An iRGD Peptide Fused Superantigen Mutant Induced Tumor-Targeting and T Lymphocyte Infiltrating in Cancer Immunotherapy

Abstract

Backgrounds: As a promising tumor immunotherapy agent, superantigen mutant ST-4 has a notable ability to directly activate T lymphocytes. To accumulate ST-4 into tumor tissue, an iRGD peptide was fused to the carboxyl terminal of ST-4 to construct a fusion protein ST-4-iRGD. Methods: The tumor cell targeting ability of ST-4-iRGD was determined by flow cytometer and Laser scanning confocal microscope. ST-4-iRGD-mediated cytotoxicity of splenocytes towards tumor cells and tumor multicellular spheroid was detected by viability assay in vitro. The iRGD-mediated tumor tissue targeting and penetration was confirmed by In Vivo Imaging System and immunofluorescence analysis of solid tumor sections. In vivo anti-tumor activity of ST-4-iRGD were assessed using subcutaneous B16F10 xenografts and orthotopic 4T1 xenografts. Findings: ST-4-iRGD showed improved tumor targeting and enhanced growth inhibition on both monolayer culturing and multicellular spheroid of integrin αv and neuropilin-1 (NRP-1) positive tumor cells. Results of in vivo studies identified an increased accumulation of ST-4-iRGD in solid tumor as well as an enhanced solid tumor inhibition effects on subcutaneous B16F10 xenografts and orthotopic 4T1 xenografts. Importantly, ST-4-iRGD induced significantly improved CD4+ and CD8 + lymphocyte infiltration in solid tumor tissues, which is crucial for the effectiveness of tumor immunotherapy. Interpretation: Our data demonstrate that ST-4-iRGD could internalize into tumor microenvironment (TME) through an integrin αv targeting and Neuropilin-1-mediated penetrating way to activate the regional immunoreaction as well as enhance lymphocyte infiltration in TME, and ST-4-iRGDmight be a potential candidate for efficient cancer immunotherapy. Funding Statement: This work was supported by Strategic Priority Research Program of the Chinese Academy of Sciences Grant (XDA12020225), Science and Technology Plan Projects of Shenyang City Grants (Z17-7-013 and RC190060), and Liaoning Revitalization Talents Program (XLYC1807226). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All animal-involved experiments were carried out in compliance with the guidelines approved by the Animal Research Committee at the Chinese Academy of Sciences (Beijing, China).