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Building a translational pathway using pharmacodynamic and syngeneic tumour models in conjunction with gene expression to enable the development of cancer immune therapies Louise Brackenbury 1 , Tommaso Iannitti 1 , Robert Nunan 1 , Louise Harvey 1 , Clio Andreae 1 and S. Rhiannon Jenkinson 1 1 Charles River Laboratories, Portishead, Bristol, UK 3 USE OF MURINE TUMOR PD MODELS DRIVES DECISIONS AND VALIDATES POTENTIAL BIOMARKERS 2 PD MODELS ALLOW THE ASSESSMENT OF T CELL FUNCTION IN VIVO AND IDENTIFICATION OF POTENTIAL BIOMARKERS 1 INTRODUCTION In order to develop therapeutics which drive the immune system to target tumour cells and eliminate tumour growth, sophisticated in vitro and in vivo models are required. We have developed models which enable us to combine data from human immune cell in vitro assays and murine pharmacodynamic (PD) and syngeneic tumour models to evaluate which pathways a therapeutic is hitting and whether it is effective at inhibiting tumour growth in vivo. We show here the powerful combination of using TCR transgenic T cell adoptive transfer and syngeneic tumour models with flow cytometry and Nanostring profiling of gene expression within the tumour microenvironment (TME) to determine the effect of therapeutic intervention. 4 TUMOR GENE EXPRESSION ANALYSIS USING NANOSTRING 5 IN VITRO TUMOR KILLING ASSAY TO EXAMINE HOW MODULATION OF HUMAN T CELL AND NK CELL FUNCTION CAN ENHANCE TUMOR KILLING OT-I CD8 T cells • To determine the effect of checkpoint inhibitors, mice bearing a defined population of ovalbumin (OVA)-specific T cells are challenged with cognate antigen (PD model). • Flow cytometric analysis of OT-I T cells following in vivo challenge with cognate antigen allows assessment of the ability of novel therapeutics to engage their target and for their ability to enhance T cell function. • Antigen specific T cells can be assessed for proliferation (CTV dilution), their expression of a panel of T cell activation markers and expression of effector molecules which drive cytotoxic T cell function. • Blockade of the checkpoint inhibitor ligand, PD-L1 and blockade of CTLA-4 enhances the OT-I CD8 T cell response to cognate antigen in vivo as shown by the increased percentage of OT-I T cells expressing IFNγ, Granzyme B and CD25. Tumor and TIL analysis Effect of adoptive cell therapy on Tumour Volume 0 500 1000 1500 2000 Time (days post-induction) Tumour volume (mm 3 ) 0 4 6 8 11 13 15 18 Alum/OVA (n=10) Alum (n=10) ACT OVA 0 500 1000 1500 2000 2500 0 2×10 4 4×10 4 6×10 4 8×10 4 Tumour volume (mm 3 ) Total OT-I Cells OT-I tumour infiltrate per mm 3 tumour d18 R 2 = 0.5 5 4 3 2 1 0 0 20 40 60 80 100 Tumour Draining Lymph Node OT-I proliferation Division Percentage of cells within each round of division Alum/OVA Alum 0 5 10 15 20 CTV+ Granzyme B+ % of OT-I CD8a+ Thy 1.2+ Alum/OVA Alum 0 20 40 60 80 CTV+ IFN-γ+ % of OT-I CD8a+ Thy 1.2+ Analysis of the tumour specific CD8 T cell response in the tumor draining lymph node • To determine the effect of checkpoint inhibitors, mice bearing a defined population of ovalbumin (OVA)-specific T cells were challenged with an OVA expressing tumour (EG7 or MC38). • OT-I T cells which recognise an OVA peptide (SIINFEKL) were transferred to allow the effect of novel therapeutics or vaccines on tumour specific T cell responses to be assessed. • In vivo activation of OT-I T cells inhibits growth of an OVA expressing tumour and results in a larger number of OT-I T cells present within the tumour microenvironment. • Analysis of the proliferation of OT-I T cells in the tumour draining lymph node of a tumour expressing the model tumour antigen OVA, demonstrates the efficiency of priming driven by antigen captured from the tumour versus that seen when mice are primed with exogenous OVA. • Proliferation of OT-I T cells is assessed by CTV dilution. The ability of a novel therapeutic or vaccine to enhance CD8 T cell effector function is assessed by expression of effector molecules (Granzyme B and IFNγ). Nanostring analysis of gene expression using the murine immune-oncology 360 panel enables a deeper analysis of the TME and acts as a powerful tool for the identification of potential PD biomarkers. • Volcano plots were generated from advanced analysis run on normalised data sets. Data displays each gene's -log10 (p-value) (y- axis) and log2 fold change (x-axis) with the selected covariate. The 40 most statistically significant genes are labelled on the plot. • Abundance of immune cell types in the TME was determined. Genes characteristic of individual cell populations were used to measure the abundance of each cell type. Cell type scores are shown as mean of the log2 expression for each set of unique cell-specific marker genes 0 24 48 72 -1000 -500 0 500 1000 T im e (Hr) SKOV3 Counts/Im age (Norm alised to t0) PBMC +PEM+IPI PBMC +IgG1/4 NK cell depleted +PEM+IPI NK Cell depleted +IgG1/4 0 24 48 72 -1000 -500 0 500 1000 T im e (Hr) SKOV3 Counts/Im age (Norm alised to t0) PBMC +PEM+IPI PBMC +IgG1/4 CD8 T cell depleted +IgG1/4 CD8 T cell depleted +PEM+IPI 0 24 48 72 0 20 40 60 80 T im e (Hr) Apoptotic Tum our cells (% 66h) NK cell depleted +IgG1/4 PBMC +PEM+IPI NK cell depleted +PEM+IPI PBMC +IgG1/4 0 24 48 72 0 20 40 60 80 T im e (Hr) Apoptotic Tum our cells (% 66h) PBMC +PEM+IPI PBMC +IgG1/4 CD8 T cell depleted +PEM+IPI CD8 T cell depleted +IgG1/4 • Tumour killing assays were also performed with human cell lines and PBMC using clinical antibodies targeting the same pathways. • The SKOV-3 tumour cell line was co-cultured with whole human PBMC or PBMC depleted of NK cells or CD8 T cells to enable measurement of the contribution of these cell types to tumour cell killing. • The ability of checkpoint inhibitors targeting CTLA-4 (Ipilimumab) and PD-1 (Pembrolizumab) to enhance immune cell mediated tumour killing was measured for each condition. • Cultures were imaged using IncuCyte Zoom software. Analysis of tumour cell growth and apoptotic cell death are shown. Data is displayed as mean values. NK depleted CD8 T cell depleted Contribution of NK and CD8 T cells to tumor cell killing in a PBMC tumour cell killing assay 6 CONCLUSION These models allow testing of novel therapeutics alongside benchmark reagents. The PD model provides a screening platform to assess enhancement of T cell function before moving into syngeneic models. Nanostring gene expression profiling may direct which in vitro assays should be used to map the immunological mechanisms underlying immune modulation. The use of in vitro and in vivo PD/efficacy models better enable assessment of novel cancer immune therapeutics and allow early identification of potential biomarkers. The study was approved by HRA NRES South West, Bristol (UK), approval number 15/SW/0029.
