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. 2024 Sep;31(9):1323-1334.
doi: 10.1038/s41417-024-00766-8. Epub 2024 Apr 6.

RevCAR-expressing immune effector cells for targeting of Fn14-positive glioblastoma

Haidy A Saleh #  1 , Nicola Mitwasi #  1 , Liliana R Loureiro  1 , Alexandra Kegler  1 , Karla Elizabeth González Soto  1 , Lydia Hoffmann  1 , Eugenia Crespo  1 , Claudia Arndt  1   2 , Ralf Bergmann  3 , Michael Bachmann  4   5   6   7 , Anja Feldmann  8   9   10   11
Affiliations

RevCAR-expressing immune effector cells for targeting of Fn14-positive glioblastoma

Haidy A Saleh et al. Cancer Gene Ther. 2024 Sep.

Abstract

In recent studies, we have established the unique adapter chimeric antigen receptor (CAR) platform RevCAR which uses, as an extracellular CAR domain, a peptide epitope instead of an antibody domain. RevCAR adapters (termed RevCAR target modules, RevTMs) are bispecific antibodies that enable the reversible ON/OFF switch of the RevCAR system, improving the safety compared to conventional CARs. Here, we describe for the first time its use for retargeting of both T and NK-92 cells. In addition, we describe the development and preclinical validation of a novel RevTM for targeting of the fibroblast growth factor-inducible 14 (Fn14) surface receptor which is overexpressed on Glioblastoma (GBM) cells, and therefore serves as a promising target for the treatment of GBM. The novel RevTM efficiently redirects RevCAR modified T and NK-92 cells and leads to the killing of GBM cells both in vitro and in vivo. Tumor cell killing is associated with increased IL-2, TNF-α and/or IFN-γ secretion. Hence, these findings give an insight into the complementary potential of both RevCAR T and NK-92 systems as a safe and specific immunotherapeutic approach against GBM.

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Conflict of interest statement

The authors declare no competing interests. The research was partially funded by the “Europäischer Fonds für regionale Entwicklung (EFRE)”. This project is co-financed by tax funds based on the budget passed by the Saxon State Parliament (SAB-project number 100544183 to AF). CA is a fellow of the Mildred Scheel Early Career Center Dresden P2 funded by the German Cancer Aid (Deutsche Krebshilfe).

Figures

Fig. 1
Fig. 1. Schematic overview of RevCAR T and NK-92 cell systems.
A Either RevCAR T cells or RevCAR NK-92 cells express reverse chimeric antigen receptors (RevCARs) that consist of an intracellular CD3z signaling domain (SD) as well as a CD28 co-stimulatory domain (CSD), in addition to a CD28 transmembrane and hinge region linked to an extracellular peptide epitope (either E5B9 or E7B6). RevCAR immune cells can be activated by RevTMs. B The novel designed RevTMs are bsAbs derived from the variable light (VL) and heavy (VH) chain domains of mAbs directed against either the TAA Fn14 or the RevCAR epitope E5B9 or E7B6. All domains are connected via peptide linkers (Li). Both RevTMs contain an N-terminal signal peptide (SP) and C-terminal Histidines (His) as well as a Strep-tag for their purification. After purification via their Strep tag, the RevTMs were separated by SDS-PAGE followed by (C) Coomassie Blue staining, or (D) immunoblotting on nitrocellulose membrane and detection via an anti-His mAb and an alkaline phophatase-conjugated anti-mouse Ab. Sor; sortase recognition site, M; molecular weight marker, BSA Std.; bovine serum albumin standard.
Fig. 2
Fig. 2. Binding of Fn14-specific RevTMs to GBM cells, RevCAR T cells and RevCAR NK-92 cells.
A Fn14 expression on luciferase (Luc)-expressing U251 Luc and U343 Luc GBM cells was determined by staining with anti-Fn14 mAb (primary Ab) and AlexaFluor647-conjugated anti-mouse IgG (secondary Ab). The number of Fn14 antigens expressed per cell was detected by a bead-based flow cytometry assay (QIFIKIT). The binding of Fn14-specific RevTMs to Fn14 on both U251 Luc and U343 Luc cell lines was tested using APC-conjugated anti-His mAb. The expression of RevCAR receptors (E5B9 or E7B6) on transduced (B) T cells or (C) NK-92 cells was confirmed by the anti-La mAb La5B9 or La7B6, respectively, and the secondary Ab AlexaFluor647-conjugated anti-mouse IgG. In addition, the number of RevCAR receptors per NK-92 cell was determined. The binding of the Fn14-specific RevTMs to the respective RevCAR T or NK-92 cells was detected using an APC-conjugated anti-His mAb. A, C Quantitative data from three different experiments are shown as mean ± SD. A–C Flow cytometry data are displayed in histograms (light lines: negative control, dark lines: stained cells).
Fig. 3
Fig. 3. Specific lysis of GBM cells by Fn14-redirected RevCAR T cells.
U251 Luc and U343 Luc cells expressing luciferase (Luc) were co-cultured with either (A) RevCAR-E5B9 or (B) RevCAR-E7B6 T cells at an effector to target (E:T) cell ratio of 1:2 in the absence or presence of RevTMs (50 nM) for 20 h. Cytotoxic activity was measured using a luminescence-based cytotoxicity assay. C Half-maximal effective concentration (EC50) of Fn14-specific RevTMs was estimated in a co-culture of tumor cells and RevCAR T cells with an E:T ratio of 1:2 (20 h of incubation). A–C Data are shown for three independent T cell donors and represented as mean ± SD (one-way ANOVA with Tukey multiple comparisons test, ****p < 0.0001; comparison to samples without (W/O) RevTM or with irrelevant RevTM).
Fig. 4
Fig. 4. Cytokine secretion of Fn14-redirected RevCAR T cells.
The pro-inflammatory cytokines IL-2, IFN-γ and TNF-α were detected using ELISA in supernatants from co-culture experiments of either (A) and (C) U251 Luc or (B) and (D) U343 Luc cells with either (A) and (B) RevCAR-E5B9 or (C) and (D) RevCAR-E7B6 T cells incubated at an E:T ratio of 1:2 in the absence or presence of matching RevTM or irrelevant RevTM (negative control) for 20 h. Results are shown as mean ± SD for three independent T cell donors (One-way ANOVA with Tukey multiple comparison test, ****p < 0.0001; comparison to samples without RevTM or with irrelevant RevTM). X; not detectable.
Fig. 5
Fig. 5. Specific lysis of GBM cells by Fn14-redirected RevCAR NK-92 cells.
U251 Luc and U343 Luc cells were co-cultured with either (A) RevCAR-E5B9 or (B) RevCAR-E7B6 NK-92 cells at E:T cell ratios of 10:1, 5:1 and 2:1 in the absence or presence of RevTMs (50 nM) for 4 h. Cytotoxic activity was measured using a luminescence-based cytotoxicity assay. C Half-maximal effective concentration (EC50) of the Fn14-specific RevTMs was estimated. In the presence of a concentration range of the tested RevTMs, RevCAR NK-92 cells were co-cultured with the target cells at an E:T ratio of 10:1 for 4 h. A–C Data are shown for three independent experiments and represented as mean ± SD (one-way ANOVA with Tukey multiple comparisons test, ****p < 0.0001; comparison to samples without RevTM or with irrelevant RevTM).
Fig. 6
Fig. 6. Cytokine secretion of Fn14-redirected RevCAR NK-92 cells.
The pro-inflammatory cytokine IFN-γ was detected using ELISA in supernatants from co-culture experiments of either U251 Luc or U343 Luc cells with either (A) RevCAR-E5B9 or (B) RevCAR-E7B6 NK-92 cells incubated at an E:T ratio of 10:1, 5:1 or 2:1 in the absence or presence of either matching Fn14-specific RevTM or irrelevant one (negative control) for 4 h. Results are shown as mean ± SD for three independent experiments. (One-way ANOVA with Tukey multiple comparison test, *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001; comparison to samples without RevTM or with irrelevant RevTM).
Fig. 7
Fig. 7. In vivo killing of GBM cells by Fn14-directed RevCAR NK-92 cells.
Three groups of NXG mice were injected with either U251 Luc cells alone, U251 Luc cells and RevCAR NK-92 cells, or U251 Luc cells and RevCAR NK-92 cells in the presence of Fn14-specific RevTM. Luminescence was measured over 4 days after intraperitoneal application of luciferin. Quantitative luminescence intensity data are presented as mean values ± SD of five animals. Two-way ANOVA with Tukey’s multiple comparisons test was performed. (***p ≤ 0.001; comparison to mice groups with U251 Luc tumor cells alone or with RevCAR NK-92 cells but without RevTM). p.i post injection.
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