Improvement in the function of self-activating chimeric antigen receptor by replacing the linker sequence

Our plain-English summary of Improvement in the function of self-activating chimeric antigen receptor by replacing the linker sequence, by T. Kouro, D. Hoshino, Y. Mano, et al (Frontiers in Immunology, 2025).

The study

• Authors: T. Kouro, D. Hoshino, Y. Mano, S. Tsuji, H. Himuro, K. Imai, T. Sasada
• Journal: Frontiers in Immunology
• Published: 2025;16:1502607
• Cited by: 2 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Chimeric antigen receptor (CAR)-T cell therapy is an effective treatment for hematological cancers; however, challenges remain in its application to solid tumors. Among these, the control of CAR-T cell exhaustion is important. The relationship between tonic signals generated by the CAR self-activation and CAR-T cell exhaustion has attracted considerable attention. The magnitude of the tonic signal is known to depend on the structure of the extracellular portion of CAR, but the role of the linker sequence of the single-chain variable region (scFv) in the tonic signal and function in CAR-T cells has not been clarified. In this study, we compared two scFv linkers, G4S and Whitlow/218, in self-activating SKM-CAR, which recognized a malignant mesothelioma-specific modified HEG1 molecule. We observed no differences in cell surface phenotypes, NFAT and NFκB signaling intensities, and gene expression profiles between SKM-CAR T cells with these different linkers. However, switching from the G4S to the Whitlow/218 linker in SKM-CAR-T cells with the CD28 co-stimulatory domain significantly altered cytokine expression after antigen stimulation and improved the in vitro tumor cell killing activity, but not the in vivo tumor control. This is the first study describing the advantages of the Whitlow/218 linker over the G4S linker for some aspects of CAR-T cell function.

Copyright © 2025 Kouro, Hoshino, Mano, Tsuji, Himuro, Imai and Sasada.

In plain English

This laboratory study tested whether changing the scFv linker sequence of a chimeric antigen receptor (CAR) altered CAR-T cells targeting a modified HEG1 molecule the authors describe as mesothelioma-specific. The authors built SKM-CARs with either the (G4S)4 or the Whitlow/218 linker, each with a CD28 (28z) or 4-1BB (BBz) co-stimulatory domain, expressed them in T cells from three healthy donors and in Jurkat reporter lines, and tested them against ACC-MESO-4 mesothelioma cells; only the two CD28 constructs were given to NSG mice, with untreated controls. Linker swapping did not change CAR expression, antigen specificity, surface clustering, exhaustion markers, or NFAT and NFkB reporter signals; without antigen stimulation, gene expression profiles were closely similar, with 16 differentially expressed genes between the 28z pair and four between the BBz pair. However, SKM-W218-28z CAR-T cells killed tumour cells better in culture and secreted significantly more of all cytokines measured than SKM-G4S-28z cells, while tumour growth in mice was not delayed. No CAR-T cells were detected in tumours or spleens at the end of the mouse experiment.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory experimental study: engineered human CAR-T cells and Jurkat reporter lines tested in vitro, plus a mouse xenograft experiment
• Who was studied: CAR-T cells made from peripheral blood mononuclear cells of three healthy volunteers; TCR-deficient Jurkat reporter cell lines; the ACC-MESO-4 malignant mesothelioma cell line; and 25 male NSG mice (6-7 per group, receiving G4S-28z CAR-T cells, W218-28z CAR-T cells, or no treatment), at Kanagawa Cancer Center, Japan
• Exposure: Comparison of two scFv linker sequences, (G4S)4 versus Whitlow/218, in SKM-CAR constructs carrying either a CD28 (28z) or 4-1BB (BBz) co-stimulatory domain; a CD19 (FMC63) CAR with the Whitlow/218 linker served as a comparison construct
• Main results:
  • CAR-T cells with either linker bound the SKM9-2 epitope peptide to a similar degree, which the authors took to show retained antigen specificity and comparable expression
  • Surface clustering of the CAR was unchanged by the linker swap, but all SKM-CARs clustered more than the CD19-CAR with the Whitlow/218 linker (12 cells measured per CAR)
  • Exhaustion markers (Tim-3, LAG3, PD-1) on SKM-W218-28z CAR-T cells were similar to those on SKM-G4S-28z CAR-T cells
  • NFAT reporter activity (tonic signal) in Jurkat cells was the same for the two 28z CARs regardless of linker
  • In vitro killing of ACC-MESO4 cells by SKM-W218-28z and SKM-W218-BBz CAR-T cells was comparable to SKM-G4S-BBz CAR-T cells, whereas SKM-G4S-28z cells killed less well
  • After antigen stimulation, SKM-W218-28z CAR-T cells secreted higher levels of every cytokine measured (IL-4, IL-5, IL-10, IL-12 p70, IL-13, GM-CSF, IFN-gamma, TNF-alpha) than SKM-G4S-28z CAR-T cells
  • RNA sequencing of CAR-T cells without antigen stimulation (three replicates) found few differentially expressed genes attributable to the linker: 16 for the 28z pair and 4 for the BBz pair
  • In NSG mice bearing subcutaneous ACC-MESO-4 tumours (6-7 per group, tumours measured to day 70), SKM-W218-28z CAR-T cells did not slow tumour growth
  • No CAR-T cells could be found in tumours or spleens of treated mice at the end of the experiment
• The authors' conclusion: The authors concluded that switching from the G4S to the Whitlow/218 linker left surface phenotype, NFAT and NFkB signalling and unstimulated gene expression profiles unchanged, but altered cytokine production and improved in vitro killing by SKM-28z CAR-T cells without improving in vivo tumour control; they described this as the first report of advantages of the Whitlow/218 linker over G4S in some aspects of CAR-T cell function
• Limitations the authors state:
  • The authors stated the underlying molecular mechanisms were not determined
  • The dose of CAR-T cells given to mice may have been too low
  • The immunodeficient mouse model may not be suitable for assessing in vivo CAR-T cell function
  • Findings were not confirmed in other self-activating CAR constructs, which the authors said would be needed for generalizability

Topics

Medical terms in this study, explained in our Lexicon: CAR-T Cell, Mesothelioma, HEG1, Cytokine.

Related studies

• Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer — J. Lv, R. Zhao, et al. (2019)
• Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors — S. Tomar, J. Zhang, et al. (2022)
• T cells expressing chimeric antigen receptors can cause anaphylaxis in humans — M.V. Maus, A.R. Haas, et al. (2013)
• Mesothelin as a target for chimeric antigen receptor-modified T cells as anticancer therapy — M. O'Hara, C. Stashwick, et al. (2016)
• Affinity-tuned mesothelin CAR T cells demonstrate enhanced targeting specificity and reduced off-tumor toxicity — Y. Yang, Y. Vedvyas, et al. (2024)
• Chimeric antigen receptor T cells engineered to recognize the P329G-mutated Fc part of effector-silenced tumor antigen-targeting human IgG1 antibodies enable modular targeting of solid tumors — S. Stock, M.R. Benmebarek, et al. (2022)

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