Background High-risk neuroblastoma (NBL) is an aggressive extra-cranial pediatric solid tumor with poor overall survival. Prior studies testing allogeneic hematopoietic stem cell transplant (allo-HSCT) for NBL patients were limited by graft-versus-host-disease (GVHD) and disease progression. Ex-vivo stimulated allogeneic Natural Killer (NK) cells can enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD. Because TIM-3 is expressed on both exhausted NK and T cells, immune checkpoint blockade may enhance the GVT effect after allo-HSCT. Here we investigate adoptive transfer of ex-vivo activated NK cells and TIM-3 checkpoint blockade after T cell depleted allo-HSCT for relapsed NBL.
Methods NK cells from C57BL/6 (B6) mice were expanded with soluble IL-15/IL-15Rα alone or with an irradiated murine neuroblastoma cell line transfected to over-express CD54, CD80, CD86 and CD137L, called AgN2a-4P, at a 1:1 ratio for 10–12 days. AgN2a-4P-stimulated allogeneic NK (Ag4P-NK) cell phenotype, cytokine production, and cytotoxic activity against murine NBL cell lines Neuro2a and NXS2 in the presence of anti-TIM-3 antibody were analyzed in vitro. B6AJF1 mice received a T cell depleted allo-HSCT from B6 donors followed by NBL inoculation to model relapse. Select groups received anti-TIM-3 antibody every 9 days and/or infusions of B6 Ag4P-NK cells on days 14, 21, and 28. In select groups, T cells or NK cells were depleted to determine their relative contribution on the GVT effect. All groups were analyzed for tumor growth, GVHD and survival.
Results NK expansion with AgN2a-4P and IL-15/IL-15Rα resulted in increased expression of Ki-67 and multiple markers of NK cell activation. Ag4P-NK cells showed enhanced cytotoxicity in vitro against NBL compared to IL-15/IL-15Rα activated NK cells, and this effect was augmented by TIM-3 blockade. Exposure to TIM-3 blockade further increased Ag4P-NK degranulation against Neuro2a compared to NXS2, due to a higher frequency of TIM-3 ligands present on Neuro2a. In vivo, the combination of Ag4P-NK cells and anti-TIM-3 after allo-HSCT resulted in significantly improved survival and reduced NBL tumor burden without inducing GVHD, as compared to mice treated with Ag4P-NK cells or anti-TIM-3 alone. Depletion of NK cells led to substantial increase in tumor burden, while depletion of T cells did not affect therapy efficacy.
Conclusions T cell-depleted allo-HSCT can be a viable treatment platform for treating relapsed NBL when combined with adoptive transfer of ex-vivo-stimulated NK cells and TIM 3 checkpoint blockade. These studies demonstrate that multi-modal approaches incorporating combination immunotherapy are needed for NBL and that immunotherapies that enhance NK cell function should be prioritized.
Acknowledgements This work was supported by grants from the NSF 1810916 WiscAMP Bridge to the Doctorate and NSF Graduate Research Fellowship Program DGE-1747503 (AEQ), NCI/NIH R01 CA215461, American Cancer Society Research Scholar grant RSG-18–104-01-LIB, St. Baldrick’s Empowering Pediatric Immunotherapies for Childhood Cancer Grant, Hyundai Hope on Wheels and the MACC Fund (C.M.C). We would like to thank the UWCCC core facilities, who are supported in part through NCI/NIH P30 CA014520.
Ethics Approval The animal study M005915 was reviewed and approved by University of Wisconsin-Madison IACUC.
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