Abstract LB508: The development of HK013, a bispecific antibody targeting MSLN and CD137, for the treatment of MSLN+ solid tumors
Authors: Xu Xing , Zhang Dayan , Liu Wenting , Shen Guodong , Cheng Liansheng 
Published: June 2022

Abstract
Mesothelin (MSLN) is a 70 KDa cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in a variety of cancers, including mesothelioma, ovarian cancer, pancreatic cancer and breast cancer. Binding of MSLN with CA125/MUC16 promotes cell proliferation, local infiltration and metastasis. CD137 is mainly expressed on the surface of activated T cells, NK cells and some other immune cells, mediating the costimulatory signal to enhance the function of T cells and improve the anti-tumor ability of immune cells. Here, we report the development of a bispecific antibody, HK013, targeting both MSLN and CD137 for the treatment of solid tumors. The bispecific antibody HK013 was generated by introducing an anti-MSLN nanobody to the C-terminus of the heavy chain of an anti-CD137 antibody. Two stages were involved: first, we compared the MSLN nanobodies with different binding epitopes to select the optimal candidate. Two nanobodies, R2G12 and R3C7, with the binding affinity of 10-9M to human MSLN were selected. R3C7 blocks the binding of MORAb-009 to MSLN, while R2G12 does not. However, the bi-specific antibody containing R2G12 exhibited stronger anti-tumor activity than that containing R3C7 in both N87 and H226 mouse xenograft models. We then determined the best isotype and format of the bispecific antibody. While IgG1 or IgG4 (FALA) were selected as the candidate isotypes, the anti-MSLN VHH were linked to the N-terminal or C-terminal of the heavy chain of anti-CD137 antibody. Results showed that in the presence of H226 cells, IFN-γ secretion of CD8+T cells was slightly higher as triggered by IgG1 isotype than that by IgG4. However, IgG1-based molecules were not as potent as those in IgG4 in N87 and H226 xenograft models. Moreover, IgG1-based molecules showed cytotoxicity on CD4+T and CD8+T cells while those in IgG4 did not. In addition, HK013 exhibited the minimal ability of non-specific activation of CD137 signaling pathway by Fc receptor in a NFκB-luciferase reporter assay.These data indicate that our bispecific antibodies containing MSLN nanobodies with different epitopes exhibit varied antitumor activity, and antibodies with similar epitopes to MORAb-009 show poor antitumor activity. Molecules in IgG1 may potentially reduce the antitumor activity due to the cytotoxicity to the effector T cells. The bispecific formats are related to the Fc receptor-mediated non-specific activation of CD137 signal, which may potentially cause safety issue. The strong antitumor activity and the lowest non-specific activation of CD137 signal of HK013 makes it the candidate worthwhile of further evaluation of its safety and efficacy.
Citation Format: Xing Xu, Dayan Zhang, Wenting Liu, Guodong Shen, Liansheng Cheng. The development of HK013, a bispecific antibody targeting MSLN and CD137, for the treatment of MSLN+ solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB508.