Engineering and Partial Characterization of an Anti-IL-23/Anti-TNF-α Bispecific Domain Antibody Fused to Human Serum Albumin Domain for Inflammatory Disease Therapy.

Interleukin-23 (IL-23) and Tumor Necrosis Factor-alpha (TNF-α) are key pro-inflammatory cytokines that regulate the progression of inflammation. Currently approved anti-inflammatory biologics involve monospecific antibodies targeting either IL-23 or TNF-α. However, in multifaceted inflammatory conditions where multiple inflammatory pathways are involved, such monospecific antibodies have proven only partially effective. Hence, there is a need to develop biologics that can target both IL-23 and TNF-α proinflammatory cytokines together. An anti-IL-23/anti-TNF-α bispecific domain antibody (BiSpekDAb) was engineered by fusing an anti-IL-23 domain antibody and an anti-TNF-α domain antibody to the 3rd domain of human serum albumin (3dHSA), using flexible linkers (GGGGS). The BiSpekDAb coding genes were transformed into Pichia pastoris using electroporation and subsequently expressed in shake-flask cultures. In-vitro antigen-binding affinity and neutralization potential were determined by ELISA and cellular assays, respectively. A lab-scale process was established for production with a yield of 7 ± 3 mg/L. Functional in vitro characterization demonstrated that BiSpekDAb bound IL-23 and TNF-α with EC50 values of 11.39 ± 2.7 nM and 12.72 ± 8.25 nM, respectively. Moreover, BiSpekDAb significantly neutralized the biological effect of both cytokines. The successful design, expression, and lab-scale process with substantial yield demonstrates suitability for early-stage development and future scalability. BiSpekDAb with nanomolar dual-binding affinity controls IL-23 and TNF-α-mediated inflammation. BiSpekDAb shows promise to be an efficacious anti-inflammatory therapeutic, with applicability in inflammatory conditions, such as pulmonary inflammation (severe asthma).