RETRACTED ARTICLE: A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function

Jeremy To,Lilin Li,Frederick Lo,Doug Quackenbush,Emily Rowell,Shane R Horman,Connor Reed

doi:10.1038/s42003-020-01565-1

Abstract

Overcoming tumor-mediated immunosuppression and enhancing cytotoxic T-cell activity within the tumor microenvironment are two central goals of immuno-oncology (IO) drug discovery initiatives. However, exploratory assays involving immune components are often plagued by low-throughput and poor clinical relevance. Here we present an innovative ultra-high-content assay platform for interrogating T-cell-mediated killing of 3D multicellular tumor spheroids. Employing this assay platform in a chemical genomics screen of 1800 annotated compounds enabled identification of small molecule perturbagens capable of enhancing cytotoxic CD8+ T-cell activity in an antigen-dependent manner. Specifically, cyclin-dependent kinase (CDK) and bromodomain (BRD) protein inhibitors were shown to significantly augment anti-tumor T-cell function by increasing cytolytic granule and type II interferon secretion in T-cells in addition to upregulating major histocompatibility complex (MHC) expression and antigen presentation in tumor cells. The described biotechnology screening platform yields multi-parametric, clinically-relevant data and can be employed kinetically for the discovery of first-in-class IO therapeutic agents.

Highlights

Aof bainotmigiemne-dtiecpaesnsdaeynpt laantftoi-rtmumfoorr tTh-eceinlltefIurCnrocgtLiaotnion Jeremy To1, Doug Quackenbush2, Emily Rowell3,6, Lilin Li3, Connor Reed4, Frederick Lo5 & T Shane R
The CEF peptide pool is a mix of 32 different peptides derived from either Cytomegalovirus (CMV), Epstein–Barr virus (EBV), or Influenza virus (Flu) and induces T-cell IFNg secretion and expansion in approximately 80% of tested donor peripheral blood mononuclear cells (PBMCs) (Supplementary Fig. 1b)
PBMC cultures were expanded in the presence of low dose interleukin-2 (IL-2) for a further 3 days to increase the proliferation of CD8+CD127+ memory T-cells18

Summary

Results

Development of the T-cell tumor spheroid-killing platform and screening workflow. In developing an assay platform to enable the identification of agents that enhance T-cell anti-tumor function the aim was to detect compounds that elicit effects on Tcells either intrinsically or extrinsically via tumor cells. On day −2, 1000 human colorectal tumor cells (HCT116 expressing the enhanced green fluorescent proteinEGFP) were plated into 1536w spheroid plates in the presence of either 1 μM CEF peptide pool or a negative control CEF Scramble pool (to act as a counter screen for antigen-dependence). HCT116 tumor spheroids were loaded with CMVpp viral antigen conjugated to a BODIPY-488nm fluorochrome and treated with either Brefeldin (a protein transport inhibitor), DMSO (vehicle control), or an unlabeled CMV competitor peptide (CMV cold competitor). Treatment of spheroids with an unlabeled CMV anti-tumor activity indicated by increased numbers of T-cells cold competitor did not affect CMV-BODIPY transport to cell observed in hit compound-treated samples versus non-hit commembranes to a noticeable degree, though we did observe some pound-treated samples (Supplementary Fig. 4b). D resting media mitigated these antigen-independent cytolytic effects enabling the identification of T-cell-enhancing therapeutics

E To ensure that antigen alone was responsible for this observed

C No T-cells

Discussion

D D19054445

It is conceivable the hypoxic environment of spheroid cores