Investigating genes and pathways that play a role in immune responses mediated by anti-CTLA-4 therapy.

Abstract

2520^ Background: Blockade of the inhibitory T cell molecule CTLA-4 with a monoclonal antibody has led to enhanced anti-tumor immune responses and clinical benefit. Ipilimumab, an anti-CTLA-4 antibody (BMS), was recently FDA-approved for the treatment of metastatic melanoma. Only a subset of patients benefit from anti-CTLA-4. In order to identify genes and pathways that are induced by anti-CTLA-4, which may be used in future studies for potential correlation with clinical outcomes or provide additional targets for therapy, we purified and analyzed CD4 T cells from patients treated with anti-CTLA-4 for changes in gene expression profile. We also obtained tumor tissues from treated patients for similar studies. Methods: On an IRB-approved Phase Ia pre-surgical clinical trial, 6 patients with localized bladder cancer were treated with two doses of Ipilimumab at 10 mg/kg at weeks 1 and 4. Blood was collected pre-therapy and post-therapy (3 weeks after each dose). CD4 T cells were enriched from peripheral blood by using the CD4 T cell isolation kit from Miltenyi Biotec (Auburn, CA). Total RNA was isolated from purified CD4 T cells using Qiagen RNeasy kit for Affymetrix microarray analyses. Microarray data were then analyzed using Ingenuity iReport (Redwood City, CA). RT-PCR, RPPA and Western blot were used to confirm significant changes in genes or pathways identified in microarray analyses. Results: Ipilimumab treatment resulted in modulation of differentially expressed genes (DEGs). After dose #1, Ipilimumab only modulated 16 DEGs >2-fold (p<0.05) in CD4 T cells. After two doses of treatment, Ipilimumab significantly changed expression of a total of 100 DEGs. Further pathway analyses indicated that Ipilimumab induced a variety of pathways involved in cell cycle control, cell proliferation, apoptosis, and immune modulation. Specifically, these pathways include PI3K/AKT, MAP/ERK, IFN/JAK-STAT, granzyme, and protein ubiquination. Conclusions: Ipilimumab treatment results in modulation of multiple genes and pathways, which likely play important roles in anti-tumor immune responses and need to be considered for future optimization of anti-CTLA-4 therapy and design of combination immunotherapy strategies.