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-γ; 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. Key Points A llama was immunized with Qa-1^b/Qdm, ligand for NKG2A, to construct a VHH library. A single-domain TCR-like Ab, EXX-1, specific for Qa-1^b/Qdm, was identified. EXX-1 selectively blocks the NKG2A/Qa-1^b axis to promote NK lysis of target cells.

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