TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant

Abstract

BackgroundHigh-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in HR-NBL patients; however, graft-versus-host-disease (GVHD) and disease progression have limited clinical application. Ex-vivo stimulated allogeneic natural killer (NK) cells represent a potential approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL. MethodsEx-vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble IL-15/IL-15R alone or with irradiated CD137L/CD54+ AgN2a-4P (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-TIM-3. Lethally irradiated B6AJF1 Mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish their contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and overall survival. ResultsCo-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Ki-67 and NKG2D, NKp46, TRAIL and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared to IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared to NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomes, TRAIL and FasL expression observed. Analysis of RNA from 15-4P NK cells exposed to TIM-3 blockade showed gene expression of chemokines, NKG2D/DAP12 signaling, non-canonical NF-{kappa}b pathway and TRAIL signaling. Blockade of NKG2D, TRAIL or FasL on 15-4P NK cells abrogated cytotoxicity. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared to NK cells or anti-TIM-3 alone, without inducing GVHD. Depletion of NK cells, but not T cells, abrogated the GVT effect. ConclusionAllo-HCT can be a platform for treating NBL using combination ex-vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD. Ethics StatementThe animal study M005915 was reviewed and approved by University of Wisconsin-Madison IACUC. WHAT IS ALREADY KNOWN ON THIS TOPICO_LIT cell depleted (TCD) allogeneic hematopoietic cells transplant (allo-HCT) has potential to be a salvage therapy for relapsed/refractory neuroblastoma (NBL) through the graft-versus-tumor (GVT) effect, however, the high incidence of graft-vs-host disease (GVHD) and disease progression has hindered widespread clinical application. Allogeneic NK cell therapy can be a safe and feasible treatment but has had limited efficacy in NBL. TIM-3 blockade has shown encouraging results for a variety of tumors but has not been explored for NBL nor used to enhance the GVT effect. C_LI WHAT THIS STUDY ADDSO_LIEx vivo stimulated allogeneic NK cells demonstrate robust expansion, proliferation, activation and cytotoxicity against NBL which is further augmented by the addition of TIM-3 checkpoint blockade. In an allo-HCT model of NBL relapse, mice treated with IL-15/IL-15R/AgN2a-4P (15-4P) stimulated NK cells and anti-TIM-3 exhibited prolonged overall survival and reduced tumor growth without exacerbating GVHD. C_LI HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYO_LIRelapse of NBL after TCD allo-HCT can be successfully treated with the combination of adoptively transferred 15-4P stimulated allogeneic NK cells and TIM-3 checkpoint blockade, representing a novel therapeutic approach that can be translated to the clinic or used as a platform to treat other solid tumors with high TIM-3 ligand expression. C_LI