A Single-Domain TCR-like Antibody Selective for the Qa-1(b)/Qdm Peptide Complex Enhances Tumoricidal Activity of NK Cells via Blocking the NKG2A Immune Checkpoint

The NKG2A/HLA-E axis is an immune checkpoint that suppresses immune effector activity in the tumor microenvironment. In mice, the ligand for the NKG2A/CD94 inhibitory receptor is the nonclassical MHC molecule Qa-1(b), the HLA-E ortholog, which presents the peptide AMAPRTLLL, referred to as Qdm (for Qa-1 determinant modifier). This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci. To broaden our understanding of Qa-1(b)/Qdm peptide complex biology and its tumor protective role, we identified a TCR-like Ab from a single domain VHH library using yeast surface display. The TCR-like Ab (EXX-1) binds only to the Qa-1(b)/Qdm peptide complex and not to Qa-1(b) alone or Qa-1(b) loaded with control peptides. Conversely, currently available Abs to Qa-1(b) bind independent of peptide loaded. Flow cytometric results revealed that EXX-1 selectively bound to Qa-1(b)/Qdm-positive B16F10, RMA, and TC-1 mouse tumor cells but only after pretreatment with IFN-gamma; no binding was observed following genetic knockdown of Qa-1(b) or Qdm peptide. Furthermore, EXX-1 Ab blockade promoted NK cell-mediated tumor cell lysis in vitro. Our findings show that EXX-1 has exquisite binding specificity for the Qa-1(b)/Qdm peptide complex, making it a valuable research tool for further investigation of the Qa-1(b)/Qdm peptide complex expression and regulation in healthy and diseased cells and for evaluation as an immune checkpoint blocking Ab in syngeneic mouse tumor models.