Facile Generation of Potent Bispecific Fab via Sortase A and Click Chemistry for Cancer Immunotherapy

Author:

Bai Xuefei,Liu Wenhui,Jin Shijie,Zhao WenbinORCID,Xu Yingchun,Zhou Zhan,Chen ShuqingORCID,Pan Liqiang

Abstract

Bispecific antibodies (BsAbs) for T cell engagement have shown great promise in cancer immunotherapy, and their clinical applications have been proven in treating hematological malignance. Bispecific antibody binding fragment (BiFab) represents a promising platform for generating non-Fc bispecific antibodies. However, the generation of BiFab is still challenging, especially by means of chemical conjugation. More conjugation strategies, e.g., enzymatic conjugation and modular BiFab preparation, are needed to improve the robustness and flexibility of BiFab preparation. We successfully used chemo-enzymatic conjugation approach to generate bispecific antibody (i.e., BiFab) with Fabs from full-length antibodies. Paired click handles (e.g., N3 and DBCO) was introduced to the C-terminal LPETG tag of Fabs via sortase A mediated transpeptidation, followed by site-specific conjugation between two click handle-modified Fabs for BiFab generation. Both BiFabCD20/CD3 (EC50 = 0.26 ng/mL) and BiFabHer2/CD3 exhibited superior efficacy in mediating T cells, from either PBMC or ATC, to kill target tumor cell lines while spared antigen-negative tumor cells in vitro. The BiFabCD20/CD3 also efficiently inhibited CD20-positive tumor growth in mouse xenograft model. We have established a facile sortase A-mediated click handle installation to generate homogeneous and functional BiFabs. The exemplary BiFabs against different targets showed superior efficacy in redirecting and activating T cells to specifically kill target tumor cells, demonstrating the robustness of sortase A-mediated “bio-click” chemistry in generating various potent BiFabs. This approach also holds promise for further efficient construction of a Fab derivative library for personalized tumor immunotherapy in the future.

Funder

National Natural Science Foundation of China

key research and development project of Zhejiang province

Fundamental Research Funds for the Central Universities

Publisher

MDPI AG

Subject

Cancer Research,Oncology

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