Isolation of Novel Fully Human Single-Domain Antibodies Targeting GUCY2C from a Heavy-Chain-Only Transgenic Mouse Platform
Pingxia Zhu, Lei He, Xiaoming Du, Pengsha Pang, Zhihua Ji, Yanyu Yang, Fengqi Cao, Quanjun WangIntroduction
Traditional natural single-domain antibodies(sdAbs) are primarily derived from camelids and sharks. However, the large size and slow reproduction of these species limit their widespread application in antibody development. In this study, we employed a fully human heavy-chain-only antibody mouse ( huNano mouse ) platform and a rapid, efficient preparation process to successfully develop fully human heavy-chain-only antibodies(HcAbs) targeting the tumor antigen guanylyl cyclase C (GC-C). These sdAbs provide high-quality candidate molecules for subsequent GC-C-based CAR-T therapy and bispecific antibody drug development.
Methods
Fully huNano mice were immunized with GC-C recombinant protein. SdAb sequences specifically binding to GC-C were selected via phage display technology and subsequently cloned into mammalian expression vectors for expression and purification. The binding activity, affinity, thermal stability, and monomeric dispersion of the antibodies were systematically evaluated using various methods, including ELISA, flow cytometry (FACS), biolayer interferometry (BLI), and size-exclusion chromatography (SEC-HPLC).
Results
Multiple sdAbs with specific recognition of GC-C were successfully obtained and exhibited strong binding affinities. After one-step purification, the monomeric purity of most antibodies exceeded 95%, with aggregate content below 5%. The nanobodies exhibited good thermal stability, with melting temperature (Tm) and aggregation onset temperature (Tagg) both falling within the range of 60–68 °C, demonstrating excellent drug development potential.
Conclusion
These results validate the capability of the huNano mouse platform to efficiently produce high-quality, fully human sdAbs.