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Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells

  • Authors:
    • Chansu Shin
    • Masaru Imamura
    • Yasushi Kasahara
    • Yuko Suzuki
    • Minori Baba
    • Nobuhiro Kubo
    • Ryosuke Hosokai
    • Haruko Iwabuchi
    • Yudai Murayama
    • Hiroyuki Kawashima
    • Akira Ogose
    • Keichiro Mihara
    • Akihiko Saitoh
    • Chihaya Imai
  • View Affiliations / Copyright

    Affiliations: Department of Pediatrics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Niigata 951‑8510, Japan, Division of Orthopedic Surgery, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Niigata 951‑8510, Japan, Department of Orthopedic Surgery, Uonuma Kikan Hospital, Minamiuonuma, Niigata 949‑7302, Japan, International Center for Cell and Gene Therapy, Fujita Health University, Toyoake, Aichi 470‑1192, Japan
    Copyright: © Shin et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 243
    |
    Published online on: July 7, 2025
       https://doi.org/10.3892/mmr.2025.13608
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Abstract

The clinical application of T cells engineered with chimeric antigen receptors (CARs) for solid tumors is challenging. A major reason for this involves tumor immune evasion mechanisms, including the high expression of immune checkpoint molecules, such as the programmed death 1 (PD‑1) ligands PD‑L1 and PD‑L2. The inducible expression of PD‑L1 in tumors has been observed after CAR‑T‑cell infusion, even in tumors natively not expressing PD‑L1. Furthermore, numerous types of pediatric cancer do not have suitable targets for CAR‑T‑cell therapy. Therefore, the present study aimed to develop novel CAR‑T cells that target PD‑L1 and PD‑L2, and to evaluate their efficacy against pediatric solid tumors. A novel CAR harboring the immunoglobulin V‑set domain of the human PD‑1 receptor as an antigen binding site (PD‑1 CAR‑T) was developed without using a single‑chain variable fragment. PD‑1 CAR‑T cells were successfully manufactured by adding an anti‑PD‑1 antibody, nivolumab, to the ex vivo expansion culture to prevent fratricide during the manufacturing process due to the inducible expression of PD‑L1 in activated human T cells. The expression of PD‑L1 (and PD‑L2 to a lesser extent) was revealed to be highly upregulated in various pediatric solid tumor cells, which displayed no or very low expression initially, on in vitro exposure to interferon‑γ and/or tumor necrosis factor‑α, which are cytokines secreted by tumor‑infiltrating T cells. Furthermore, PD‑1 CAR-T cells exhibited strong cytotoxic activity against pediatric solid tumor cells expressing PD‑L1 and PD‑L2. Conversely, the effect of PD‑1 CAR‑T cells was significantly attenuated against PD‑L1‑positive cells coexpressing CD80, suggesting that the toxicity of PD‑1 CAR‑T cells to normal immune cells, including antigen presenting cells, can be minimized. In conclusion, PD‑1 ligands are promising therapeutic targets for pediatric solid tumors. PD‑1 CAR‑T cells, either alone or in combination with CAR‑T cells with other targets, represent a potential treatment option for solid tumors.
View Figures

Figure 1

Expression of (PD-1) ligands in
pediatric tumor cell lines. (A) Histogram plots showing the
expression of PD-L1 (upper panels) and PD-L2 (lower panels) in
SaOS2 cells with or without treatment with cytokines or CM. (B)
Expression of PD-L1 and PD-L2 in SaOS2 with or without treatment
with cytokines or CM. (C) Expression of PD-L1 and PD-L2 in various
tumor cell lines treated with CM. *P<0.05, **P<0.01. PD-1,
programmed death 1; PD-L, programmed cell death 1 ligand; CM,
conditioned medium; IFN-γ, interferon-γ; TNF-α, tumor necrosis
factor-α; MFI, mean fluorescence intensity.

Figure 2

Generation of PD-1 CAR-T cells. (A)
Gene constructs of PD-1 CAR. PD-1 TR represents a chimeric receptor
lacking signaling domain to serve as a non-signaling control
receptor. (B) Cell surface expression of PD-1 CAR and PD-1 TR in
retrovirally transduced T cells. (C) Expression of programmed cell
death 1 ligand 1 (PD-L1) in T cells before and after activation
with CD3/CD28 beads. PD-L1 was not expressed before activation;
however, expression was significantly upregulated 24 h after T cell
activation and declined 14 days. (D) Absolute cell counts of CAR-T
cells in the expansion culture. PD-1 CAR-T cells exhibited
significantly poorer growth compared with mock-transduced T cells
or PD-1 TR T cells that proliferated exponentially. By adding the
anti-PD-1 Ab nivolumab to the culture during the first week, we
were able to produce PD-1 CAR T cells with growth rates comparable
to mock or PD-1 TR control T cells. *P<0.05 vs. PD-1, programmed
death 1; CAR, chimeric antigen receptor; PD-1 TR, truncated PD-1
CAR; Ab, antibody; EC, extracellular domain; TM, transmembrane
domain; IC, intracellular signaling domain; GFP, green fluorescent
protein.

Figure 3

Cytotoxic effects of PD-1 CAR-T cells
against tumor cell lines (A) 4-h cytotoxicity of PD-1 CAR-T cells
against osteosarcoma cell line U2OS with modest expression of PD-1
ligands, with or without pretreatment with CM. (B) 4-h cytotoxicity
of PD-1 CAR-T cells against osteosarcoma cell line SaOS2 that does
not express PD-1 ligands, with or without pretreatment with CM. (C)
Real-time monitoring of SaOS2 cytolysis. SaOS2 were incubated with
PD-1 CAR-T at a low E:T ratio (E:T=0.25:1). SaOS2 cells were
compared with SaOS2 cells with pretreatment with CM (SaOS2-CM). (D)
Time series of histogram plots of PD-L1 expression in SaOS2 by flow
cytometry. After co-culture with PD-1 CAR-T cells, PD-L1 expression
of residual SaOS2 was gradually upregulated. *P<0.05 vs. PD-1
CAR. PD-1, programmed death 1; CAR, chimeric antigen receptor; CM,
conditioned medium; E:T, effector-to-target; PD-L1, programmed cell
death 1 ligand 1; PD-1 TR, truncated PD-1 CAR.

Figure 4

Dual targeting CAR-T cells. (A) The
proposed scheme for dual-targeting CAR. PD-1 CAR and anti-CD19 CAR
were transduced in the same T cells. (B) Surface expression of
anti-CD19 CAR and PD-1 CAR in transduced T cells. T cells showed
strong expression of both PD-1 CAR and anti-CD19 CAR. (C) Surface
expression of CD19 and PD-L1 in HeLa cells retrovirally transduced
with CD19 gene (19-HeLa). 19-Hela cells stimulated with CM
expressed both CD19 and PD-L1. (D) Long-term cytotoxicity of
dual-targeting CAR-T cells against 19-HeLa with pretreatment with
CM. Anti-CD19 CAR-T cells were used as control. PD-1, programmed
death 1; CAR, chimeric antigen receptor; CM, conditioned medium;
PD-L1, programmed cell death 1 ligand 1; GFP, green fluorescent
protein; E:T, effector-to-target.

Figure 5

CD80 inhibits PD-1 CAR binding to
PD-L1 and attenuates cytotoxicity. (A) Surface expression of CD80
in 19-HeLa cells after transduction of CD80. (B) Surface expression
of PD-L1 after treatment with CM in 19-HeLa cells and
CD80-transduced 19-HeLa cells. Anti-PD-L1 antibody binding was
modestly attenuated in CD80-expressing 19-HeLa cells. (C) Schematic
diagram of the experiment. Cytotoxicity of anti-CD19 CAR-T cells or
PD-1 CAR-T cells against CM-pretreated, CD80-transduced 19-HeLa
(that simultaneously expresses CD80, CD19, and PD-L1) were
evaluated. (D) Cytotoxic effects of PD-1 CAR-T cells against
CM-pretreated 19-HeLa with or without CD80 overexpression. Cells
were cocultured for 24 h. (E) Long-term cytotoxicity of PD-1 CAR-T
cells and anti-CD19 CAR-T cells against CM-pretreated 19-HeLa cells
with or without CD80 overexpression (lower and upper panels). PD-1,
programmed death 1; CAR, chimeric antigen receptor; CM, conditioned
medium; PD-L1, programmed cell death 1 ligand 1; GFP, green
fluorescent protein; E:T, effector-to-target.

Figure 6

Proposed concept of the study. (A)
Conventional CAR-T cells are inhibited by immune checkpoint
molecules, such as PD-1 ligands. (B) Loss of the target antigen
occurs after exposure to CAR-T cells as one of immune evasion
mechanisms by cancer cells. (C) PD-1 CAR-T cells can target PD-1
ligands. (D) Loss of PD-1 ligands in cancer cells occurring as an
immune evasion mechanism after attack by PD-1 CAR-T cells leads to
the restoration of the T-cell inhibiting microenvironment. (E) PD-1
CAR-T cells using the PD-1 extracellular domain developed in this
study have minimal impact on normal immune cells that express both
CD80 and PD-L1 (e.g. dendritic cells). PD-1, programmed death 1;
CAR, chimeric antigen receptor; PD-L1, programmed cell death 1
ligand 1.
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Copy and paste a formatted citation
Spandidos Publications style
Shin C, Imamura M, Kasahara Y, Suzuki Y, Baba M, Kubo N, Hosokai R, Iwabuchi H, Murayama Y, Kawashima H, Kawashima H, et al: Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells. Mol Med Rep 32: 243, 2025.
APA
Shin, C., Imamura, M., Kasahara, Y., Suzuki, Y., Baba, M., Kubo, N. ... Imai, C. (2025). Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells. Molecular Medicine Reports, 32, 243. https://doi.org/10.3892/mmr.2025.13608
MLA
Shin, C., Imamura, M., Kasahara, Y., Suzuki, Y., Baba, M., Kubo, N., Hosokai, R., Iwabuchi, H., Murayama, Y., Kawashima, H., Ogose, A., Mihara, K., Saitoh, A., Imai, C."Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells". Molecular Medicine Reports 32.3 (2025): 243.
Chicago
Shin, C., Imamura, M., Kasahara, Y., Suzuki, Y., Baba, M., Kubo, N., Hosokai, R., Iwabuchi, H., Murayama, Y., Kawashima, H., Ogose, A., Mihara, K., Saitoh, A., Imai, C."Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells". Molecular Medicine Reports 32, no. 3 (2025): 243. https://doi.org/10.3892/mmr.2025.13608
Copy and paste a formatted citation
x
Spandidos Publications style
Shin C, Imamura M, Kasahara Y, Suzuki Y, Baba M, Kubo N, Hosokai R, Iwabuchi H, Murayama Y, Kawashima H, Kawashima H, et al: Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells. Mol Med Rep 32: 243, 2025.
APA
Shin, C., Imamura, M., Kasahara, Y., Suzuki, Y., Baba, M., Kubo, N. ... Imai, C. (2025). Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells. Molecular Medicine Reports, 32, 243. https://doi.org/10.3892/mmr.2025.13608
MLA
Shin, C., Imamura, M., Kasahara, Y., Suzuki, Y., Baba, M., Kubo, N., Hosokai, R., Iwabuchi, H., Murayama, Y., Kawashima, H., Ogose, A., Mihara, K., Saitoh, A., Imai, C."Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells". Molecular Medicine Reports 32.3 (2025): 243.
Chicago
Shin, C., Imamura, M., Kasahara, Y., Suzuki, Y., Baba, M., Kubo, N., Hosokai, R., Iwabuchi, H., Murayama, Y., Kawashima, H., Ogose, A., Mihara, K., Saitoh, A., Imai, C."Chimeric PD‑1 receptor redirects primary T cells against childhood solid tumors but not to PD‑1 ligand‑positive CD80‑coexpressing cells". Molecular Medicine Reports 32, no. 3 (2025): 243. https://doi.org/10.3892/mmr.2025.13608
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