Modulation of NKp44-PCNA Immune Checkpoint Using a Novel Monoclonal Antibody Against Membrane-Associated PCNA

Introduction- Suppression of immunity within the tumor microenvironment was added as an emerging hallmark of cancer. Cancers developed to harness certain immune-checkpoint pathways as a major mechanism of immune resistance. Blocking immune checkpoints involving the CTLA4-B7 and the PD1-PDL1 inhibitory axes was proven to have a therapeutic benefit for cancer patients, mostly through enhancing T cell-based adaptive responses. Natural Killer (NK) cells play a major role in anti-cancer immunity. It has been reported by us and others that Proliferating Cell Nuclear Antigen (PCNA), expressed on the surface of cancer cells act as an inhibitory ligand for the NK cell activating receptor, NKp44. To block this NKp44-PCNA immune checkpoint-mediated inhibition of NK cell function; we developed a novel monoclonal antibody against membrane-associated PCNA, named 14-25-9. Treatment with 14-25-9 induced activation of NK cells interacting with cancer cells both in vitro and in vivo.

Materials and Methods- We used hybridoma technology along with FACS- and ELISA-based high throughput screening to produce 14-25-9 mAb. Characterization of mAbs was done using ELISA, Western blot and Surface Plasmon Resonance. FACS and ImageStream were employed to quantify the membrane-associated and cytoplasmic PCNA staining by 14-25-9. Immunohistochemistry of human cancer FFPE tissues were done with 14-25-9. NK functions were measured using ELISA-based IFN-γ secretion assay and FACS-based Killing assay. In-vivo experiments were done on PDX-bearing NSG mice. Experiments were done according to BGU’s IACUC, permit- IL-80-12-2015).

Results and discussion- In Western blot 14-25-9 binds to endogenous PCNA similarly to commercial anti-PCNA mAb (clone PC10). In ELISA, 14-25-9, but not PC10, showed inhibition of binding of chimeric NKp44 receptor to PCNA. The mAb 14-25-9 can recognize cell surface PCNA, whereas clone PC10 showed almost no surface staining in cancer cells. 14-25-9 recognized cytoplasmic PCNA while PC10 recognized nuclear PCNA, as confirmed by confocal and image stream analysis. NK92-NKp44 cell line and primary human NK cells showed increased in IFN-γ release upon co-incubation with 14-25-9 and different solid tumor and leukemia cell lines. Treatment with 14-25-9 also increased the lysis activity of both NK92-NKp44 and primary human NK. Further, in PDX mouse model intravenous administration of mAb 14-25-9 increased the CD107a expression of the intratumorally injected patient autologous / allogeneic NK cells.

Conclusion- Our study represents a novel immune checkpoint blocker antibody against the NKp44-PCNA immune checkpoint, which can enhance NK cell anti-tumor activity both in vitro and in vivo.