Document resource
Background CAR-T therapy has demonstrated potent anti-tumor efficacy in treating a range of B-cell malignancies. CAR-T constructs containing a CD28 costimulatory domain and CD3ζ signaling domain are efficacious through strong activation but lead to CAR-T exhaustion over time, limiting their persistence. CD3ζ contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that contribute to activation and overall function of engineered CAR-T products. Modification of these ITAMs can improve the efficacy and persistence of CAR-T cells in vivo. 1–4 CD3ε, a subunit of the TCR complex, contains a basic-rich stretch (BRS), proline-rich sequence (PRS), and a single ITAM driving its distinctive signaling and functio.5 We hypothesized that the unique properties of CD3ε may optimize CAR signaling to improve CAR-T functionality and persistence.Methods Using a lentiviral vector and healthy human donor T cells, we generated anti-CD19 CAR-T cells, containing the CD28 costimulatory domain and either the CD3ζ or CD3ε signaling domain. The final products were manufactured and characterized via immunophenotyping and were functionally assessed in vitro and in vivo. For tumor rechallenge efficacy studies, CAR-T cells were added followed by multiple injections of Nalm6 tumor cells, and tumor burden and T cell expansion were examined. Spleens were collected and subjected to single-cell RNA sequencing upon study completion. For assessing antigen-independent expansion, CAR-T cells were injected into tumor-free NSG mice and T cell expansion was tracked. Nalm6 tumor cells were injected on Day 50 and tumor burden was measured.Results In vitro, CD3ε CAR-T cells could recognize and kill antigen-positive tumor cells equivalently to the CD3ζ benchmark. However, in an in vivo disseminated Nalm6 tumor model, CD3ε CAR-T cells mediated greater anti-tumor activity with more than 50-fold expansion compared to the CD3ζ benchmark. Unlike CD3ζ which relapsed after the first tumor challenge, CD3ε CAR-T cells controlled tumor growth after 5 tumor rechallenges ( figure 1). These persisting CD3ε CAR-T cells were primarily effector memory T cells. In a subsequent study, CD3ε CAR-T cells survived in the absence of antigen, while retaining anti-tumor function (figure 2).Conclusions This innovative approach leveraging the unique signaling properties of CD3ε achieved complete tumor eradication at low CAR-T doses with enhanced CAR-T expansion and persistence, while promoting survival of effector memory T cells in vivo. With optimized signaling and increased potency and persistence, CD3ε CAR-T cells have the potential to provide deeper and more durable responses in patients.References Feucht J, Sun J, Eyquem J, Ho YJ, Zhao Z, Leibold J, Dobrin A, Cabriolu A, Hamieh M, Sadelain M. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nat Med. 2019 Jan;25(1):82-88.Duan Y, Chen J, Meng X, Liu L, Shang K, Wu X, Wang Y, Huang Z, Liu H, Huang Y, Zhou C, Gao X, Wang Y, Sun J. Balancing activation and co-stimulation of CAR tunes signaling dynamics and enhances therapeutic potency. Mol Ther. 2023 Jan 4;31(1):35-47.Schoutrop E, Poiret T, El-Serafi I, Zhao Y, He R, Moter A, Henriksson J, Hassan M, Magalhaes I, Mattsson J. Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models. J Immunother Cancer. 2023 Feb;11(2):e005691.Smagghe BJ, Carter MG, Yi KR, Nash JSS, Grant TJ, Miller DS, Moe ST, Smith DD, Nash MJ, Miller NK, Agarkov LL, Marquez JP, Stewart AK, Bamdad CC. Effective CAR T-cell targeting of an MUC1 cleavage product. J Immunother Cancer. 2025 May 30;13(5):e010577.Wu W, Zhou Q, Masubuchi T, Shi X, Li H, Xu X, Huang M, Meng L, He X, Zhu H, Gao S, Zhang N, Jing R, Sun J, Wang H, Hui E, Wong CC, Xu C. Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy. Cell. 2020 Aug 20;182(4):855-871.e23.Ethics Approval Mice were maintained and used for conducting experiments in adherence to an Institutional Animal Care and Use Committee (IACUC)-approved protocol in an AAALAC-approved facility.Abstract 249 Figure 1CD3ε CAR-T cells demonstrate greater potency and persistence after multiple tumor rechallenges in vivo NSG mice with Nalm6 tumors were treated with CD19 CD3ζ or CD3ε CAR-T cells. (A) Tumor burden by BLI. (B) CAR-T cells in blood by ddPCR. (C) Rechallenged groups received additional Nalm6 tumor cells at indicated times. Tumor burden was measured by BLIAbstract 249 Figure 2CD3ε CAR-T cells persist and retain functionality without antigen stimulation in vivo. NSG mice without tumors received CD19 CD3ζ or CD3ε CAR-T cells to assess persistence without antigen and function upon tumor challenge. (A) T cells in blood by flow cytometry. (B) Nalm6 tumor cells were added on day 50. Tumor burden was measured by BLI