Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation

• Published in: Journal of biological engineering Vol. 18; no. 1; pp. 23 - 12
• Main Authors: Go, Eun Byeol, Lee, Jae Hun, Cho, Jeong Haeng, Kwon, Na Hyun, Choi, Jong-il, Kwon, Inchan
• Format: Journal Article
• Language: English
• Published: London BioMed Central 04.04.2024; BioMed Central Ltd; BMC
• Subjects: Affinity; Albumin; Amino acids; Analysis; Antibodies; Antigens; Applied Microbiology; Binding; Biological Techniques; Biomedical Engineering and Bioengineering; Biotechnology; Breast cancer; Cancer therapies; Conjugation; Diels-Alder reaction; Diels-Alder reactions; Engineering; Environmental Engineering/Biotechnology; ErbB-2 protein; Ethylenediaminetetraacetic acid; Growth factors; Half-life; Half-life extension; Health aspects; Human serum albumin; Immune system; Kinases; Macromolecules; Molecular weight; Monoclonal antibodies; Nucleic Acid Chemistry; Pharmacokinetics; Phenylalanine; Proteins; Serum albumin; Single-chain variable fragment; Site-specific conjugation; Tetracycline; Tetracyclines; Therapeutic antibody; Therapeutic applications; Thiols; Transfer RNA; Trastuzumab; Tumors; Viral antibodies; South Korea; Seoul South Korea; United States > US; Germany; Single-chain variable fragment; Human serum albumin; Site-specific conjugation; Therapeutic antibody; Half-life extension
• ISSN: 1754-1611; 1754-1611
• DOI: 10.1186/s13036-024-00418-3

Background The use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties. Results In this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv. Conclusions The antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.