PTK7 is a catalytically defective receptor protein tyrosine kinase upregulated in various cancers, including esophageal squamous cell carcinoma (ESCC). In previous studies, we observed a positive correlation between PTK7 expression levels and tumorigenicity in various ESCC cell lines and xenograft mice with ESCC KYSE-30 cells. In this study, we analyzed the effects of anti-PTK7 monoclonal antibodies (mAbs) on the tumorigenic activity in KYSE-30 cells and in mouse xenograft models. PTK7 mAb-32 and mAb-43 bind with a high affinity to the extracellular domain of PTK7. PTK7 mAbs significantly reduced three-dimensional cell proliferation, adhesion, wound healing, and migration. PTK7 mAbs also reduce chemotactic invasiveness by decreasing MMP-9 secretion. PTK7 mAbs decreased actin cytoskeleton levels in the cortical region of KYSE-30 cells. PTK7 mAbs reduced the phosphorylation of ERK, SRC, and FAK. In a mouse xenograft model of ESCC using KYSE-30 cells, PTK7 mAbs reduced tumor growth in terms of volume, weight, and the number of Ki-67-positive cells. These results demonstrated that PTK7 mAbs can inhibit the tumorigenicity of ESCC at the cellular level and in vivo by blocking the function of PTK7. Considering the anticancer activities of PTK7 mAbs, we propose that PTK7 mAbs can be used in an effective treatment strategy for PTK7-positive malignancies, such as ESCC.

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.