Unveiling a novel NKG2A inhibitor based on monoclonal antibody to HLAE-peptide complex.

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e14677 Background: HLAE is a non-classical MHC class I molecule complexed with the VMAPRTLFL nonapeptide, derived from the leader sequence of HLAG (HLAEpHLAG). HLAEpHLAG is overexpressed across a wide type of tumors prominently on Multiple Myeloma (MM). HLAEpHLAG complex is the ligand to CD94-NKG2A in the immune system which negatively regulates the activation of natural killer (NK) cells as well as NKG2A positive T cells. Harnessing of the HLAE-NKG2A axis by the cancer cells leads to evasion from the immune system attack resulting in cancer progression. Currently, several Immune Checkpoint Inhibitors (ICIs) target the NKG2A receptor which has a low specificity to cancer and high toxicity to the patient. Engineering a new ICI that targets the HLAEpHLAG complex can potentially yield improved outcomes in cancer therapy. Methods: Using the hybridoma system, a new mouse monoclonal antibody (mAb) called 4D7 that targets the HLAEpHLAG complex was developed. mAb 4D7 affinity was assessed in vitro with recombinant proteins and a panel of cell lines that expressed zero to high levels of HLAE. The functional impacts of the mAb 4D7 was examined in a system where various MM cell lines were separately co-cultured with primary NK (pNK) cells. pNK were isolated from the peripheral blood of 5 healthy donors, and their activity was examined in the presence of mAb 4D7 by CD107a degranulation assay. Also, the functional impacts of the mAb 4D7 on allogenic pNK and MM blast cells, isolated from the bone marrow of 6 MM patients were assessed. Results: mAb 4D7 was determined to be an IgG-kappa isotype showing a high specific binding to recombinant HLAEpHLAG. Cell lines with overexpression of membranal HLAEpHLAG: RPMI8226, U266, 721.221 HLAE, and 721.221 HLAG showed positive staining with the mAb 4D7. While cell lines with low to zero expression of HLA-E like 721.221 WT showed low or negative staining with the mAb 4D7. The degranulation of NKG2A+ pNK subset cells against MM target cells increased in presence of mAb 4D7. However, adding mAb 4D7 to NKG2A -pNK subset cells co-cultured with MM target cells did not affect the pNK activity. Similar results were obtained when the target and effector cells were isolated from the bone marrow of active treatment-naive MM patients. Conclusions: MAb 4D7 demonstrates a remarkably high affinity for binding to the HLAEpHLAG complex, ensuring the safety of its application. The ability of 4D7 mAb to effectively block the HLAEpHLAG complex and counteract the inhibition signal in NK cells. mAb 4D7 is a promising monoclonal antibody with substantial potential to serve as an ICI for clinical use in MM patients.