Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells

Abstract

Introduction: Malignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy.

Methods: To evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice.

Results: FAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice.

Conclusion: FAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells.

Figures

Figure 1

IHC of fresh-frozen malignant pleural mesothelioma patient sections. A, c and e were stained with the FAP recognizing murine F19 antibody, whereas b, d, f represent the secondary system only. All three histosubtypes of MPM have been analyzed: epithelioid (a, b), sarcomatoid (c, d) and bi-phasic (e, f). Regions marked with * indicate areas of predominant tumor cell location.

Figure 2

Transduction efficacy and purity of CD8+ T cells (a-c) and functional activity of CAR transduced T cells in vitro (d, e, f). Transduction efficacy was assessed by staining the CH2/3 immunoglobulin linker domain with anti-hIgG mAb and with anti-CD8 mAb (n = 10). A shows non-transduced T cells, whereas b displays T cells transduced with the FAP-specific anti-FAP-F19-∆CD28/CD3ζ CAR and c with the NY-ESO-1 specific anti-NY-ESO-1-T1-∆CD28/CD3ζ CAR. 24 h co-cultivation of re-directed T cells with the target cell lines HT1080FAP-luc and T2-1B was analyzed for IFNγ release (n = 5) (d). Comparison of IL-2 production of anti-FAP-F19-∆CD28/CD3ζ and anti-FAP-F19-CD28/CD3ζ after 24 h of co-cultivation with HT1080FAP-luc (n = 2) (e). Proliferation of anti-FAP-F19-∆CD28/CD3ζ re-directed T cells after stimulation with recombinant human FAP (1 μg/ml) in the absence and presence of IL-2 (100 IU/ml) at indicated time points (n = 2) (f). Error bars were calculated as mean + SD (***p < 0.001, **p < 0.01 as calculated by Student’s unpaired 2 tailed t-tests).

Figure 3

Antigen-specific cytolysis of anti-FAP-F19-∆CD28/CD3ζ re-directed T cells. Re-directed T cells recognizing FAP (anti-FAP-F19-∆CD28/CD3ζ (square) and NY-ESO-1157-165 peptide in the HLA-A*02:01 context (anti-NY-ESO-1-T1-∆CD28/CD3ζ) (circle) were cultured with MSTO-211H cells (a, FAP positive), HT1080FAP-luc cells (b, FAP positive), primary fibroblasts (c, FAP positive), HT1080PA-luc cells (d, FAP negative) and T2-1B cells (e, FAP negative but HLA-A*02:01, NY-ESO-1157-165 peptide positive) at different effector to target ratios and analyzed after 4 h of incubation in a europium release assay (n = 2). Error bars were calculated as mean + SD.

Figure 4

Xenograft model of disseminated i.p. tumors. Anti-FAP-F19-∆CD28/CD3ζ or anti-NY-ESO-1-T1-∆CD28/CD3ζ re-directed T cells were co-injected i.p. with 1*106 HT1080FAP-luc tumor cells at an effector to target ratio of 5:1. HT1080FAP-luc tumor cells were recorded by bioluminescence imaging in the same mouse at the indicated days starting day 4 after cell injection (a). A total of 11 mice per experimental setting were treated with re-directed T cells from 2 different donors (donor 1: 6 mice per group, donor 2: 5 mice per group). The median survival of mice was determined by Kaplan-Meier analysis (b). Significance between cohorts was determined by log rank test.