Development Of 4-1BB And CTLA-4 Targeted Bispecific Antibodies Based On RenLite Mice

Tumorigenesis is cogently associated with immune escape from immune surveillance.Regulatory T cells(Tregs),as immune suppressive cells,are involved in tumor development and progression by inhibiting antitumor immunity through several mechanisms.Tregs can inbibition of costimulatory signals by competitively bind to B7 ligands on Antigen-presenting cell(APC)via surface-highly expressed Cytotoxic T lymphocyte associated antigen 4(CTLA-4),or consumption of interleukin-2(IL-2)by high affinity of CD25 expression,and the release of immunosuppressive cytokines that inhibit the activation and response of T cells.A high infiltration by Tregs in tumor microenvironment(TME)is associated with poor survival in various types of cancer.Therefore,it is feasible to deplete tumor-infiltrating Tregs and promote T cell activation as a therapeutic strategy.CTLA-4,also known as CD152,is a well-known immunosuppressive checkpoint.Ipilimumab(Yervoy),which is an anti-CTLA-4 monoclonal antibody,was the first immune checkpoint inhibitor to be approved by the US Food and Drug Administration.It achieves anti-tumor efficacy by blocking the CTLA-4/B7 axis,but subsequent studies further demonstrated potent antitumor activity,with CTLA-4 monoclonal antibodies capable of efficiently depleting tumor Tregs through Fc-mediated effector functions such as ADCC activity.However,CTLA-4 is expressed on Tregs in a variety of normal tissue;thus,therapies that depletion of CTLA-4 expressing Tregs may have unanticipated adverse effects.4-1BB(CD137)is a costimulatory receptor that belongs to the tumor necrosis factor(TNF)receptor superfamily,is also highly expressed in tumor-infiltrating Treg cells.Coexpression of 4-1BB and Fox P3 was associated with poorer survival outcomes,again across multiple cancer types.Unlike CTLA-4,4-1BB in mainly express on tumor-infiltrating Tregs rather than normal tissue.Bispecific antibody(Bs Ab)can target two different antigens or different epitopes of the same antigen.Bs Ab based immunotherapies have gained momentum in preclinical and clinical investigations.Therefore,develop a bs Ab targeting CTLA-4 and 4-1BB can be a promising strategy to specifically target tumor-infiltrating Tregs,thereby enhance the anti-tumor activity,reducing off-target toxicity and improving safety.Objective:To develop a CTLA-4/4-1BB Bs Ab targeting Tregs in the tumor microenvironment.Methods:The Bs Abs plasmid of CTLA-4/4-1BB were constructed then transiently transfected by liposome and expressed in CHO cells.After purified by protein A affinity chromatography,the concentration was determined by microplate reader and analysis by SDS-PAGE.The binding affinity of bs Abs were measured by surface plasmon resonance and flow cytometry.The bioactivity of 4-1BB activate function and ADCC function were assessed by luciferase reporter assay.The antitumor activity of bs Abs were explored in B-h CTLA-4/h4-1BB double-humanized mice.Results:CTLA-4/4-1BB Bs Abs were successfully constructed and expressed,then exhibited binding affinity to dual targets.In the reporter cell line of 4-1BB agonist activity assay and the ADCC function assay,the results showed that the CTLA-4/4-1BB Bs Abs can activate 4-1BB signaling by CTLA-4 as well as FcγRIIB cross-linking.However,Urelumab-analog showed 4-1BB activation activity without cross-linking,which indicated that CTLA-4/4-1BB Bs Abs had more specific activation activity than Urelumab-analog.Meanwhile,the CTLA-4/4-1BB Bs Abs exhibited stronger ADCC activity than the Ipilimumab-analog.In vivo efficacy assay,the result demonstrated that the CTLA-4/4-1BB Bs Abs had more potent antitumor efficacy than Ipilimumab-analog.Furthermore,Fc-mediated effector function play an important role in the antitumor activity of CTLA-4/4-1BB Bs Abs.Conclusion:In this study,we design and construct the CTLA-4/4-1BB Bs Abs based on common light chain mice platform.This Bs Abs exhibits 4-1BB simulation when CTLA-4 and FcγRIIB are engaged,also shows stronger ADCC activity than the Ipilimumab-analog.Moreover,CTLA-4/4-1BB Bs Ab exhibits potent antitumor activity in h CTLA-4/h4-1BB double-humanized/MC38 tumor model,and depends on the Fc-mediated effector function.In conclusion,our data suggest that CTLA-4/4-1BB Bs Ab targeting the Tregs in tumor microenvironment might be a promising immunotherapeutic agent against human cancers.