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

SIMPLE SUMMARY: The formats of bispecific antibody have been investigated for many years to enhance the stability of the structure and anti-tumor efficacy. One of the formats combining two Fabs at their C termini provides unmodified variable region and comparable activity to other fragment-based bis...

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Detalles Bibliográficos
Autores principales: Bai, Xuefei, Liu, Wenhui, Jin, Shijie, Zhao, Wenbin, Xu, Yingchun, Zhou, Zhan, Chen, Shuqing, Pan, Liqiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8467688/
https://www.ncbi.nlm.nih.gov/pubmed/34572769
http://dx.doi.org/10.3390/cancers13184540
Descripción
Sumario:SIMPLE SUMMARY: The formats of bispecific antibody have been investigated for many years to enhance the stability of the structure and anti-tumor efficacy. One of the formats combining two Fabs at their C termini provides unmodified variable region and comparable activity to other fragment-based bispecific antibodies that are usually combined in a head-to-tail manner. However, the current strategy to produce the BiFab molecule is limited to a semisynthetic method that introduces unnatural amino acid to antibodies’ sequences during production. To improve the application of BiFab format in investigational biodrugs, we have applied sortase A-mediated “bio-click” chemistry to generate BiFab, for facile assembly of Fab molecules that have been expressed and stored as BiFab module candidates. The BiFabs made by our method stimulate T cell proliferation and activation with favorable in vitro and in vivo anti-tumor activit. Our results indicate that BiFab made by sortase A-mediated click chemistry could be used to efficiently generate various BiFabs with high potency, which further supports personalized tumor immunotherapy in the future. 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., N(3) 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 BiFab(CD20/CD3) (EC(50) = 0.26 ng/mL) and BiFab(Her2/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 BiFab(CD20/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.