Osteoarthritis (OA) is the most common joint disease that causes local inflammation and pain, significantly reducing the quality of life and normal social activities of patients. Currently, there are no disease-modifying OA drugs (DMOADs) available, and treatment relies on pain relief agents or arthroplasty. To address this significant unmet medical need, we aimed to develop monoclonal antibodies that can block the osteoclast-associated receptor (OSCAR). Our recent study has revealed the importance of OSCAR in OA pathogenesis as a novel catabolic regulator that induces chondrocyte apoptosis and accelerates articular cartilage destruction. It was also shown that blocking OSCAR with a soluble OSCAR decoy receptor ameliorated OA in animal models. In this study, OSCAR-neutralizing monoclonal antibodies were isolated and optimized by phage display. These antibodies bind to and directly neutralize OSCAR, unlike the decoy receptor, which binds to the ubiquitously expressed collagen and may result in reduced efficacy or deleterious off-target effects. The DMOAD potential of the anti-OSCAR antibodies was assessed with in vitro cell-based assays and an in vivo OA model. The results demonstrated that the anti-OSCAR antibodies significantly reduced cartilage destruction and other OA signs, such as subchondral bone plate sclerosis and loss of hyaline cartilage. Hence, blocking OSCAR with a monoclonal antibody could be a promising treatment strategy for OA.

The protein therapeutics market, including antibody and fusion proteins, has experienced steady growth over the past decade, underscoring the importance of optimizing amino acid sequences. In our previous study, we developed a fusion protein, R31, which combines retinol-binding protein (RBP) with albumin domains IIIA and IB, linked by a sequence (AAAA), and includes an additional disulfide bond (N227C-V254C) in IIIA. This fusion protein effectively inhibited hepatic stellate cell activation. In this study, we further optimized the sequence. The G176K mutation at the C-terminus of RBP altered the initiation site of the first α-helix in domain IIIA, shifting it from P182 to K176, and promoted polar interactions between K176 and adjacent residues, enhancing the rigidity of the RBP/IIIA interface. The introduction of an additional disulfide bond (V231C/Y250C) connecting helices 3 and 4 in IIIA resulted in a three-fold increase in productivity and a 2 °C improvement in thermal stability compared to R31. Furthermore, combining the G176K mutation with V231C/Y250C further enhanced both productivity and anti-fibrotic activity. These findings suggest that the enhanced stability of domain IIIA, conferred by V231C/Y250C, along with the increased rigidity of the RBP/IIIA interface, optimizes interdomain distance and alignment, facilitating proper protein folding.

Antibody–drug conjugates (ADCs) have received renewed attention in oncology following the clinical and commercial success of agents such as trastuzumab deruxtecan (Enhertu^®), which underscored the clinical importance of coordinated optimization of the antibody, linker, payload, and conjugation strategy. Among these parameters, conjugation strategy strongly influences drug-to-antibody ratio (DAR) distribution, pharmacokinetics, manufacturability, and safety. Over the past two decades, ADC conjugation technologies have evolved from first-generation stochastic modification approaches to second-generation site-specific engineering strategies and, more recently, to third-generation site-selective platforms applicable to native antibodies. First-generation approaches remain the most clinically validated and have the strongest regulatory precedent despite their intrinsic heterogeneity. Second-generation approaches improve positional control and DAR homogeneity, but often increase demands on antibody engineering, expression, and process complexity. Third-generation approaches aim to preserve controlled conjugation while reducing sequence-engineering requirements, yet remain limited by a restricted number of accessible sites and less extensive clinical and regulatory experience. In this review, we summarize the evolution of ADC conjugation technologies, highlight representative clinical and translational examples, and discuss the major trade-offs of each platform. We also propose a practical selection framework based on payload properties, required DAR control, antibody stability, manufacturability, and regulatory considerations, with relevance to next-generation ADCs including dual-payload and bispecific formats. Overall, conjugation strategy should be selected according to the scientific, manufacturing, and regulatory requirements of each ADC program rather than on the basis of a presumed technological hierarchy.