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. 2025 Mar 6;74(4):136.
doi: 10.1007/s00262-025-03979-4.

Prostaglandin F2 receptor negative regulator as a potential target for chimeric antigen receptor-T cell therapy for glioblastoma

Hideki Kuroda  1 , Noriyuki Kijima  2 , Tetsuro Tachi  1 , Shunya Ikeda  3 , Koki Murakami  1 , Tomoyoshi Nakagawa  1 , Moto Yaga  4 , Kanji Nakagawa  1 , Reina Utsugi  1 , Ryuichi Hirayama  1 , Yoshiko Okita  1 , Naoki Kagawa  1 , Naoki Hosen  5   6 , Haruhiko Kishima  1
Affiliations

Prostaglandin F2 receptor negative regulator as a potential target for chimeric antigen receptor-T cell therapy for glioblastoma

Hideki Kuroda et al. Cancer Immunol Immunother. .

Abstract

Background: Chimeric antigen receptor (CAR)-T cell therapy targeting novel glioblastoma (GBM)-specific cell surface antigens is a promising approach. However, transcriptome analyses have revealed few GBM-specific target antigens.

Methods: A library of monoclonal antibodies (mAbs) against tumor cell lines derived from patients with GBM was generated. mAbs reacting with tumor cells in resected tissues from patients with GBM but not with nonmalignant human brain cells were detected. The antigens that were recognized were identified through expression cloning. CAR-T cells derived from a candidate mAb were generated, and their functionality was tested in vitro and in vivo.

Results: Approximately 3,200 clones were established. Among them, 5E17 reacted with tumor cells in six of seven patients with GBM, but not with nonmalignant human brain cells. Prostaglandin F2 receptor negative regulator (PTGFRN) was identified as an antigen recognized by 5E17. CAR-T cells derived from 5E17 produced cytokines and exerted cytotoxicity upon co-culture with tumor cells from patients with GBM. Furthermore, intracranial injection of 5E17-CAR-T cells demonstrated antitumor effects in an orthotopic xenograft murine model with patient-derived GBM cells.

Conclusions: Cell surface PTGFRN is a candidate target for intracranial CAR-T cell therapy for GBM. On-target off-tumor toxicity in alternative normal tissues needs to be carefully tested.

Keywords: CAR-T cell therapy; Expression cloning; Glioblastoma (GBM); Monoclonal antibodies; PTGFRN.

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Conflict of interest statement

Declarations. Conflict of interests: The authors have no relevant financial or non-financial interests to disclose. Ethical approval: This study adhered to the tenets of the Declaration of Helsinki (2013, as amended) and was approved by the Ethics Review Committee of Osaka University Graduate School of Medicine (Suita, Osaka, Japan) (approval number: 20561). All animal experiments were approved by the Institutional Animal Care and Use Committee at Osaka University Graduate School of Medicine (approval numbers: 03–071-000 and 04–028-002) and were performed according to the animal use guidelines of the Animal Experiment Committee of Osaka University Graduate School of Medicine. Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent to publish: Not applicable.

Figures

Fig. 1
Fig. 1
Identification of 5E17 as a GBM-specific mAb. A Strategy for the identification of the GBM-specific mAb 5E17. B Representative flow cytometry findings of 5E17 mAb bound to GBM and nonmalignant brain cells from patients. Analyses of live (PI-negative) cells are shown. The results of staining with the isotype or negative control instead of anti-5E17 mAb, CD31, CD45, or CD90 are shown to draw the gate for 5E17-positive cells. Analyses of the other patients are shown in Supplementary Fig. 2. Blue histogram indicates isotype control. SSC, side scatter. C Mean fluorescence intensity (means ± standard error of the mean) of flow cytometry findings bound to GBM (n = 7) and nonmalignant brain cells (n = 6) from patients. Each plot shows the mean fluorescence intensity. GBM cells were assessed as CD31-CD45-CD90 + cells, and nonmalignant tumor cells were assessed as CD31-CD45- cells. *p < 0.05, calculated using a Mann–Whitney U-test
Fig. 2
Fig. 2
5E17 recognized PTGFRN. A Procedure for expression cloning to identify antigens recognized by 5E17 monoclonal antibody. B Flow cytometry plots showing the process of 5E17-positive cell enrichment in the expression cloning of the 5E17 antigen. SSC, side scatter. C Flow cytometry analysis of 5E17 (top row) and commercially available anti-PTGFRN antibody (R&D systems, inc.) (bottom row) reactivity to wild/5E17 overexpressed Ba/F3 cells and wild/PTGFRN KO U87MG cells. WT, wild type; KO, knock out
Fig. 3
Fig. 3
Development of CAR-T cells for the 5E17 antigen. A Establishment of CAR-T cells using the 5E17 monoclonal antibody in the indicated construct. VH, variable heavy; VL, variable light. B Flow cytometry plots of 5E17 CAR or Mock transduction efficiency in human T cells 10 days after CAR transduction. SSC, side scatter. C Growth of 5E17 CAR-T or Mock T cells during in vitro culture
Fig. 4
Fig. 4
5E17 CAR-T cells are activated and have antitumor effects in vitro. A ELISA assay of IFN-γ and IL-2 released by 5E17 CAR-T cells or control CAR-T cells after co-culture with the indicated cells. Irrelevant antibody-transduced (CD19) T cells were used as controls. All experiments were performed in technical-quintuplicate wells. IFN-γ, interferon gamma; IL, interleukin; WT, wild type; KO, knock out. B 51Cr release assay to measure specific lysis of the indicated target cells by 5E17 or control CAR-T cells (CD19). All experiments were performed in technical-triplicate wells. Data are expressed as means ± standard error of the means. WT, wild type; KO, knock out. **p < 0.01, *p < 0.05, n.s, not significant, calculated using a Mann–Whitney U-test
Fig. 5
Fig. 5
5E17 CAR-T cells are activated and have antitumor effects in vivo. A Experimental design. B Bioluminescence imaging of mice every 2 weeks after injection with 5E17 and control CAR-T cells (CD19) (n = 5 per group). p, photons; s, second; sr, steradian; Min, minimum; Max, maximum. C Quantification of the brain luminescence. Avg, average. D Kaplan–Meier curve of the mice infused with either 5E17 or control CAR-T cells (CD19). p = 0.16; calculated using a log rank test
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