Background The inhibitory receptor NKG2A and the activating receptor NKG2C modulate the function of NK and CD8 T cells by recognizing the same ligand, HLA-E. NKG2A is selectively expressed on lymphocytes with cytolytic function, including NK cells, NKT cells, and a subset of CD8 T cells. The NKG2C - expressing subset of NK cells, also called ‘adaptive NK,’ displays an altered receptor profile and is associated with chronic virus infections. Likewise, the NKG2C + CD8 T cell population also expands in CMV-infected patients. Moreover, the NKG2C ‘engager’ has the potential to generate a strong antitumor response against various tumors. NKG2A blockade, in combination with other PD-1 antibody, has demonstrated some positive clinical activity; However, as a standalone therapy, inhibition of NKG2A and HLA-E appears to be poorly effective both in mouse tumor models and in clinical trials. Thus, we developed a dual targeting antibody ES015 that recognizes both NKG2A and NKG2C, by simultaneously inhibiting NKG2A function yet promoting NKG2C action, leading to better anti-tumor response.

Methods NKG2A family member binding and cross-species reactivity were evaluated by ELISA and FACS. Binding affinity was determined by Octet Red. Blocking activity was determined by competition assay. In vitro functional activity was determined by NK cytotoxicity assay. The NKG2C - mediated activation was evaluated in Jurkat-NFAT reporter assay and NK activation. Epitope analysis was performed by competitive ELISA and HDX-MS. Lead clone was humanized via CDR grafting and back mutation screening. The effects of dual acting ES015 on the antitumor immune response were investigated in PBMC humanized mouse model.

Results We report the generation and preclinical characterization of a novel mAb ES015 with dual targeting of NKG2A and NKG2C. ES015 binds NKG2A with a sub-nM affinity and reverses the inhibitory effect of the NKG2A/HLA-E interaction. We also show that ES015 increased the killing sensitivity in different presenting antigen systems by additional activation of NKG2C signaling. As a single agent ES015 enhanced the killing activity and cytokine secretion of NK when in co-culture with cancer cell lines. ES015 in combination with PD-1/L1 blocking agents resulted in superior anti-tumor activity in vivo.

Conclusions ES015 appears to be a first-in-class antibody with dual targeting of NKG2A (inhibition) and NKG2C (activation). Our preclinical data suggested that dual target of NKG2A and NKG2C is more efficacious than targeting NKG2A alone. The dual targeting agent ES015, once entering the clinical trials, is hence expected to demonstrate superior clinical response in clinical trials.