Agglutinin Chip Screening of B Cell Surface Biomarkers in Hashimoto's Thyroiditis for Therapeutic Targeting

Jun-ling Ren¹Xiao-ming Wang^2*

• ¹Anhui Medical University, Hefei, China
• ²The first affiliated hospital of Wannan Medical College, Wuhu, China

Background and objectives: Hashimoto’s thyroiditis (HT) is a chronic autoimmune thyroid disorder characterized by B lymphocyte dysregulation and the production of autoantibodies. This study aimed to evaluate a targeted B cell depletion strategy by identifying and validating a disease-associated membrane glycoprotein selectively expressed on B cells. Methods: B lymphocytes were isolated from peripheral blood samples of 32 individuals with HT and 40 age- and sex-matched healthy controls (HC). Membrane glycoprotein expression was profiled using a 38-lectin microarray, followed by differential analysis via liquid chromatography-tandem mass spectrometry. Tetraspanin-33 (TSPAN33) was identified for further investigation based on its expression pattern. Recombinant TSPAN33 protein was used as the target in a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process to generate high-affinity DNA aptamers. This aptamer was chemically conjugated with a CD20 monoclonal antibody (rituximab) using bismaleimide crosslinking to generate a bispecific complex capable of selectively depleting pathogenic B lymphocytes. Binding specificity, complement-dependent cytotoxicity (CDC), and B cell depletion efficacy were evaluated using flow cytometry, confocal microscopy, and in vitro functional assays. Results: The TSPAN33 aptamer–rituximab conjugate exhibited selective binding to activated B lymphocytes and induced significant cell lysis via CDC. The observed B cell depletion supports the therapeutic potential of this bispecific approach for modulating aberrant B cell activity in HT and other B cell–mediated autoimmune conditions. Conclusion: The development of a bispecific aptamer–antibody conjugate targeting TSPAN33 offers a promising strategy for selective B cell depletion. Its therapeutic efficacy depends on the expression of the target glycoprotein. Further studies are warranted to optimize the conjugate design, assess in vivo functionality, and evaluate complement activation for clinical application.