Investigating combination benefit of PD1 and LAG3 co-blockade using an engineered cellular bioassay
Bhagyashree Bhagwat1, Rene de Waal Malefyt2, Aarron Willingham1
1Discovery Biologics, Merck & Co., Inc, South San Francisco, CA, USA.
International Immunopharmacology
|April 12, 2023
Summary
Combined blockade of Lymphocyte-activation gene 3 (LAG3) and Programmed cell death protein 1 (PD1) enhances immune activation more than individual treatments. This study reveals distinct downstream signaling effects of LAG3 and PD1 blockade, offering a new assay for immune pathway research.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Immunotherapy
Background:
- Lymphocyte-activation gene 3 (LAG3) and Programmed cell death protein 1 (PD1) are key inhibitory receptors on T cells, contributing to an immunosuppressive tumor microenvironment.
- While combined PD1 and LAG3 blockade shows clinical promise, the precise downstream signaling pathways of LAG3 remain poorly understood.
- Understanding these pathways is crucial for optimizing cancer immunotherapy strategies.
Purpose of the Study:
- To investigate the downstream signaling transduction effects of simultaneous PD1 and LAG3 pathway blockade using a co-culture system.
- To compare the immune activation profiles induced by individual versus combined PD1 and LAG3 blockade.
- To explore the role of specific signaling molecules and pathways, including ERK and CD28, in LAG3 and PD1 signaling.
Main Methods:
- Utilized an engineered Jurkat (T cell) and Raji (Antigen-presenting cell) co-culture system.
- Employed RNA-sequencing (RNA-Seq) to analyze gene expression changes upon antibody treatment.
- Assessed extracellular signal-regulated kinase (ERK) phosphorylation and Interleukin-2 (IL-2) levels.
- Investigated the impact of a phosphatase inhibitor (NSC87877) and CD28 knockout on immune activation.
Main Results:
- Combination treatment of PD1 and LAG3 blocking antibodies resulted in a 5-fold increase in gene upregulation compared to individual treatments.
- Key costimulatory genes (CD28, CD5, CD6) and intracellular signaling molecules (LCP2, ITK) were upregulated.
- Combined blockade led to prolonged ERK phosphorylation, and LAG3 blockade was necessary for immune activation by the phosphatase inhibitor, which mimicked combination therapy benefits when combined with PD1 blockade.
- CD28 knockout affected IL-2 response but did not abolish the combination benefit, suggesting complex signaling interactions.
Conclusions:
- This reductionist co-culture system effectively highlights distinct downstream signaling effects of PD1 and LAG3 blockade.
- The findings suggest synergistic immune activation upon combined PD1 and LAG3 inhibition.
- The developed assay holds potential for dissecting signaling pathway convergence and augmenting studies in primary immune cells for improved immunotherapy development.
