Research Projects

Human NLRP3 inflammasome pathway confers innate immunity against infections and metabolic distress by promoting the release of inflammatory cytokines and inducing the PANoptosis, a programmed inflammatory cell death. However, the dysregulation of NLRP3 inflammasome pathway leads to severe inflammatory and autoimmune diseases. Although various small-molecule inhibitors have been developed to inhibit the abnormal activation of the inflammasome pathway, none of them were successful due to their off-target effects and non-specific nature. Thus, the development of very specific inhibitors of NLRP3 inflammasome pathway is essential to treat the inflammasome associated diseases. Nanobody engineering is the rapidly emerging technologies for the development of diagnostics and therapeutics for various diseases. Nanobodies, the single domain antibodies usually raised in camelids, are very small in size (15 kDa) and have higher stability, specificity, and tissue penetration over monoclonal antibodies (150 kDa). In addition, the production costs of nanobodies are very less due to their recombinant production in E. coli. Nanobody engineering enhances the natural potential of nanobodies by designing effective nanobody-based therapeutics. This research proposal is aimed to apply the nanobody engineering strategies to develop the humanized bispecific nanobody therapeutics for inhibiting the abnormal activation of NLRP3 inflammasome pathway in inflammatory and autoimmune diseases. The recently generated inhibiting nanobodies of ASC and GSDMD proteins, the key mediators of inflammasome pathway will be taken for humanization followed by fusing them together with linker sequence to develop bispecific nanobody with an enhanced therapeutic potential. Nanobody humanization will be carried out using a universal humanized nanobody scaffold and rational design. Next, these humanized nanobodies will be used for developing bispecific nanobodies by testing various fusion sequences to link them together. In addition, the generated potent humanized bispecific nanobody will be taken for structural characterization followed by protein engineering for further improving its affinity and stability. The engineered nanobodies generated in this proposal will be recombinantly produced in E. coli, purified to homogeneity and evaluated for their affinities by nanobody ELISA and surface plasmon resonance, and tested for their efficiency of inflammasome inhibition in human macrophage cell line. Thus, this proposal results in development of bispecific nanobody therapeutics for effective inhibition of abnormal activation of human NLRP3 inflammasome pathway in inflammatory and autoimmune diseases.