Improved Calreticulin Nanobody by Framework Engineering

Calreticulin is an emerging cancer biomarker, but current detection methods rely on expensive monoclonal antibodies that suffer from inefficient protein production, pharmacokinetic challenges and poor tissue penetration. Cal3, a calreticulin-specific nanobody, was constructed by replacing the complementarity-determining region 2 (CDR2) of a soluble, clinically validated HER2 nanobody with a calreticulin-specific CDR2 isolated from a phage display library. However, the poor solubility and low yield of Cal3 limit its usefulness. In this study, we engineered the CALR nanobody 02 by adapting the core of Cal3 to a consensus framework sequence for stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a soluble protein with an increased thermal stability and a five-fold higher calreticulin binding affinity (KD: 25 nM) compared with Cal3. These results reveal a strategy for quickly altering the specificity of a stable nanobody and provide an improved calreticulin-binding reagent for future diagnostic, imaging, and therapeutic applications.