Institute of Biomedical Sciences

Title

Engineering the TGFβ receptor to Enhance the Therapeutic Potential of Natural Killer Cells as an Immunotherapy for Neuroblastoma

Poster Number

6

Document Type

Poster

Status

Graduate Student - Doctoral

Abstract Category

Cancer/Oncology

Keywords

immunotherapy, neuroblastoma, natural killer cell, immunosuppression

Publication Date

Spring 2018

Abstract

High-grade neuroblastomas (NB) have a poor prognosis, and there is a need to explore novel therapies. Natural killer (NK) cells rapidly lyse target cells without prior exposure, and serve as a promising source for adoptive cell therapy, however availability from adult donors is limited. Cord blood (CB) derived NK cells have promise as an ideal source for “off the shelf” therapy as they can be selected for optimally mismatched donors. However, CB NK cell efficacy is limited by the immunosuppressive microenvironment in solid tumors such as NB, where high levels of secreted TGFβ actively impairs NK cell activity. To overcome this, we genetically-modified NK cells to express a modified TGFβ receptor which contains an activation domain, and is thus able to engage with TGFβ while potentially converting downstream signals to enhance NK cell function.

NK cells were isolated from umbilical cord donors, expanded, and transduced to express an engineered TGFβ receptor: truncated TGFβRII external domain fused to the intracellular DAP12 motif. Cellular phenotype and proliferation was determined by flow cytometry, and cell function determined with in vitro cytotoxicity assays, cell signaling assays, and in vivo murine studies.

CB NK cells were transduced with a modified TGFβ receptor (Trans-NKs; mean transduction 42.5±5.5%). Trans-NK had no variations in their expression of activating receptors NKG2D, NKp44, or NKp30 (p>0.05) or proliferative capacity (p>0.05) vs. non-transduced (NT) NK cells. Unlike NT NK cells, pre-treatment of trans-NKs with TGFβ resulted in increased Erk1/2 phosphorylation, consistent with NK cell activation. NT NK cells exhibited dose-dependent cytotoxicity against K562 target cells (45.2±4.69% killing); however following pre-treatment with TGFβ, NT NK cells had impaired cytotoxicity (34.6±4.58% killing). In contrast, trans-NKs exhibited dose-dependent cytotoxicity (40.2±4.42% killing), which was enhanced following exposure to TGFβ (47.2±5.17% killing). In a xenograft model of human NB, administration of trans-NKs delayed tumor progression by >14 days.

We can successfully generate modified NK cells, which are phenotypically and functionally superior at eradicating NB in a TGFβ-rich environment. This strategy provides preclinical evidence for the establishment of “off the shelf” gene-modified NK cells as a treatment for patients with NB and other malignancies that utilize TGFβ secretion for immune evasion.

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Engineering the TGFβ receptor to Enhance the Therapeutic Potential of Natural Killer Cells as an Immunotherapy for Neuroblastoma

High-grade neuroblastomas (NB) have a poor prognosis, and there is a need to explore novel therapies. Natural killer (NK) cells rapidly lyse target cells without prior exposure, and serve as a promising source for adoptive cell therapy, however availability from adult donors is limited. Cord blood (CB) derived NK cells have promise as an ideal source for “off the shelf” therapy as they can be selected for optimally mismatched donors. However, CB NK cell efficacy is limited by the immunosuppressive microenvironment in solid tumors such as NB, where high levels of secreted TGFβ actively impairs NK cell activity. To overcome this, we genetically-modified NK cells to express a modified TGFβ receptor which contains an activation domain, and is thus able to engage with TGFβ while potentially converting downstream signals to enhance NK cell function.

NK cells were isolated from umbilical cord donors, expanded, and transduced to express an engineered TGFβ receptor: truncated TGFβRII external domain fused to the intracellular DAP12 motif. Cellular phenotype and proliferation was determined by flow cytometry, and cell function determined with in vitro cytotoxicity assays, cell signaling assays, and in vivo murine studies.

CB NK cells were transduced with a modified TGFβ receptor (Trans-NKs; mean transduction 42.5±5.5%). Trans-NK had no variations in their expression of activating receptors NKG2D, NKp44, or NKp30 (p>0.05) or proliferative capacity (p>0.05) vs. non-transduced (NT) NK cells. Unlike NT NK cells, pre-treatment of trans-NKs with TGFβ resulted in increased Erk1/2 phosphorylation, consistent with NK cell activation. NT NK cells exhibited dose-dependent cytotoxicity against K562 target cells (45.2±4.69% killing); however following pre-treatment with TGFβ, NT NK cells had impaired cytotoxicity (34.6±4.58% killing). In contrast, trans-NKs exhibited dose-dependent cytotoxicity (40.2±4.42% killing), which was enhanced following exposure to TGFβ (47.2±5.17% killing). In a xenograft model of human NB, administration of trans-NKs delayed tumor progression by >14 days.

We can successfully generate modified NK cells, which are phenotypically and functionally superior at eradicating NB in a TGFβ-rich environment. This strategy provides preclinical evidence for the establishment of “off the shelf” gene-modified NK cells as a treatment for patients with NB and other malignancies that utilize TGFβ secretion for immune evasion.