Humanization of the antigen-recognition domain does not impinge on the antigen-binding, cytokine secretion, and antitumor reactivity of humanized nanobody-based CD19-redirected CAR-T cells

Abstract

Abstract Background The immunogenicity of the antigen-recognition domains of chimeric antigen receptor (CAR)-T cells leads to immune responses that may compromise the antitumor effects of the adoptively transferred T cells. Herein, we attempt to humanize a CD19-specific VHH (named H85) using in silico techniques and investigate the impact of antigen-recognition domain humanization on CAR expression and density, cytokine secretion, and cytolytic reactivity of CAR-T cells based on the humanized VHH. Methods H85 was humanized (named HuH85), and then HuH85 was compared with H85 in terms of conformational structure, physicochemical properties, antigenicity and immunogenicity, solubility, flexibility, stability, and CD19-binding capacity using in silico techniques. Next, H85CAR-T cells and HuH85CAR-T cells were developed and CAR expression and surface density were assessed via flow cytometry. Ultimately, the antitumor reactivity and secreted levels of IFN-γ, IL-2, and TNF-α were assessed following the co-cultivation of the CAR-T cells with Ramos, Namalwa, and K562 cells. Results In silico findings demonstrated no negative impacts on HuH85 as a result of humanization. Ultimately, H85CAR and HuH85CAR could be surface-expressed on transduced T cells at comparable levels as assessed via mean fluorescence intensity. Moreover, H85CAR-T cells and HuH85CAR-T cells mediated comparable antitumor effects against Ramos and Namalwa cells and secreted comparable levels of IFN-γ, IL-2, and TNF-α following co-cultivation. Conclusion HuH85 can be used to develop immunotherapeutics against CD19-associated hematologic malignancies. Moreover, HuH85CAR-T cells must be further investigated in vitro and in preclinical xenograft models of CD19+ leukemias and lymphomas before advancing into clinical trials.