CRISPR-Based UPR Reporter Reveals Mechanism of Antibody Homeostasis in Hybridomas

Prof. Jae Seong Lee at the KAIST Graduate School of Engineering Biology, in collaboration with Zhejiang University and international partners, has uncovered a key mechanism governing antibody homeostasis in hybridoma cells and developed a novel reporter platform that enables real-time monitoring of antibody production stability. By integrating quantitative proteomics with CRISPR/Cas9 genome editing, the team demonstrated that the unfolded protein response (UPR) pathway plays a critical role in regulating immunoglobulin synthesis and quality control in antibody-producing cells. The study was published in Advanced Science on January 20, 2026.

Understanding the Cellular Mechanism of Antibody Instability

Monoclonal antibodies are among the most important classes of biotherapeutics and are widely used in medicine, diagnostics, and biotechnology. However, hybridoma cells, which have long served as the primary platform for antibody discovery and production, often lose the ability to produce immunoglobulin chains during prolonged cultivation. This loss of antibody expression disrupts antibody homeostasis and reduces production efficiency, posing a major challenge for stable antibody manufacturing and research applications.

To investigate this problem, the international research team systematically analyzed hybridoma subclones with impaired antibody production. Through quantitative proteomic profiling, the researchers identified significant changes in proteins associated with endoplasmic reticulum stress and the UPR pathway. Further functional validation using CRISPR/Cas9 genome editing revealed that Xbp1s, a key transcription factor downstream of UPR signaling, is essential for maintaining efficient antibody production. Disruption of this pathway resulted in reduced immunoglobulin expression and impaired antibody secretion, highlighting the critical role of cellular protein quality control mechanisms in antibody-producing cells.

Development of a CRISPR-Based UPR Reporter System

Building on these findings, the research team developed a novel intracellular reporter system that enables real-time monitoring of UPR activation in living cells. Using CRISPR/HDR-mediated genome editing, the researchers inserted a fluorescent mGFP tag into the endogenous Hspa5 (BiP) gene, a master regulator of the UPR pathway. This engineered reporter system allows visualization of cellular stress responses associated with antibody production.

Using this platform, the researchers discovered a strong negative correlation between intracellular UPR activation and IgG production levels, revealing that elevated ER stress is closely linked to disrupted antibody homeostasis. In addition, fluorescence-activated cell sorting of cells with high reporter signals enabled the identification of dysfunctional hybridoma subclones that failed to secrete antibodies. This demonstrates that the reporter system can serve as a powerful tool for tracking antibody production stability and isolating productive antibody-producing cells.

Future Impact for Antibody Engineering

This internationally collaborative research provides new insights into the cellular mechanisms that regulate immunoglobulin synthesis and stability in hybridoma cells. The CRISPR-engineered UPR reporter system offers a powerful platform for monitoring antibody production dynamics and identifying dysfunctional cell populations during antibody development. The technology is expected to contribute to the development of more stable antibody-producing cell lines and may support advances in therapeutic antibody manufacturing, protein quality control research, and next-generation cell engineering.

This work was supported by the National Research Foundation of Korea (RS-2024-00397714).