The fetal form of the insulin receptor (IRA) is predominantly expressed in many cancers, where IR signaling promotes progression of the disease. Alternatively, the adult IR isoform (IRB) is predominantly expressed in normal tissues. Previous attempts to target IR indiscriminately via dual IR/IGF1R tyrosine kinase inhibitors frequently failed due to on-target off-tumor effects. However, an IRA-specific therapeutic has the potential to specifically target cancer while sparing normal tissues. Furthermore, an IRA-specific binder would advance research regarding this disease-associated isoform. Yet, isoform-specific IR binders have not been developed. Herein, we engineer six high-affinity, IRA-specific protein binders from a synthetic miniprotein scaffold. These miniprotein variants have IRA KDs ranging from 0.98-7.6 nM with no appreciable IRB binding at 100 nM ligand. Five of the six variants are synergistic agonists. One variant (V1) is a passive, IRA-specific binder with a KD = 6.3 nM and no IRB binding at 1 μM ligand as well as no IGF1R binding at 100 nM, which provides compelling potential as a targeted therapeutic. Additionally, we present the discovery process for these molecules, a yeast display directed evolution campaign utilizing a combination of sorting techniques. Retrospective analysis of the discovery process revealed the importance of alternating selections for target affinity and isoform specificity to generate binders with a favorable combination of these properties.
Bacteriophage M13 substrate libraries are a useful tool to discover novel protease substrates. In this study, we have mapped 7-mer protease substrates cleaved by native Escherichia coli proteases during phage outgrowth. Cleavage was monitored by the loss of a biotinylated AviTag at the N-terminus of the recombinant M13 minor coat protein, protein III. Using this method, we isolated 120 phage-displayed substrates that contain dibasic amino acid motifs (Lys-Lys, Arg-Arg, Lys-Arg, and Arg-Lys), of which the Lys-Lys motif has not been previously described using substrate phage. Since dibasic motifs are known to be substrates of the outer membrane protein proteases OmpP and OmpT, the phage library was transferred into a mutant strain deficient in both protease activities, and the selection process was repeated. Sequencing of recovered clones revealed no enrichment of the four dibasic motifs, demonstrating that all four motifs are substrates of Omptin proteases. These findings indicate that phage propagation in Omptin-expressing bacteria leads to the loss of dibasic amino acid motifs from phage-displayed combinatorial peptide, protein, and antibody libraries. This suggests that the presence of dibasic residues in proteins and peptides displayed on bacteriophage M13 should necessitate the use of OmpT- and OmpP-deficient bacterial hosts.
Protein bioconjugates have a wide range of applications, including for targeted drug delivery and in vivo diagnostics. Expanding the chemistry available to link protein to cargo remains a critical consideration for the development of novel bioconjugates for unmet needs. Currently, most FDA-approved protein bioconjugates are based on the antibody structure, which constrains the available conjugation chemistries and applications. Alternative scaffold proteins have potential as targeting molecules that advance the synthetic possibilities. Here, we report two methods of high-yield incorporation of azide-containing groups into an Fn3 domain protein scaffold to enable click chemistry conjugation. One approach incorporates noncanonical amino acid azidohomoalanine during recombinant protein expression, and the other method develops a linker system using a unique thiol residue in the Fn3 structure. These synthetic approaches enabled orthogonal, dual labeling. The resulting conjugates retain functionality in receptor binding assays, validating these methods for diverse applications in engineering scaffold proteins for bioconjugate applications.
Generative models have transformed structural bioinformatics, enabling antibody and nanobody design against protein epitopes; however, nanobody engineering for small molecule sensing remains largely experimental. In this work, we evaluate seven state-of-the-art structure predictors, AlphaFold3, Chai-1, Boltz-2x, RoseTTAFold3, Protenix, FlowDock and OmegaFold, on nanobody-small molecule complexes. Most predictors accurately reproduced nanobody and CDR geometries but struggled with ligand placement and orientation, although co-folding improved overall accuracy. Contact analysis revealed that CDR1, rather than CDR3, was predominantly involved in ligand binding. Intrinsic confidence scores correlated poorly with experimental accuracy and showed limited power in distinguishing binders from non-binders. Increasing the number of samples and seeds yielded modest gains in accuracy, whereas additional recycles did not. These findings highlight both the strengths and limitations of structure prediction methods for nanobody-small molecule complexes.
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 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.
