Refining Immune Checkpoints for Natural Killer-based immunotherapy

The cancer-associated immunome, particularly within the cancer microenvironment, is associated with cancer prognosis. The presence and phenotype of intratumoral natural killer (NK) cells in the cancer microenvironment is tightly associated with cancer prognosis. NK cell activity is a balance between signals delivered by inhibitory and activating receptors. The issue of altered expression of activating/inhibitory isoforms of immune-associated genes needs will be presented; in particular, the splicing-enabled paradoxical role of the NK receptor, NKp44/NCR2. The NKp44-PCNA immune checkpoint will be detailed with references to cancer and pregnancy. Monoclonal antibody based blocking of immune checkpoints involving the CTLA4-B7 and the PD1-PDL1 inhibitory axes enhance T cell-based adaptive responses in cancer patients. Similarly, anti-tumor responses by Natural Killer (NK) cells can also be enhanced by checkpoint blocking mAb against Proliferating Cell Nuclear Antigen (PCNA) which is expressed on the surface of cancer cells and acts as an inhibitory ligand for the NK cell receptor, NKp44-isoform1. Hybridoma technology followed by FACS- and ELISA-based screenings were done to generate the specific mAb, 14-25-9. FACS and ImageStream based staining of cell lines and immunohistochemistry of human cancer FFPE tissues were done to test for cytoplasmic and membrane-associated PCNA. NK functions were measured using ELISA-based IFN-γ secretion assays and FACS-based killing assays. In vivo efficacy was evaluated on patient-derived xenografts (PDX)-bearing NSG mice. The 14-25-9 mAb effectively inhibits binding of chimeric NKp44 receptor to PCNA and stains mostly the cytoplasm and membrane of tumor cells, whereas commercial antibody (clone PC10) stains nuclear PCNA. The NK92-NKp44-1 cell line and primary human NK cells showed increased IFN-γ release upon co-incubation with mAb 14-25-9 and various solid tumor cell lines and leukemia. Treatment with 14-25-9 also increased the NK cytotoxic activity. In PDX-bearing mice, intravenous administration of mAb 14-25-9 increased degranulation (CD107a expression) of intratumorally-injected patient-autologous or allogeneic NK cells as well as inhibited tumor growth when treated long term. This study represents a novel mAb against the NKp44-PCNA innate immune-checkpoint, which can enhance NK cell anti-tumor activity both in vitro and in vivo.