Background
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. 75% of the patients are diagnosed in advanced stage with an overall survival rate of about 30%. Immunotherapeutic approaches with checkpoint inhibitors did not have prognostic impact so far. Accordingly, for development of highly needed novel therapeutic strategies, investigation of the tumor microenvironment (TME) and tumor infiltrating lymphocytes (TILs) as well as identification of immune checkpoints (ICPs) and tumor associated antigens (TAAs) appears indispensable.
Materials and Methods
Hence, we analyzed primary EOC tumor tissue of 45 patients with primary advanced EOC. The tumor tissue was mechanically and enzymatically dissociated into a single cell suspension. CD45+ TILs were magnetically isolated. Multiplex flow cytometry (MPFC) was used to characterize the composition of the TME and to determine ICP expression (PD-1, TIGIT, TIM3) on T cells. In addition, T-cell function of TILs was compared to healthy donor (HD) T cells in an established cytotoxicity assay system using OCI-AML3 cells and a T-cell bispecific antibody (TCB) in a co-culture setup for 7 days. To overcome the dysfunction of TILs, a combination of the TCB and an anti-CD28 monoclonal antibody (mAb) or IL-2 was performed. Furthermore, T-cell metabolism was evaluated using the Seahorse XF analyzer. Moreover, potential TAAs (mesothelin (MSLN), mucin 1 (MUC1), EpCAM) and ICPs (B7H3 and CD47) were assessed on EOC cells.
Results
The CD45+ TILs consisted mainly of T cells (67.7%), followed by a monocyte/macrophage population (15.6%) and B cells (10.3%) as well as NK cells (5.9%) and pDCs (1%). The CD4:CD8 T-cell ratio varied widely within patients. Within the monocyte/macrophage population we predominantly detected M2-like polarized cells (CD163+ and CD206+). There was a high (co)-expression of PD-1, TIGIT and TIM3 on T cells. In co-culture with OCI-AML3 cells and a TCB, TILs-derived T cells showed an exhausted phenotype with reduced proliferation, cytokine secretion, and killing capacity compared to HD-derived T cells (94.7% vs. 55.1% specific lysis). The cytotoxic capacity was rescued in combination with anti-CD28 mAb (16.4% vs 37.7% specific lysis) and IL-2 (16.4% vs 85.0% specific lysis). The metabolic capacity was also diminished in comparison to HD-derived T cells. The TAAs MSLN (median MFI ratio 2.2), MUC1 (median MFI ratio 22.9), and EpCAM (median MFI ratio 51.2), and the ICPs B7H3 (median MFI ratio 2.5) and CD47 (median MFI ratio 49) were expressed on primary EOC cells.
Conclusions
In summary, the immune contexture of EOC samples was dominated by immunosuppressive type 2 macrophages and TILs with an exhausted phenotype, as detected by surface expression but also functional data. Albeit TAAs could be identified, the data highlights that immunotherapeutic strategies must integrate immunomodulatory agents, to counteract the immunosuppressive TME of EOC.
A. Leutbecher: None. S. Geweniger: None. G. Hänel: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. A. Neumann: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. B. Czogalla: None. F. Trillsch: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; AstraZeneca, Roche, SAGA diagnostics. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; AstraZeneca, Roche, MSD, Eisai, GSK. F. Consultant/Advisory Board; Modest; AstraZeneca, Roche, Eisai, MSD, GSK. A. Burges: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; AstraZeneca, Tesaro, Roche. F. Consultant/Advisory Board; Modest; AstraZeneca, Tesaro, Roche. M. von Bergwelt-Baildon: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Gilead, Miltenyi Biotec, MSD Sharpe & Dohme, Roche, Mologen, Novartis, Astellas, BMS. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Gilead, Miltenyi Biotec, MSD Sharpe & Dohme, Roche, Mologen, Novartis, Astellas, BMS. M. Subklewe: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Amgen, Gilead, Miltenyi Biotec, Morphosys, Roche, Seattle Genetics. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen, Celgene, Gilead, Janssen, Pfizer. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. F. Consultant/Advisory Board; Modest; Amgen, Celgene, Gilead, Janssen, Novartis, Pfizer, Seattle Genetics, BMS. A. Reischer: None.