Background: In principle, T cells can target cancer cells by recognizing tumor-specific peptides presented by HLA proteins. HLA downregulation is one of the most common immune escape mechanisms and frequently observed in patients with high-risk neuroblastoma (NB) in whom it is associated with an adverse prognosis. Interferon-gamma (IFNγ) has been shown to rescue HLA expression on NB cells, however no strategies exist for the targeted delivery of IFNγ to upregulate HLA on tumor cells. We here present a novel approach to induce HLA expression in NB cells using a targeted cellular therapy.

Methods: We genetically engineered GD2-specific T cells expressing synthetic Notch (synNotch) receptors. In our receptors, an internal Notch domain is cleaved upon binding of the NB-specific target antigen GD2, which releases a transcription factor driving expression of IFNγ (IFNγ-synNotch). We analyzed co-cultures of IFNγ-synNotch T cells and human NB cell lines by flow cytometry, enzyme-linked immunosorbent assay (ELISAs), enzyme-linked immunospot (ELISpot) assay, and luminescence-based cytotoxicity assays.

Results: Treatment with recombinant IFNγ led to a strong induction of HLA expression in 6/6 NB cell lines. Next, we generated 13 synNotch receptors targeting NB-specific antigen GD2, and one CD19-specific synNotch receptor. One of the 13 GD2-targeting synNotch receptors showed strong surface expression and no basal IFNγ expression. T cells expressing this construct (GD2- IFNγ-synNotch T cells) produced IFNγ only in the presence of GD2-positive NB cells and not GD2-negative cells. In vitro, GD2-IFNγ-synNotch T cells efficiently and rapidly upregulated HLA on NB cell lines SK-N-DZ and NB-1643. HLA upregulation was maintained for 7 days after removing GD2-IFNγ-synNotch T cells from the co-culture. In contrast, treatment with GD2-IFNγ-synNotch T cells did not increase PD-L1 on NB cells or PD-1 on T cells. In vivo, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were first engrafted with the human NB cell line Kelly. Once tumors reached a diameter of 5 mm, GD2-IFNγ-synNotch T cells were injected intratumorally. Three days after treatment, mice were euthanized, and their tumors were harvested and analyzed by immunohistochemistry for HLA induction. Mice treated with GD2-IFNγ-synNotch T cells, but not CD19-IFNγ-synNotch T cells, showed HLA upregulation in their tumors. Finally, GD2-IFNγ-synNotch T cells significantly enhanced the killing of HLA-A*02+NB cell lines by T cell receptor (TCR)-transgenic T cells targeting the tumor antigens NY-ESO-1 and PRAME.

Conclusions: Induction of HLA on tumor cells using engineered IFNγ-synNotch T cells is feasible and increases T cell-mediated tumor cell killing. Treatment with GD2-IFNγ-synNotch T cells alone or in combination with TCR-transgenic T cells represents a promising approach to improve anti-tumor immunity in solid tumors.

Citation Format: Fiorella Iglesias, Siani Weston, Djordje Atanackovic, Tim Luetkens. Enhancement of anti-tumor immunity by the adoptive transfer of T cells engineered to restore HLA expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2171.