Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed "public" cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope's presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01-restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.

Granulocytes play a key role in the defense against invading pathogens. To study granulocyte functions, the isolation of a pure and active cell population from fresh blood is required. Anticoagulants and red blood cells (RBCs) lysis used in the isolation procedure may influence cell harvest, cell marker expression, and pre-activation of cells. In this study, the influence of the anticoagulants K3EDTA or lithium heparin and the effect of different RBCs lysis methods on bovine granulocyte population from fresh blood of healthy cows after density gradient centrifugation were investigated. Venous blood from healthy cows was collected in K3EDTA and lithium heparin tubes. Density gradient centrifugation to separate granulocytes from other cells was conducted using Biocoll. Then, RBCs were lysed with hypotonic water or 0.2% sodium chloride (NaCl). Immediately after isolation, harvest, viability, size, granularity, purity, and CD11b expression as a marker for granulocytes was analyzed by flow cytometry. In addition, as a marker for activation and reactivity of the granulocytes, we stimulated cells with phorbol-myristate-acetate to evaluate the release of reactive oxygen species. Furthermore, extracellular trap (ET) formation was investigated by confocal immunofluorescence microscopy in untreated control cells and cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin. We did not find a significant difference in percentage of dead cells when comparing the two anticoagulants or the different RBCs lysis methods. However, the percentage of granulocytes in the harvested population was significantly less using lithium heparin blood as anticoagulant compared to K3EDTA. The granulocytes harvested from lithium heparin blood and water lysis exhibited higher clumping and pre-activation of unstimulated control cells as indicated by isolation of doublet cells, increased CD11b expression, and increased oxidative burst and higher amount of ET-releasing cells. Furthermore, the combination of K3EDTA as anticoagulant and NaCl as RBCs lysis method revealed the lowest variability and highest difference between untreated and methyl-β-cyclodextrin-treated cells when quantifying ET formation. In conclusion, density gradient centrifugation of K3EDTA blood resulted in higher purity of bovine granulocytes compared to lithium heparin blood. In contrast to water lysis, NaCl lysis method is recommended to avoid pre-activation of cells which may occur during hypotonic water lysis.