Generation of aptamers for the selective detection and neutralization of soluble lymphotoxin alpha

Lymphotoxin alpha (LTα) is a potent inflammatory cytokine implicated in the pathophysiology of numerous human autoimmune and inflammatory diseases. Existing as a soluble homotrimer (LTα 3 ) or membrane-bound heterotrimer (LTα 1 β 2 ), the differential and distinct functions of lymphotoxin signaling have meant that selective targeting of the cytokine with traditional pharmacological agents has proven difficult. While monoclonal antibodies that can neutralize human LTα 3 in vivo do exist (e.g., pateclizumab), their efficacy and subsequent use within the clinic have been limited. This may be in part perhaps due to cross-reactivity between the homotrimeric and heterotrimeric forms, leading to LTα 3 -independent effects. Herein, we implement a counter-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol to enrich aptamers targeting LTα 3 but not LTα 1 β 2. Through a combination of in silico and in vitro tests, we also refine the aptamer sequences and test their ability to limit LT⍺ 3 -TNFR1 engagement and subsequent cellular cytotoxicity in L929 fibroblasts. We highlight the generation of 4 aptamer candidates that can selectively detect LTα 3 but not LTα 1 β 2 . Using rational design, we optimize the sequences and show that LTa1 and LTa5 can significantly reduce LTa 3 -TNFR1 engagement-associated cytotoxicity in vitro , highlighting their future therapeutic potential. These data highlight aptamers for the future investigation of lymphotoxin signaling, limiting off-target impacts on other LT⍺ 3 -dependent mechanisms.