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

Removing the overexpressed TNF-α by hemoperfusion positively affects clinical treatments for diseases such as autoimmune disease and sepsis. However, clearance ratios of adsorbents targeting TNF-α were limited by the extremely low concentration of TNF-α (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-α, 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-α 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.

Nanobody-functionalized conduit with built-in static mixer for specific elimination of cytokines in hemoperfusion.

Removing excessively produced cytokines is of paramount significance in blood purification therapy for hypercytokinemia-associated diseases. In this study, we devised a conduit that is modified with nanobodies (Nb) and incorporates static mixers (Nb-SMC) to eliminate surplus cytokines from the bloodstream. The low-pressure-drop (LPD) static mixer, with each unit featuring two 90°-crossed blades, was strategically arranged in a tessellated pattern on the inner wall of the conduit to induce turbulent mixing effects during the flow of blood. This arrangement enhances mass transfer and molecular diffusion, thereby assisting in the identification and elimination of cytokines. By utilizing computational fluid dynamics (CFD) studies, the Nb-SMC was rationally designed and prepared, ensuring an optimal interval between two mixer units (H/G = 2.5). The resulting Nb-SMC exhibited a remarkable selective clearance of IL-17A, reaching up to 85 %. Additionally, the process of Nb immobilization could be adjusted to achieve the simultaneous removal of multiple cytokines from the bloodstream. Notably, our Nb-SMC displayed good blood compatibility without potential adverse effects on the composition of human blood. As the sole documented static mixer-integrated conduit capable of selectively eliminating cytokines at their physiological concentrations, it holds promise in the clinical potential for hypercytokinemia in high-risk patients. STATEMENT OF SIGNIFICANCE: High-efficient cytokines removal in critical care still remains a challenge. The conduit technique we proposed here is a brand-new strategy for cytokines removal in blood purification therapy. On the one hand, nanobody endows the conduit with specific recognition of cytokine, on the other hand, the build-in static mixer enhances the diffusion of antigenic cytokine to the ligand. The combination of these two has jointly achieved the efficient and specific removal of cytokine. This innovative material is the only reported artificial biomaterial capable of selectively eliminating multiple cytokines under conditions close to clinical practice. It has the potential to improve outcomes for patients with hypercytokinemia and reduce the risk of adverse events associated with current treatment modalities.

Nanobodies as solubilization chaperones for the expression and purification of inclusion-body prone proteins.

Here, we report a new protocol for enhancing the soluble expression of inclusion body (IB)-prone proteins in E. coli using nanobodies (Nbs) as a molecular-specific chaperone. The specific intracellular binding between the cognate-Nbs and the antigen is successfully achieved and enables the formation of a soluble Nb-antigen complex in E. coli. We further expand this method by adding an epitope tag (EPEA-tag) to the target proteins, and the anti-EPEA Nb was intended to act as the chaperone for in vivo binding with the EPEA tag. Such substitution may develop a "multi-specific" Nb-chaperone that can simultaneously and effectively cope with different IB proteins of interest.

Nanobody-loaded immunosorbent for highly-specific removal of interleukin-17A from blood.

Elimination of overproduced cytokines from blood can relieve immune system disorders caused by hypercytokinemia. Due to the central roles of interleukin-17A (IL-17A) plays in regulating the immunity and inflammatory responses in humans, here, a novel immunosorbent containing anti-IL-17A nanobodies (Nbs) was constructed for IL-17A removal from blood. The theoretical maximum adsorption capacity estimated from the Langmuir isotherm is up to 11.55 mg/g gel, which is almost consistent with the saturated adsorption capacity determined in dynamic adsorption. The in vitro plasma perfusion test demonstrated a remarkable adsorptive performance of the Nb-coupled sorbent since more than 75% IL-17A could be eliminated under the plasma/sorbent ratio of 1000:1. These results indicated the Nb-loaded immunosorbent can provide a simple and economic platform technology for immunoaffinity depletion of single or even multiple cytokines from plasma.