A novel antibody targeting the human MICA α3 domain enhances NK cell activity in vitro

The major histocompatibility complex-class I chain related proteins A and B (MICA/B) are expressed by many human cancers as a result of genomic damage, facilitating immune surveillance by cytotoxic lymphocytes. Tumors evade immune recognition via proteolytic shedding of MICA/B proteins from the cell surface. This study developed a mouse monoclonal antibody (m1226AD) against the proteolytic shedding site of the MICA α3 domain, which was subsequently humanized to generate 1226AD. The binding affinity of m1226AD was characterized by ELISA and flow cytometry. The functional activity of 1226AD was evaluated in NK cell cytotoxicity assays using K562 target cells, with parallel measurement of IFN-γ secretion and soluble MICA levels. Antibody-epitope interactions were delineated using AlphaFold structural modeling and compared to those of the clinically relevant antibody CLN-619. m1226AD exhibited high-affinity binding to the isolated MICA α3 domain (EC 50 = 4.9 ng/ml), as well as to full-length MICA/B domains (EC 50 = 5.7 and 3.2 ng/ml, respectively). The humanized antibody 1226AD enhanced NK cell-mediated cytotoxicity in vitro by inhibiting MICA shedding from tumor cells and promoting IFN-γ secretion. IL-15 synergized with 1226AD to augment tumor cell killing, whereas an Fc effectorless mutant (1226ADmu) failed to enhance cytotoxicity. Structural modeling revealed that 1226AD binds a unique epitope on the MICA α3 domain distinct from that of CLN-619. Our findings demonstrate that 1226AD effectively augments NK cell-mediated cytotoxicity and that multi-epitope engagement within this domain is essential for inhibiting tumor immune evasion and restoring anti-tumor immunity.