Saturday, May 18, 2024
5/18/2024
Abstract Streptavidin (SA) and biotin have the strongest known binding interaction in nature, with a KD in the femtomolar range (4.8x10-14 M). This extraordinary binding affinity has led to its ubiquitous use in biomedical research and diagnostics. Though SA/biotin enjoys success in many applications, abundant endogenous biotin attenuates assay sensitivity. Moreover, SA is a highly immunogenic foreign protein, which limits its use in therapeutic applications such as pretargeted immunotherapy (PTI). Mirror-image SA and biotin (D-SA/L-biotin) offer an elegant solution to these problems. For example, D- Proteins are inert to L-proteases and therefore cannot be digested for MHC presentation to the immune system. This property means that D-SA will have greatly decreased immunogenicity and increased half-life compared to L-SA. Additionally, symmetry dictates that the mirror-image pair (D-SA/L-biotin) will have the exact same exceptional affinity as the natural pair (L-SA/D-biotin). Importantly, we have discovered that D-biotin has minimal binding to D-SA. Therefore, we propose that D-SA/L-biotin can be used as a biotin orthogonal streptavidin system (BOSS). We hypothesize that the orthogonality of BOSS, along with D-SA’s low immunogenicity, will overcome the limitations of natural SA/biotin. We will chemically synthesize D-SA using solid-phase peptide synthesis with D-amino acids and native chemical ligation. We will then replicate a previously reported SA PTI method using BOSS. We will attach this D-SA to the C-terminus of the antibody fragment (scFv) used in the previous study (scFv-D-SA) and will recombinantly express its L-counterpart (scFv-L-SA). This scFv binds to GD2, a cell-surface glycosphingolipid that is upregulated in neuroblastoma (NB). We will first measure the efficacy of scFv-D-SA in NB cells using fluorescence microscopy. We will attach a red fluorophore to D-biotin and magenta fluorophore to L-biotin to determine the relative binding of scFv-D-SA and scFv-L-SA to the NB cells and a panel of control cells. We will then test our BOSS PTI method in mice xenografted with NB cells using fluorescence-based full-body imaging to look for enhanced fluorescence localized around the tumor. We expect that BOSS will dramatically improve current pretargeting efforts. Moreover, given the ubiquity of biotin/SA-based systems throughout biomedical science, we also expect BOSS to be widely applicable and relevant to proximity labeling, diagnostic testing, and any method that suffers from SA immunogenicity and biotin interference.
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