Targeted therapy based on protein-drug conjugates has attracted significant attention owing to its high efficacy and low side effects. However, efficient and stable drug conjugation to a protein binder remains a challenge. Herein, a chemoenzymatic method to generate highly stable and homogenous drug conjugates with high efficiency is presented. The approach comprises the insertion of the CaaX sequence at the C-terminal end of the protein binder, prenylation using farnesyltransferase, and drug conjugation through an oxime ligation reaction. MMAF and an EGFR-specific repebody are used as the antitumor agent and protein binder, respectively. The method enables the precisely controlled synthesis of repebody-drug conjugates with high yield and homogeneity. The utility of this approach is illustrated by the notable stability of the repebody-drug conjugates in human plasma, negligible off-target effects, and a remarkable antitumor activity in vivo. The present method can be widely used for generating highly homogeneous and stable PDCs for targeted therapy.

Targeted therapyb ased on protein-drug conjugates has attracted significant attention owing to its high efficacy and low side effects.However,efficient and stable drug conjugation to ap rotein binder remains ac hallenge.H erein, ac hemoenzymatic method to generate highly stable and homogenous drug conjugates with high efficiency is presented. The approach comprises the insertion of the CaaX sequence at the C-terminal end of the protein binder,p renylation using farnesyltransferase,a nd drug conjugation through an oxime ligation reaction. MMAF and an EGFR-specific repebody are used as the antitumor agent and protein binder,r espectively. The method enables the precisely controlled synthesis of repebody-drug conjugates with high yield and homogeneity. The utility of this approach is illustrated by the notable stability of the repebody-drug conjugates in human plasma, negligible off-target effects,a nd ar emarkable antitumor activity in vivo. The present method can be widely used for generating highly homogeneous and stable PDCs for targeted therapy.

Early detection of human cardiac markers is regarded as a gold standard for diagnosis of acute myocardial infarction (AMI). Here we demonstrate the increase in the detection sensitivity of a lateral flow assay for a cardiac marker troponin I (cTnI) by oriented immobilization of antibodies on magnetic beads. For the construction of sensitive magnetic labels, orientated-immobilization of anti-troponin I antibodies on magnetic beads was attempted using protein G. Magnetic beads were first conjugated with protein G followed by immobilization of anti-cTnI antibodies. The factors affecting the detection sensitivity in the lateral flow assay system were investigated. The lateral flow assay of cTnI was carried out using the magnetic beads and a Giant magnetoresistive (GMR) sensor at the optimized conditions, and compared the results with using a conventional random immobilization method. Magnetic labels with oriented immobilization of antibodies enabled the detection of cTnI up to 0.01 ng/mL, showing significantly increased sensitivity compared with conventional immobilization method. This result clearly shows that the magnetic labels with oriented immobilization of antibodies can find wide applications in the lateral flow assays of target analytes.

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Nanoparticle clusters (NPCs) have attracted significant interest owing to their unique characteristics arising from their collective individual properties. Nonetheless, the construction of NPCs in a structurally well-defined and size-controllable manner remains a challenge. Here we demonstrate a strategy to construct size-controlled NPCs using the DNA-binding zinc finger (ZnF) protein. Biotinylated ZnF was conjugated to DNA templates with different lengths, followed by incubation with neutravidin-conjugated nanoparticles. The sequence specificity of ZnF and programmable DNA templates enabled a size-controlled construction of NPCs, resulting in a homogeneous size distribution. We demonstrated the utility of magnetic NPCs by showing a three-fold increase in the spin-spin relaxivity in MRI compared with Feridex. Furthermore, folate-conjugated magnetic NPCs exhibited a specific targeting ability for HeLa cells. The present approach can be applicable to other nanoparticles, finding wide applications in many areas such as disease diagnosis, imaging, and delivery of drugs and genes.