Biparatopic Nanobody-Based Immunosorbent for the Highly Selective Elimination of Tumor Necrosis Factor-

Removing the overexpressed TNF-alpha by hemoperfusion positively affects clinical treatments for diseases such as autoimmune disease and sepsis. However, clearance ratios of adsorbents targeting TNF-alpha were limited by the extremely low concentration of TNF-alpha (mostly <1000 ng/L in sepsis) and hydrophobic interactions. In this work, biparatopic nanobodies (NbC21) with a high affinity of 19.9 pM, which bind to two distinct sites of TNF-alpha, were constructed as high-affinity ligands for the immunosorbent. The theoretical maximum adsorption capacity estimated from the Langmuir isotherm was up to 18.22 mg/g gel. The prepared immunosorbent (NbC21-sorbent) had an outstanding TNF-alpha clearance ratio of approximately 96% during the dynamic adsorption test, with a sorbent-to-serum ratio of 1:1000. Additionally, it demonstrated favorable hemocompatibility and a prolonged storage capability. The results indicated that the biparatopic nanobody immunosorbent exhibited significant potential for clinical applications as it met the stringent criteria for both efficacy and safety.