Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Neutralization of the adaptor protein PAG by monoclonal antibody limits murine tumor growth

doi: 10.1016/j.omtm.2022.10.012

Figure Lengend Snippet: Clone 7M16A binds to the extracellular domain of PAG (A) ELISA with immobilized PAG peptide (human, mouse, or cynomolgus) using hybridoma supernatants. (B) 7M16A staining of non-permeabilized wild-type C57BL6 or PAG knockout (KO) murine splenocytes shows specificity of binding. (C) Western blot of A549 cells made to stably overexpress PAG-GFP. (D) Western blot of fractionated cell lysates showing that PAG-GFP is expressed at high levels at the cell membrane. Confocal imaging showing the localization of PAG-GFP at the plasma membrane. (E) 7M16A staining of non-permeabilized A549 cells shows binding to PAG-GFP on the surface. (F) ELISA with immobilized human PAG peptide using purified monoclonal antibodies. (G) ELISA of IL-2 secretion from primary human CD3 + T cells stimulated as indicated for 48 h. n = 3; ∗p ≤ 0.05 and ∗∗p ≤ 0.01. (H and I) Jurkat T cells stably express PAG-GFP with or without PD-1-SNAP incubated with Raji B cells in the presence of SEE. Cells were imaged using confocal microscopy to assess the location of PAG and PD-1 relative to the immune synapse. White arrow indicates the center of the immune synapse where PAG was excluded with 7M16A pretreatment. White star indicates the point in the cell with enrichment of PD-1 either at or away from the immune synapse. Quantification of the number of cells per phenotype. ∗p ≤ 0.05.

Article Snippet: Primary human CD3 + T cells were purified from peripheral blood using RosetteSep CD3 + Negative Selection (catalog no. 15021; Stemcell) followed by Lymphoprep separation.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Knock-Out, Binding Assay, Western Blot, Stable Transfection, Membrane, Imaging, Clinical Proteomics, Purification, Bioprocessing, Incubation, Confocal Microscopy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Neutralization of the adaptor protein PAG by monoclonal antibody limits murine tumor growth

doi: 10.1016/j.omtm.2022.10.012

Figure Lengend Snippet: PAG antibody and PD-1 antibody in combination enhance T cell infiltration into MC38 tumors Immunohistochemistry of dissected tumors at the study endpoint. Sections were stained with anti-CD3 from 2 tumors per condition; representative images shown (40×) (A). (B) Quantification of CD3 + cells per high-power field (HPF; 20×) from 2 tumors per condition. ∗p ≤ 0.05.

Article Snippet: Primary human CD3 + T cells were purified from peripheral blood using RosetteSep CD3 + Negative Selection (catalog no. 15021; Stemcell) followed by Lymphoprep separation.

Techniques: Immunohistochemistry, Staining