InVivoMAb anti-human HLA-E

Immunopeptidomics enables the identification of peptides presented by major histocompatibility complex (MHC) molecules, offering insights into antigen presentation and immune recognition. Understanding these mechanisms in hypoxic conditions is crucial for deciphering immune responses within the tumor microenvironment. Current immunopeptidomics approaches do not capture hypoxia-induced changes in the repertoire of MHC-presented peptides. This protocol describes the isolation of MHC class I-bound peptides from in vitro hypoxia-treated cells, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. It describes optimized steps for cell lysis, immunoaffinity purification, peptide elution, and MS-compatible preparation under controlled low-oxygen conditions. The method is compatible with various quantitative mass spectrometry approaches and can be adapted to different cell types. This workflow provides a reliable and reproducible approach to studying antigen presentation under hypoxic conditions, thereby enhancing physiological relevance and facilitating deeper immunological insights. Key features • Enables isolation of MHC class I-bound peptides from cells cultured under hypoxic conditions. • Designed for low-input samples and optimized for maintaining cell viability during extended hypoxic exposure. • Compatible with label-free LC-MS/MS for detailed immunopeptidome analysis. • Adaptable to all human and murine cell lines commonly used in cancer and immunology research.

HLA-E presenting the HLA-G leader peptide VMAPRTLFL (HLA-E[pHLA-G]) on tumor cells plays a crucial role in suppressing natural killer (NK) and cytotoxic CD8+ T cells through NKG2A interaction. While blocking HLA-E:NKG2A is a promising immune checkpoint (IC) approach in cancer therapy, toxicity remains a major clinical concern. We developed a novel IC inhibitor that selectively prevents HLA-E:NKG2A interaction, a monoclonal antibody that selectively targets the HLA-E[pHLA-G] complex, distinguishing cancerous from noncancerous cells. In clinical bone marrow samples from patients with multiple myeloma (MM), 4D7 specifically recognized tumor-associated HLA-E-peptide complexes. Using NK cells from healthy donors, 4D7 effectively blocked the HLA-E:NKG2A interaction, and enhanced NKG2A-positive NK cell activity in autologous MM cell cocultures. Importantly, 4D7 did not inhibit NKG2C-positive NK cells, preserving their activity, even though NKG2C also interacts with HLA-E. In MM-bearing mice treated with human NK cells, 4D7 significantly reduced tumor growth. This targeted approach activates NK cells only against tumor cells presenting HLA-E-peptide complexes, potentially minimizing toxicity compared with current NKG2A inhibitors. The development of 4D7 highlights a promising advancement in immunotherapy for hematologic malignancies, offering improved outcomes for patients with MM, and a foundation for broader application across cancer types.

Naturally occurring T cells that recognize microbial peptides via HLA-E, a nonpolymorphic HLA class Ib molecule, could provide the foundation for new universal immunotherapeutics. However, confidence in the biological relevance of putative ligands is crucial, given that the mechanisms by which pathogen-derived peptides can access the HLA-E presentation pathway are poorly understood. We systematically interrogated the HIV proteome using immunopeptidomic and bioinformatic approaches, coupled with biochemical and cellular assays. No HIV HLA-E peptides were identified by tandem mass spectrometry analysis of HIV-infected cells. In addition, all bioinformatically predicted HIV peptide ligands (>80) were characterized by poor complex stability. Furthermore, infected cell elimination assays using an affinity-enhanced T cell receptor bispecific targeted to a previously reported HIV Gag HLA-E epitope demonstrated inconsistent presentation of the peptide, despite normal HLA-E expression on HIV-infected cells. This work highlights the instability of the HIV HLA-E peptidome as a major challenge for drug development.

The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with 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 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 naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.