Larimer, B. M. | Search bioRxiv

bioRxiv2026

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 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.

bioengineering↗

bioRxiv2025

Generation of a Synthetic Single Domain Antibody Library for Radiopharmaceutical Ligand Discovery

Single domain antibodies, often known as nanobodies, are versatile molecules with therapeutic and diagnostic applications, but they are primarily developed through immunization of camelids. This approach is not scalable by automation, not effective for non-immunogenic or toxic antigens, and prevents the use of modified scaffolds for altered pharmacokinetic properties. Synthetic libraries allow for pre-selection of a single domain framework tailored to its intended downstream use. One area of interest for these biologic vectors is radiopharmaceuticals. Ideal radiopharmaceutical pharmacokinetic properties differ from most traditional therapeutics, as short plasma circulation and rapid kidney clearance are necessary to avoid dose-limiting organ radiation. Although there are a growing number of nanobody radiopharmaceuticals in clinical trials, their frameworks and corresponding pharmacokinetic properties vary. One potential method for improving the development of novel single domain antibody radiopharmaceuticals is through synthetic libraries based on nanobodies with proven clinically acceptable pharmacokinetics. We developed a modular synthetic nanobody phage display vector based on the scaffold of the 2Rs15d nanobody that allows for manipulation of the binding and framework regions. Using this vector, we created a library of nanobodies with a randomized CDR2 containing over 1.7x106 unique sequences/{micro}L. As a proof-of-concept, we panned the library for nanobodies binding calreticulin (CALR), a protein critical in immunogenic cell death. One isolated clone, Cal3, has a measured affinity of 140 nM for CALR and is cross-reactive with mouse and human CALR. Using positron emission tomography (PET) imaging, the radiolabeled 64Cu-NOTA-Cal3 demonstrated CALR binding in vivo, representing the first reported synthetic nanobody characterized by PET imaging. This study demonstrates the feasibility of building and panning synthetic libraries for high-affinity radiopharmaceutical nanobodies as an alternative to immunized camelid libraries.

bioengineering↗