Background: Interleukin-10 (IL-10) has demonstrated utility in aiding CD8 T cell-mediated tumor control in mouse models but phase 2 clinical trials with pegylated IL-10 have so far not been entirely successful. More recent studies revealed that another half-life-extended form of IL-10 (IL10-Fc) can rejuvenate terminally-exhausted CD8+ T cells to boost anti-tumor activity. We investigated whether combination of a PDL-1-blocking antibody with a C-terminal IL-10 fusion may enhance anti-tumor immunity, and possibly achieve synergistic effects.
Methods: Recombinant proteins were produced using an expiCHO system (Thermo Fisher). PDL-1 blocking activity was evaluated by ELISA, PD-1/PDL-1 signaling (Eurofins) and mixed lymphocyte reaction assays. IL-10 activity was evaluated by MC/9 cell proliferation and CD8 T cell activation and effector assays. Mouse CT26 colon cancer and EMT6 breast cancer studies employed the services of Oneness Biotech and Crown Bioscience, respectively.
Results: To develop a bifunctional anti-PDL-1 antibody/IL-10 fusion protein, anti-human PDL-1 antibodies were first developed by screening an in-house human variable domain phage display library. Three different antibodies were then C-terminally-fused with human IL-10 polypeptide. All three showed high binding affinity to PDL-1 and blocked PD-1 signaling through PD-1/PDL-1 interaction in a dose-dependent manner. In CD4 T cell/dendritic cell co-cultures, the lead anti-PDL1/IL10 protein enhanced IL-2 and IFNγ production, and it enhanced IFNγ and granzyme B production from activated CD8 T cells as well as proliferation of MC/9 cells in an IL-10-dependent manner. Also, a mouse ‘surrogate’ version (BF11, using avelumab anti-PDL-1) showed superior efficacy compared to either IL10-Fc or anti-PDL1/TGFbR in a mouse CT26 colon tumor model, and correlated with increased serum levels of CXCL9 and IL-18. BF11 also allowed greater EMT6 tumor control than anti-PDL-1 alone or anti-PDL1/TGFbR fusion protein. Such BF11-treated mice also showed long-lasting immunity against a secondary EMT6 tumor challenge. This ability of BF11 to enhance tumor control was in part dependent on increased activity of tumor-resident cells as co-treatment with FTY720, which inhibits lymphocyte egress from secondary lymphoid organs, did not appear to reduce anti-tumor activity of BF11 in some, albeit not all, EMT6-tumor-bearing mice.
Conclusions: Anti-PDL1/IL10 fusion protein not only targets PDL-1 for checkpoint blockade but also activates the IL-10 pathway to boost T cell response, presumably including revitalization of exhausted T cells as recently reported. Effective anti-tumor activities of a mouse surrogate fusion protein, BF11, are demonstrated in CT26 and EMT6 tumor models. Taken together, our findings support this dual mechanism-strategy to potentiate anti-tumor immunity.
Citation Format: Huey-Wen Hsiao, Chih-Lun Hsiao, Pandelakis Koni, Hung-Kai Chen. A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4169.