Construction of Switch Modules for CAR-T Cell Treatment Using a Site-Specific Conjugation System.

Chimeric antigen receptor T-cell (CAR-T cell) therapy has become a promising treatment option for B-cell hematological tumors. However, few optional target antigens and disease relapse due to loss of target antigens limit the broad clinical applicability of CAR-T cells. Here, we conjugated an antibody (Ab) fusion protein, consisting of an Ab domain and a SpyCatcher domain, with the FITC-SpyTag (FITC-ST) peptide to form a bispecific safety switch module using a site-specific conjugation system. We applied the safety switch module to target CD19, PDL1, or Her2-expressing tumor cells by constructing FMC63 (anti-CD19), antiPDL1, or ZHER (anti-Her2)-FITC-ST, respectively. Those switch modules significantly improved the cytotoxic effects of anti-FITC CAR-T cells on tumor cells. Additionally, we obtained the purified CD8+ T cells by optimizing a shorter version of the CD8-binding aptamer to generate anti-FITC CD8-CAR-T cells, which combined with the CD4-FITC-ST switch module (anti-CD4) to eliminate the CD4-positive tumor cells in vitro and in vivo. Overall, we established a novel safety switch module by site-specific conjugation to enhance the antitumor function of universal CAR-T cells, thereby expanding the application scope of CAR-T therapy and improving its safety and efficacy.

Construction of a versatile fusion protein for targeted therapy and immunotherapy.

Antibody (Ab)-based drugs have been widely used in targeted therapies and immunotherapies, leading to significant improvements in tumor therapy. However, the failure of Ab therapy due to the loss of target antigens or Ab modifications that affect its function limits its application. In this study, we expanded the application of antibodies (Abs) by constructing a fusion protein as a versatile tool for Ab-based target cell detection, delivery, and therapy. We first constructed a SpaC Catcher (SpaCC for short) fusion protein that included the C domains of Staphylococcal protein A (SpaC) and the SpyCatcher. SpaCC conjugated with SpyTag-X (S-X) to form the SpaCC-S-X complex, which binds non-covalently to an Ab to form the Ab-SpaCC-S-X protein complex. The "X" can be a variety of small molecules such as fluoresceins, cell-penetrating peptide TAT, Monomethyl auristatin E (MMAE), and DNA. We found that Ab-SpaCC-S-FITC(-TAT) could be used for target cell detection and delivery. Besides, we synthesized the Ab-SpaCC-SN3-MMAE complex by linking Ab with MMAE by SpaCC, which improved the cytotoxicity of small molecule toxins. Moreover, we constructed an Ab-DNA complex by conjugating SpaCC with the aptamer (Ap) and found that Ab-SpaCC-SN3-Ap boosted the tumor-killing function of T-cells by retargeting tumor cells. Thus, we developed a multifunctional tool that could be used for targeted therapies and immunotherapies, providing a cheap and convenient novel drug development strategy.

Application of a Novel Aptamer Beacon for Rapid Detection of IgG1 Antibody Drugs.

Antibody drugs have been widely used to treat many diseases and are the fastest-growing drug class. IgG1 is the most common type of antibody because of its good serum stability; however, effective methods for the rapid detection of IgG1-type antibodies are lacking. In this study, we designed two aptamer molecules derived from the reported aptamer probe that has been proven to bind to the Fc fragment of the IgG1 antibody. The results showed that Fc-1S could specifically bind to the human IgG1 Fc proteins. In addition, we modified the structure of Fc-1S and constructed three aptamer molecular beacons that could quantitatively detect IgG1-type antibodies within a short time. Furthermore, we unveiled that the Fc-1S37R beacon has the highest sensitivity for IgG1-type antibodies with a detection limit of 48.82813 ng/mL and can accurately detect serum antibody concentrations in vivo with consistent results to ELISA. Therefore, Fc-1S37R is an efficient method for the production monitoring and quality control of IgG1-type antibodies to enable the large-scale production and application of antibody drugs.