Background NKG2A (Natural Killer Group 2A), an inhibitory immune checkpoint, forms a heterodimer receptor with CD94. NKG2A/CD94 is often expressed on subsets of natural killer (NK) cells, CD8+ tumor-infiltrating T (CD8+ TIL) cells and γδ T cells, etc. Its ligand HLA-E, a non-classical MHC-I molecule, is frequently up-regulated in many solid tumors or hematological malignancies, protecting from NKG2A-expressing immune cell mediated killing. Several NKG2A/CD94 blocking mAb (e.g. Monalizumab) are being tested in clinic, demonstrating preliminary antitumor activity in certain solid tumors including head and neck carcinoma (HNSCC) and non-small cell lung carcinoma (NSCLC).
Methods A humanized anti-NKG2A/CD94 mAb HX017 was recently constructed from mouse hybridoma screening. SPR/ELISA and flow cytometry were used to assay the binding of HX017 to human NKG2A/CD94 proteins; The blockade activity was investigated by flow cytometry as HX017 competitive binding to NKG2A/CD94-expressing 293T cells in the presence of hHLA-E-peptide tetramer (ligand blocking) or biotin-labelled Monalizumab (epitope binning); HX017-mediated NK activations were determined by 1) CD107α-expression using NK/CD4+ autoblast co-culture from different donors and 2) NKG2A/CD94+ Jurkat reporter cell (luciferase) when co-cultured with the APC expressing HLA-E/β2m; K562 target cell killing by IL-15 treated NKL cells (NKG2A/CD94+) in the presence of HLA-B7 signaling peptide was determined (CytoTox-Glo™). Another luciferase reporter assay using NKG2A/CD94+ PD-1+ Jurkat cell and APC expressing HLA-E/β2m/PD-L1 was assessed to investigate combination with anti-PD-1 in vitro. In vivo tumor growth inhibition by HX017 were evaluated using two types of humanized models.
Results HX017 was shown to have high binding affinity to NKG2A/CD94 (EC50 ~0.22 nM) and ligand-blocking (IC50 ~0.63 nM). HX017 efficiently triggered NK activation and enhanced NK-cytotoxicity to target cells in vitro, alone or in combination with anti-PD1 mAb. HX017 showed antitumor activity, as a single agent or, in particular, in combination with PD1 mAb, in two types of humanized mice: 1) MC38-hHLA-E/hPD-L1 HuGEMM model where hPD-1/hPD-L1/hCD94/hNKG2A were knocked-in, and A431 tumor in huPBMC reconstituted NSG-IL-15 mice model. Finally, HX017 has good stability.
Conclusions HX017 could potentially be developed as a promising candidate cancer immunotherapy, particularly in combination with other ICIs.
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