Anti-TGF-β/PD-L1 Bispecific Antibody Reprograms Immune Microenvironment And Exerts Synergistic Antitumor Effects With Manganese

Objective:Therapeutic antibodies targeting PD-1/PD-L1 pathway induce potent and durable antitumor immunity in various cancers.However,only a portion of patients could benefit from anti-PD-1/PD-L1 treatment.TGF-βis a negative regulator of antitumor immune response,which hampers the effect of anti-PD-1/PD-L1 and promotes drug resistance.This part focused on the construction,expression,and antitumor activity of the anti-TGF-β/PD-L1 bispecific antibody.Methods:Based on the Check-BODY^TM development platform,we designed and constructed the anti-TGF-β/PD-L1 bispecific antibody named YM101.We evaluated the bioactivity of the anti-TGF-βmoiety by Smad-luciferase reporter assay.The bioactivity of the anti-PD-L1 moiety was assessed by CFSE dilution and flow cytometry.The antitumor effect of YM101 was explored in EMT-6,CT26,and 3LL models.RNA-seq,flow cytometry,and immunohistochemical staining were used to investigate the alterations in the tumor microenvironment（TME）.Results:YM101 specifically bound to three TGF-βisoforms and murine PD-L1.In vitro experiments demonstrated YM101 effectively blocked TGF-βand PD-1/PD-L1 signaling transduction.Besides,in vivo experiments showed YM101 had more potent antitumor activity than did anti-TGF-βand anti-PD-L1 monotherapy.Mechanistically,YM101 promoted the transformation toward inflamed tumors:elevating the quantities of tumor-infiltrating dendritic cells and lymphocytes,increasing M1/M2 ratio,improving cytokine production.The normalized tumor microenvironment and boosted antitumor immunity contribute to the enhanced antitumor activity of YM101.Conclusion:YM101 could simultaneously block TGF-βand PD-1/PD-L1 signaling,with superior antitumor activity relative to the anti-TGF-βor anti-PD-1/PD-L1 monotherapies.Objective: Our previous work demonstrated that YM101 effectively relieved anti-PD-L1 resistance in immune-excluded tumors.However,the effect of YM101 was hampered in immune-desert models.As a natural stimulator of interferon genes（STING）agonist,bivalent manganese(Mn²⁺)could enhance antigen presentation of dendritic cells（DCs）,which might improve the activity of YM101.Methods:Mn²⁺-mediated STING activation was confirmed by enzyme-linked immunosorbent assay and western blotting.DC maturation was determined by flow cytometry.The in vitro synergistic effect of Mn²⁺ with YM101 was validated by one-way mixed lymphocyte reaction,cytokine detection,and CFSE dilution assay.The efficacy of Mn²⁺ combined with YM101 was evaluated in multiple murine models.RNA-seq,flow cytometry,and immunofluorescent staining were utilized to explore the changes in the TME.Results:Mn²⁺ activated STING pathway and promoted DC maturation.In vitro experiments indicated that Mn²⁺ synergized with YM101 in T cell activation.Moreover,in multiple noninflamed tumor models,Mn²⁺ combined with YM101 treatment exhibited a potent antitumor effect and prolonged survival.Compared to monotherapy,Mn²⁺ plus YM101 had a broader spectrum and higher magnitude of anticancer activities.Mechanistically,Mn²⁺ plus YM101 strategy simultaneously regulated multiple factors in the TME and drove the transformation from non-inflamed to immune-inflamed tumors.The investigation in the TME indicated Mn²⁺ plus YM101 strategy activated innate and adaptive immunity,enhanced cancer antigen presentation,and upregulated the densities and functions of tumor-infiltrating lymphocytes.This combination normalized TME and reinvigorated antitumor immunity in both immuneexcluded and immune-desert tumors.Conclusion: Mn²⁺ combined with YM101 has enhanced antitumor activity,effectively retarding tumor growth and prolonging survival in multiple murine tumor models.This novel combination therapy might be a universal strategy for inflamed and non-inflamed tumors in the future.