ABSTRACT: IL-21-αHSA combined with TIGIT blockade induces an effective anti-tumor immune response, indicated by the significant enrichment of pathways associated with anti-tumor immune response, as well as significantly increased expression of key genes associated with CD8+ T and NK cell cytotoxicity To further understand the mechanisms by which IL-21-αHSA enhances the benefit of TIGIT blockade, we analyzed MC38 tumor tissues 20 days after five doses of both single agent and combination treatments by RNA sequencing (RNA-seq) of 28,758 genes. Compared to isotype control treatment, combination treatment resulted in 764 genes with differential expression (defined as P < 0.05 and fold change > 2.0), while IL-21-αHSA treatment resulted in 402 DEGs and anti-mTIGIT treatment resulted in 528 DEGs (data not shown). These findings suggest that the combination treatment had a more dramatic effect on gene expression than IL-21-αHSA or anti-mTIGIT treatment alone. To evaluate the pathways underlying the synergistic anti-tumor efficacy induced by the combination treatment, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significance enrichment analysis (defined as P adjust < 0.05). In comparison to the isotype control treatment, 41 KEGG pathways were significantly enriched in the combination treatment group (Table S1) and 31 pathways were significantly enriched in the IL-21-αHSA treatment group (Table S2), of which 28 KEGG enrichment pathways overlapped. Additionally, only 8 KEGG pathways were significantly enriched in the anti-mTIGIT treatment group (Table S3), of which 5 KEGG enrichment pathways overlapped with those of the combination treatment group. These findings suggest that IL-21-αHSA in combination with anti-mTIGIT treatment enriched more KEGG pathways with differential gene expression than for monotherapy, and most of the KEGG pathways significantly enriched by the combination treatment were also differentially enriched by IL-21-αHSA treatment alone. The top 15 pathways, ranked in order of significance, are shown in Fig. 7A. As expected, most are associated with classical anti-tumor immunity signal pathways, including the cytokine-cytokine receptor interaction pathway, the viral protein interaction with cytokine and cytokine receptor pathway, the Th1 and Th2 cell differentiation pathway, the antigen processing and presentation pathway, the T cell receptor signaling pathway, and the PD-L1 expression and PD-1 checkpoint pathway in cancer (P adjust < 0.0001). The natural killer cell mediated cytotoxicity pathway was also significantly enriched (P adjust = 0.0059) in the combination treatment group compared to isotype control treatment group, but was not among the top 15 pathways. Overall, these findings suggest that IL-21-αHSA in combination with TIGIT blockade treatment significantly affects the anti-tumor immunity signal in the tumor micro-environment. For further verification, we analyzed the gene expression levels of key effector molecules, including Interferon-γ (IFNG), Granzyme A (Gzma), Granzyme B (Gzmb), and Perforin 1 (Prf1), each of which are critical mediators of CD8+ T cell and NK cell cytotoxicity and are known to play critical roles in anti-tumor response. As shown in Fig. 7B-E, the combination treatment significantly increased the expression of all four genes (P < 0.05) relative to isotype control treatment, though only IFNG and Gzma were differentially expressed in the combination treatment group relative to the anti-mTIGIT group (P < 0.05). Collectively, these results suggest that IL-21-αHSA in combination with TIGIT blockade induces an effective anti-tumor immune response, indicated by the significant enrichment of pathways associated with anti-tumor immune response, as well as significantly increased expression of key genes associated with CD8+ T and NK cell cytotoxicity.
