PD-1-CD28 fusion protein strengthens mesothelin-specific TRuC T cells in preclinical solid tumor models

Abstract

Background: T cell receptor fusion constructs (TRuC) consist of an antibody-based single chain variable fragment (scFv) fused to a T cell receptor chain (TCR) and allow recognition of cancer cells in an HLA-independent manner. Unlike chimeric antigen receptors (CAR), TRuC are integrated into the TCR complex resulting in a functional chimera with novel specificity, whilst retaining TCR signaling. To further enhance anti-tumor function, we expressed a PD-1-CD28 fusion receptor in TRuC T cells aiming to prevent tumor-induced immune suppression and T cell anergy.

Methods: The activation level of engineered T cells was investigated in co-culture experiments with tumor cells followed by quantification of released cytokines using ELISA. To study T cell-mediated tumor cell lysis in vitro, impedance-based real-time tumor cell killing and LDH release was measured. Finally, two xenograft mouse cancer models were employed to explore the therapeutic potential of engineered T cells.

Results: In co-culture assays, co-expression of PD-1-CD28 enhanced cytokine production of TRuC T cells. This effect was dependent on PD-L1 to PD-1-CD28 interactions, as blockade of PD-L1 amplified IFN-γ production in unmodified TRuC T cells to a greater level compared to TRuC-PD-1-CD28 T cells. In vivo, PD-1-CD28 co-expression supported the anti-tumor efficacy of TRuC T cells in two xenograft mouse cancer models.

Conclusion: Together, these results demonstrate the therapeutic potential of PD-1-CD28 co-expression in TRuC T cells to prevent PD-L1-induced T cell hypofunction.

Keywords: Adoptive cell transfer; Immunosuppression; PD-1-CD28 fusion protein; T cell hypofunction; TRuC T cells.

Fig. 1

Design and expression of TRuC and PD-1-CD28. a, b. The anti-mesothelin scFv was fused to CD3ε assembling a mesothelin-specific TRuC when expressed in human primary T cells. The PD-1-CD28 construct consists of an extracellular PD-1 domain and an intracellular CD28 domain. c, d. Following retroviral transduction, all constructs were stably expressed in human T cells as confirmed by flow cytometry. TRuC + PD-1-CD28 T cells were generated by transducing cells with two retroviruses simultaneously. In case of differential expression levels, transduction efficiencies were titrated for downstream analyses. Flow data representative for > 10 independent transductions

Fig. 2

Cytokine release and killing kinetics of TRuC and TRuC + PD-1-CD28 T cells. a. Real-time lysis of MIA PaCa-MSLN-PD-L1 (upper panel) or SUIT-MSLN-PD-L1 tumor cells (lower panel) by transduced T cells based on impedance measurements. Effector-to-target ratio 10:1. b. Transduced T cells were stimulated with MIA PaCA-MSLN-PD-L1 (upper panel) or SUIT-MSLN-PD-L1 (lower panel) target cells in presence of an anti-PD-L1 blocking antibody or a corresponding isotype control antibody. Cytokine levels were quantified 48 h following stimulation using ELISA. Experiments show mean values ± SEM of duplicates and are representative of two independent experiments (two different T cell donors). For statistical analysis one-way ANOVA was used.

Fig. 3

In vivo anti-tumor activity of TRuC and TRuC + PD-1-CD28 T cells. a, b. NSG mice were subcutaneously inoculated with SUIT-MSLN-PD-L1 target cells and treated with T cells when tumors were established. Tumor growth (a) and survival (b) was monitored for 54 days. n = 5 mice per group. c, d. NSG mice were subcutaneously inoculated with MSTO-MSLN target cells and treated with T cells when tumors were established. Tumor growth (c) and survival (d) were monitored for > 100 days. n = 15 mice for UTD or TRuC and n = 14 mice for TRuC + PD-1-CD28. Given the duration of the experiment (> 140 days), several mice developed GvHD and had to be censored towards the end of the experiment. Experiments show mean values ± SEM. Data shown in panel a and b were obtained by performing one single experiment with one T cell donor, panel c and d show pooled data of three independent experiments (three different T cell donors). Analyses of differences between groups were performed using unpaired, two-way Student’s t test (a, c), Log-rank test (b) or Gehan-Breslow-Wilcoxon test (d)