Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the na?ve B cell antibody pool have the capacity to enhance NK cell cytotoxicity. Subject terms: NK cells, Antibody therapy, Antibodies, X-ray crystallography The identification and structural analysis of HLA-E-VL9-targeting antibodies that block a natural killer (NK) cell receptor pathway and regulate NK function in vitro. Introduction Natural killer (NK) cells play critical roles in immune surveillance by discriminating normal from altered cells, and function by killing non-self malignant or pathogen-infected cells and by producing inflammatory cytokines1C3. the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG Brivudine form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from na?ve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the na?ve B cell antibody pool have the capacity to enhance NK cell cytotoxicity. Subject terms: NK cells, Antibody therapy, Antibodies, X-ray crystallography The identification and structural analysis of HLA-E-VL9-targeting antibodies that block a natural killer (NK) cell receptor pathway and regulate NK function in vitro. Introduction Natural killer (NK) cells play critical roles in immune surveillance by discriminating normal from altered cells, and function by killing non-self malignant Brivudine or pathogen-infected cells and by producing inflammatory cytokines1C3. Specific recognition of abnormal cells by NK cells relies on a series of activating and inhibitory receptors, including the killer immunoglobulin-like receptor (KIR) family and NKG2/CD94 heterodimeric receptors3,4. NK cell inhibitory receptors ligate human lymphocyte antigen (HLA) or major histocompatibility complex (MHC) class I molecules expressed on healthy cells as self. Conversely, cells lacking MHC class I are recognized by NK cells as missing-self and are sensitive to NK cell-mediated killing5,6. In humans, KIRs recognize classical HLA class Ia molecules7C9, whereas the inhibitory NKG2A/CD94 heterodimeric receptor interacts RNF57 with the nonclassical HLA class Ib molecule HLA-E and is balanced by an activating receptor NKG2C/CD9410C12. While KIR expression is heterogeneous, NKG2A/CD94 is expressed on ~40% of human NK cells9,13,14. Unlike classical HLA class I molecules, HLA-E has limited polymorphism with only two expressed variants, HLA-E*01:01 and HLA-E*01:03, that differ only in residue 107, which is outside the peptide-binding groove15. The NKG2A/CD94/HLA-E pathway is considered to be an important immune checkpoint and has recently become a focus for NK cell-based immunotherapeutic strategies4,16C19. A subset of CD8+ T cells also express NKG2A/CD94, and inhibition of the NKG2A/CD94CHLA-E interaction has similar application in CD8+ T cell-based immunotherapy4,20. HLA-E engages with NKG2A/CD94 via a restricted subset of peptides VMAPRT(L/V) (V/L/I/F)L (designated VL9) that derive from the leader sequence of HLA-A, -C, -G and a third of HLA-B molecules10,12,21,22. HLA-E binds VL9 peptides, which stabilize HLA-E surface expression10,12 on healthy host cells in which HLA-Ia expression is not perturbed and initiate recognition by NKG2A/CD94 or NKG2C/CD94 on NK cells. The binding affinity of HLA-E-VL9 peptide complexes for NKG2A/CD94 is greater than that for NKG2C/CD94, such that the inhibitory signal dominates to suppress aberrant NK cell-mediated cytotoxicity and cytokine production12,23C26. In addition, Brivudine HLA-E and its murine or rhesus macaque homologs are capable of binding to a range of other host peptides and pathogen-derived peptides, including heat-shock protein 60 (Hsp60)-derived peptides27, Mycobacterium tuberculosis (Mtb) peptides28,29, and simian immunodeficiency virus (SIV) Gag peptides30,31. However, only VL9 peptide-loaded HLA-E can engage CD94/NKG2A and protect cells from NK cell cytotoxicity27,32,33. Hence, leader sequence VL9 peptides are essential not only for stabilizing HLA-E surface expression but also for mediating the role of HLA-E/NKG2A/CD94 in regulating NK cell self-recognition. However, it remains unclear if interruption of this pathway by specifically targeting Brivudine HLA-E-peptide complexes on target cells can enhance NK cell activity. Here, we isolated a murine IgM monoclonal antibody (mAb) 3H4 that bound to HLA-E-VL9 on target cells and enhanced NK cytotoxicity mediated by an NKG2A+ NK cell line. Crystallographic analysis of an HLA-E-VL9/3H4 antigen-binding fragment (Fab) co-complex indicated that, due to steric clashes, 3H4 and CD94/NKG2A cannot simultaneously bind to these overlapping recognition surfaces on HLA-E-VL9. Surprisingly, the Ig V(D)J residues at the 3H4-HLA-E-VL9 binding interface were germline-encoded. While 3H4 mAb enhanced NK cytotoxicity as an IgM, the IgG form of the antibody did not enhance NK cytolytic activity. To address this, we developed 3H4 IgG variants with enhanced HLA-E-VL9 binding affinity by high-throughput screening of antibody libraries. Optimized 3H4 IgG Abs contained mutations in their CDR-H3 loops, bound HLA-E/VL-9 ~220 times tighter than the WT mAb and.
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