The mean nearest distance was scaled by the distance between the corresponding tumor border to tumor center . RNA-seq assay The total RNA of EMT-6 tumors was extracted by Trizol, as previously described . the Check-BODY? technology platform, we developed an anti-TGF-/PD-L1 bispecific antibody YM101. The bioactivity of the anti-TGF- moiety was determined by Smad-luciferase reporter assay, transwell assay, western blotting, CCK-8, and circulation cytometry. The bioactivity of the anti-PD-L1 moiety was measured by T cell activation assays. EMT-6, CT26, and 3LL tumor models were used to investigate the anti-tumor activity of YM101 in [Ser25] Protein Kinase C (19-31) vivo. RNA-seq, immunohistochemical staining, and circulation cytometry were utilized to analyze the effect of YM101 around the tumor microenvironment. Results YM101 could bind to TGF- and PD-L1 specifically. In vitro experiments showed that YM101 effectively counteracted the biological effects of TGF- and PD-1/PD-L1 pathway, including activating Smad signaling, inducing epithelial-mesenchymal transition, and immunosuppression. Besides, in vivo experiments indicated the anti-tumor activity of YM101 was superior to anti-TGF- and anti-PD-L1 monotherapies. Mechanistically, YM101 promoted the formation of warm tumor: increasing the numbers of tumor infiltrating lymphocytes and dendritic cells, elevating the ratio of M1/M2, and enhancing cytokine production in T cells. This normalized tumor immune microenvironment and enhanced anti-tumor immune response might contribute to the strong anti-tumor effect of YM101. Conclusion Our results exhibited that YM101 could simultaneously block TGF- and PD-L1 pathways and experienced a superior anti-tumor effect compared to the monotherapies. gene expression is higher in the nonresponders tumor tissues . Correspondingly, the dual blockade of PD-1/PD-L1 and TGF- has a synergistic anti-tumor activity [42, 43]. Given that the immunosuppressive effects of the PD-1/PD-L1 axis and TGF- are impartial and complementary, it is rational to block the TGF- transmission to enhance the efficacy of anti-PD-1/PD-L1 and overcome treatment resistance . To enhance the anti-tumor activity of anti-PD-1/PD-L1 therapies, we developed an anti-TGF-/PD-L1 bispecific antibody YM101, which could simultaneously block the PD-1/PD-L1 and TGF- pathways. Check-BODY? platform is designed by Wuhan YZY Biopharma Co., Ltd for the development of symmetric tetravalency bispecific antibodies. Check-BODY? platform is characterized by high production yield, easy purification, and high structural stability. YM101 is constructed based on the Check-BODY? technology platform. In the present study, [Ser25] Protein Kinase C (19-31) we explored the biochemistry characteristics of YM101 in vitro and assessed its anti-tumor activity in vivo. Materials and methods Cell lines and antibodies CT26 (murine colon cancer cell), EMT-6 (murine breast malignancy cell), 4T1 (murine breast malignancy cell), A549 (human lung malignancy cell), and NCI-H358 (human lung malignancy cell) were cultured in RPMI-1640 (Gibco) made up of 10% fetal bovine serum (FBS) (Biological Industries). HT-2 (murine T cell) and CTLL-2 (murine T cell) were cultured in RPMI-1640 (ATCC modification, made up of glutathione and vitamins) (A10491-01, Gibco) with 10% FBS and 200?IU/ml interleukin-2 (IL-2, Beijing Fourrings). Main murine T cells were isolated from C57BL/6 mouse-derived splenocytes and cultured in RPMI-1640 made up of 10% FBS. NF639 (murine breast malignancy cell) and 3LL (murine lung malignancy cell) were cultured PPARGC1 in DMEM (Gibco) with 10% FBS. The therapeutic antibodies and isotype control antibody used in the present study included YM101, human IgG, anti-TGF-, and anti-PD-L1. The anti-TGF- antibody was constructed based on GC1008 . The anti-PD-L1 antibody was constructed based on the sequence of a chicken anti-PD-L1 single chain variable fragments (scFv) (developed by Jeremy et al.) . All therapeutic antibodies and the human IgG were provided by Wuhan YZY Biopharma Co., Ltd. Reduced and non-reduced sodium dodecyl sulfateCpolyacrylamide gel electrophoresis (SDS-PAGE) The prepared YM101 was analyzed using SDS-PAGE and Coomassie Amazing Blue staining. To verify the purity and molecular excess [Ser25] Protein Kinase C (19-31) weight of YM101, reduced and non-reduced SDS-PAGE were conducted as previously explained . After Coomassie Amazing Blue staining and decolorization, the images of the SDS-PAGE gels were captured with ChemiDoc MP [Ser25] Protein Kinase C (19-31) Imaging system (Bio-Rad). Capillary electrophoresis with sodium dodecylsulfate Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) assay was performed following the standard protocol . For the non-reduced CE-SDS, 200?g sample was mixed with 5?l Iodoacetamide (0.5?M) and 1?l 10 KD Internal Standard. After incubation at room heat for 30?min, the prepared combination was diluted.