Distinct antibody clones detect PD-1 checkpoint expression and block PD-L1 interactions on live murine melanoma cells
Christina Martins1, Mariana Silva1, Erik Rasbach1,2
1Department of Dermatology, Harvard Skin Disease Research Center, Brigham and Women's Hospital, Harvard Medical School, HIM Building, Suite 671, 77 Avenue Louis Pasteur, Boston, MA, 02115, USA.
Abstract:
Monoclonal antibodies (abs) targeting the programmed cell death 1 (PD-1) immune checkpoint pathway have revolutionized tumor therapy. Because T-cell-directed PD-1 blockade boosts tumor immunity, anti-PD-1 abs have been developed for examining T-cell-PD-1 functions. More recently, PD-1 expression has also been reported directly on cancer cells of various etiology, including in melanoma. Nevertheless, there is a paucity of studies validating anti-PD-1 ab clone utility in specific assay types for characterizing tumor cell-intrinsic PD-1. Here, we demonstrate reactivity of several anti-murine PD-1 ab clones and recombinant PD-L1 with live B16-F10 melanoma cells and YUMM lines using multiple independent methodologies, positive and negative PD-1-specific controls, including PD-1-overexpressing and PD-1 knockout cells. Flow cytometric analyses with two separate anti-PD-1 ab clones, 29F.1A12 and RMP1-30, revealed PD-1 surface protein expression on live murine melanoma cells, which was corroborated by marked enrichment in PD-1 gene (Pdcd1) expression. Immunoblotting, immunoprecipitation, and mass spectrometric sequencing confirmed PD-1 protein expression by B16-F10 cells. Recombinant PD-L1 also recognized melanoma cell-expressed PD-1, the blockade of which by 29F.1A12 fully abrogated PD-1:PD-L1 binding. Together, our data provides multiple lines of evidence establishing PD-1 expression by live murine melanoma cells and validates ab clones and assay systems for tumor cell-directed PD-1 pathway investigations.
Insights
This study confirms programmed cell death 1 (PD-1) is expressed on live melanoma cells. Researchers validated antibodies and assays for investigating this tumor cell-intrinsic PD-1 pathway in cancer research.
Area of Science:
- Immunology
- Cancer Biology
- Oncology
Background:
- Monoclonal antibodies targeting the programmed cell death 1 (PD-1) immune checkpoint have transformed cancer therapy.
- PD-1 is expressed on T cells, but recent findings suggest expression on cancer cells, including melanoma.
- Limited studies validate antibody utility for characterizing tumor cell-intrinsic PD-1.
Purpose of the Study:
- To validate anti-murine PD-1 antibody clones and assay systems for detecting PD-1 on live melanoma cells.
- To confirm PD-1 protein and gene expression in murine melanoma models.
- To assess the blockade of PD-1:PD-L1 interaction on melanoma cells.
Main Methods:
- Flow cytometry using anti-PD-1 antibody clones (29F.1A12, RMP1-30) on live B16-F10 and YUMM melanoma cells.
- Analysis of PD-1 gene (Pdcd1) expression.
- Immunoblotting, immunoprecipitation, and mass spectrometry to confirm PD-1 protein.
- Functional assay using recombinant PD-L1 and antibody blockade.
Main Results:
- Two anti-PD-1 antibody clones detected PD-1 surface protein on live murine melanoma cells.
- PD-1 gene expression was significantly enriched in melanoma cells.
- Mass spectrometry confirmed PD-1 protein expression.
- Anti-PD-1 antibody 29F.1A12 blocked PD-1:PD-L1 binding on melanoma cells.
Conclusions:
- This study provides robust evidence for PD-1 expression on live murine melanoma cells.
- Validated antibody clones and assay systems are established for investigating tumor cell-intrinsic PD-1.
- These findings support further research into targeting the PD-1 pathway directly on cancer cells.
