Improved Calreticulin Nanobody by Framework Engineering

ABSTRACT 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 complimentary determining region 2 (CDR2) of a soluble, clinically validated 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 CALR-Nb02 by adapting the core of Cal3 to a partial consensus framework sequence of stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a predominantly monomeric, soluble protein that exhibits an increased thermal stability and a higher calreticulin binding affinity (KD: 25–50 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.