Abstract
The inhibition of the PD1/PDL1 pathway has led to remarkable clinical success for cancer treatment in some patients. Many, however, exhibit little to no response to this treatment. To increase the efficacy of PD1 inhibition, additional checkpoint inhibitors are being explored as combination therapy options. TSR-042 and TSR-033 are novel antibodies for the inhibition of the PD1 and LAG3 pathways, respectively, and are intended for combination therapy. Here, we explore the effect on cellular interactions of TSR-042 and TSR-033 alone and in combination at the single-cell level. Utilizing our droplet microfluidic platform, we use time-lapse microscopy to observe the effects of these antibodies on calcium flux in CD8+ T cells upon antigen presentation, as well as their effect on the cytotoxic potential of CD8+ T cells on human breast cancer cells. This platform allowed us to investigate the interactions between these treatments and their impacts on T-cell activity in greater detail than previously applied in vitro tests. The novel parameters we were able to observe included effects on the exact time to target cell killing, contact times, and potential for serial-killing by CD8+ T cells. We found that inhibition of LAG3 with TSR-033 resulted in a significant increase in calcium fluctuations of CD8+ T cells in contact with dendritic cells. We also found that the combination of TSR-042 and TSR-033 appears to synergistically increase tumor cell killing and the single-cell level. This study provides a novel single-cell-based assessment of the impact these checkpoint inhibitors have on cellular interactions with CD8+ T cells.
Highlights
The checkpoint pathway is an integral component of the immune system, maintaining self-tolerance and preventing unnecessary inflammation and cytotoxicity
We observed a significant increase in the cytotoxicity of T cells towards triple-negative breast cancer (TNBC) cells with anti-PD1 treatment that was further enhanced with the co-administration of antibodies, but a lack of increase in cytotoxicity towards ovarian cancer cells
Checkpoint receptor expression on CD8+ T cells and ligand expression of MDA-MB-231 breast cancer cells To better understand the extent to which TSR-042 and TSR-033 may interact with CD8+ T cell and the target cells used in these experiments, checkpoint receptor and ligand expression were assessed by flow cytometry (Fig. 2)
Summary
The checkpoint pathway is an integral component of the immune system, maintaining self-tolerance and preventing unnecessary inflammation and cytotoxicity. It presents a mechanism of immune evasion utilized by many cancers via high expression of checkpoint ligands, inducing exhaustion and anergy in cytotoxic immune cells that might otherwise recognize and kill the tumor cells[1]. Sullivan et al Cell Death and Disease (2020)11:979 activity in vitro[11]. It is unclear, if the tumorkilling observed in rats will be matched in humans
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