models aimed at harnessing the likely synergy between TGF- β signaling blockade and immunotherapy are lacking. Using the TGF- β producing, acute lymphoblastic leukemia/lymphoma cell line EL4, we undertook to characterize how TGF-β affects the leukemic microenvironment and the outcome of adoptive immunotherapy. After inoculation, EL4 cells form large tumor masses that attract a wide variety of leukocytes, including mono-myeloid cells and T lymphocytes (5–10% of tumor cellularity). In order to assess whether TGF-β contributed to shape the leukemic microenvironment, we administered the pan anti-TGF-β antibody 1D11 or isotype control to EL4 bearing mice. Antibodies were administered after leukemic cell inoculation for a period of three weeks (300 μg three times a week). The systemic administration of 1D11 altered the EL-4 leukemia microenvironment. Notably, the concentration of inflammatory cytokines IL-2, GM-CSF and MIP-1α increased along with a trend in the abundance of CD44 positive CD4 T cells and myeloid cells infiltrating the tumors. However, the administration of anti-TGF-β antibody failed to alter tumor growth kinetics or vasculogenesis. These results 1) imply that the mobilization of immune effectors cells following TGF-β neutralization is insufficient and 2) correlate with our in vitro data which showed that TGF-β blockade have no impact on EL4 cells growth and apoptosis. Although insufficient by itself, could the use of TGF-β neutralizing strategies nonetheless improve the outcome of cancer immunotherapies? In order to investigate how inhibition of TGF-β signaling in the microenvironment can alter the outcome of adoptive immunotherapy, we designed an autologous adoptive immunotherapy model of leukemia. We have generated specific anti-EL4 responses using a vaccination and an in vitro restimulation system. Following in vitro stimulation with EL4 cells vs. splenocyte lysate, T cell effectors induced 34.42 % and 4.3% specific lysis respectively against EL4 targets. The injection of these effectors in sub-lethally irradiated leukemia bearing syngeneic hosts led to 50% survival at 120 days post adoptive transfer (n=6) compared to 0% survival in unprimed host. In order to assess whether concomitant TGF-β blockade improves this response, we are currently testing the in vivo efficacy of our strategy in the context of TGF-β neutralization in terms of survival and leukemia infiltration by T cells. The possibility to harness the combined effects of TGF- β signaling blockade and current immunotherapeutic approaches has immediate translational relevance given the numerous anti-TGF modalities currently being developed.