Introduction: Glioblastoma (GBM) presents with a poor prognosis and dismal survival rates. While cell-based immune therapies have shown success in other malignancies, there is currently little evidence that GBM is as responsive to current approaches. Novel therapies for these patients are therefore urgently needed. The antitumor effect may be limited by GBM’s rapid progression, its extensive intertumoral heterogeneity, and its immune suppressive microenvironment. For cell-based therapies to be effective, they need to be manufactured rapidly or in advance, recognize multiple targets on GBM, and resist immune suppression. We therefore sought to develop “off-the-shelf” cord blood-derived NK cell therapies capable of recognizing multiple stress ligands on tumor cells but are protected from the microenvironment by expression of a dominant negative receptor to TGFβ (abrogating the negative effects of the cytokine) and secretion of IL15 (overcoming negative signals from the microenvironment). We investigate the therapeutic efficacy of NK cells engineered to express IL15-and/or TGFβ-DNR in glioblastoma model in-vitro and in-vivo.

Methods: NK cells were isolated from umbilical cord blood by magnetic selection, and subsequently modified to express IL-15, and/or TGFβ-DNR. Engineered NK cells were expanded with feeder cells. Retroviral transduction efficiency was confirmed by flow cytometry. IL15 expression was quantified by ELISA. To determine the cytotoxicity activity of our engineered NK cells against glioblastoma, we measured lysis of the U87MG cell line. To determine in vivo efficacy of our engineered NK cells, we used xenograft NSG mouse models that were orthotopically engrafted with U87MG in the brain. Tumor growth was measured by bioluminescence.

Results: CAR-NK cells were successfully transduced (with a transduction efficiency ranging from 66-83% (mean 74.55% + 10.6, n>3) from umbilical cord blood. NK cells engineered to secrete IL15 secreted a mean of 288.3 pg/mL. After exposure to TGFβ in vitro, NK cells transduced with TGFβ-DNR had a increase of killing [mean 58.25 % + 4.51 at 20:1 (E:T) 48.57% + 11.55 without TGFβ, n=3] compared to un-transduced NK cells [mean 11.98 % + 4.51 at 20:1 (E:T) 49.04% + 5.34 without TGFβ, n=3]. Mice injected with IL15 and TGFβ DNR showed better control of tumor (3.99 x 105 + 0.0007 bioluminescence Vs 1.673 x 107 + 0.160 in mice given mock-transduced NK cells and 8.44 x 109 + 28.62 in control mice).

Conclusion: Our preliminary results show that cord blood NK cells modified to secrete IL15 and express TGFβ-DNR can recognize and lyse GBM cells in vitro and orthotopically implanted GBM tumors in vivo.

Citation Format: Nethaji Muniraj, Kajal Chaudhry, Joshua Luke Terao, Vipin Suri, Luke Barron, Catherine Bollard, Conrad Russell Cruz. Genetically modified TGF-β dominant negative receptor and IL15 enhance Natural Killer (NK) cell mediated cytolytic activity in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3191.