PD-1 and CTLA-4 antibodies offer great hope for cancer immunotherapy. However, many patients are incapable of responding to PD-1 and CTLA-4 blockade and show low response rates due to insufficient immune activation. The combination of checkpoint blockers has been proposed to increase the response rates. Besides, antibody drugs have disadvantages such as inclined to cause immune-related adverse events and infiltration problems. In this study, we developed a cyclic peptide C25 by using Ph.D.-C7C phage display technology targeting LAG-3. As a result, C25 showed a relative high affinity with human LAG-3 protein and could effectively interfere the binding between LAG-3 and HLA-DR (MHC-II). Additionally, C25 could significantly stimulate CD8⁺ T cell activation in human PBMCs. The results also demonstrated that C25 could inhibit tumor growth of CT26, B16 and B16-OVA bearing mice, and the infiltration of CD8⁺ T cells was significantly increased while FOXP3⁺ Tregs significantly decreased in the tumor site. Furthermore, the secretion of IFN-γ by CD8⁺ T cells in spleen, draining lymph nodes and especially in the tumors was promoted. Simultaneously, we exploited T cells depletion models to study the anti-tumor mechanisms for C25 peptide, and the results combined with MTT assay confirmed that C25 exerted anti-tumor effects via CD8⁺ T cells but not direct killing. In conclusion, cyclic peptide C25 provides a rationale for targeting the immune checkpoint, by blockade of LAG-3/HLA-DR interaction in order to enhance anti-tumor immunity, and C25 may provide an alternative for cancer immunotherapy besides antibody drugs.

Tryptophan 2,3-dioxygnease 2 (TDO2) is specific for metabolizing tryptophan to kynurenine (KYN), which plays a critical role in mediating immune escape of cancer. Although accumulating evidence demonstrates that TDO2 overexpression is implicated in the development and progression of multiple cancers, its tumor-promoting role in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we observed that TDO2 was overexpressed in ESCC tissues and correlated significantly with lymph node metastasis, advanced clinical stage, and unfavorable prognosis. Functional experiments showed that TDO2 promoted tumor cell proliferation, migration, and colony formation, which could be prevented by inhibition of TDO2 and aryl hydrocarbon receptor (AHR). Further experimentation demonstrated that TDO2 could promote the tumor growth of KYSE150 tumor-bearing model, tumor burden of C57BL/6 mice with ESCC induced by 4-NQO, enhance the expression of phosphorylated AKT, with subsequent phosphorylation of GSK3β, and polarization of M2 macrophages by upregulating interleukin-8 (IL-8) to accelerate tumor progression in the tumor microenvironment (TME). Collectively, our results discovered that TDO2 could upregulate IL-8 through AKT/GSK3β to direct the polarization of M2 macrophages in ESCC, and suggested that TDO2 could represent as an attractive therapeutic target and prognostic marker to ESCC.

A novel peptide targeting Clec9a on dendritic cell for cancer immunotherapy

Background: Metastasis is the leading cause of mortality in human cancers, including esophageal squamous cell carcinoma (ESCC). As a pro-inflammatory cytokine, IL-32 was reported to be a poor prognostic factor in many cancers. However, the role of IL-32 in ESCC metastasis remains unknown. Methods: ESCC cells with ectopic expression or knockdown of IL-32 were established and their effects on cell motility were detected. Ultracentrifugation, Transmission electron microscopy and Western blot were used to verify the existence of extracellular vesicle IL-32 (EV-IL-32). Coculture assay, immunofluorescence, flow cytometry, and in vivo lung metastasis model were performed to identify how EV-IL-32 regulated the crosstalk between ESCC cells and macrophages. Results: Here, we found that IL-32 was overexpressed and positively correlated to lymph node metastasis of ESCC. IL-32 was significantly higher in the tumor nest compared with the non-cancerous tissue. We found that IL-32β was the main isoform and loaded in EV derived from ESCC cells. The shuttling of EV-IL-32 derived from ESCC cells into macrophages could promote the polarization of M2 macrophages via FAK-STAT3 pathway. IL-32 overexpression facilitated lung metastasis and was positively correlated with the proportion of M2 macrophages in tumor microenvironment. Conclusions: Taken together, our results indicated that EV-IL-32 derived from ESCC cell line could be internalized by macrophages and lead to M2 macrophage polarization via FAK-STAT3 pathway, thus promoting the metastasis of ESCC. These findings indicated that IL-32 could serve as a potential therapeutic target in patients with ESCC.