Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185 HER-2-ECD antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185 HER-2-ECD -expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.reactive oxygen species ͉ phototoxicity ͉ KillerRed

A bioengineering method for self-assembly of multifunctional superstructures with in-advance programmable properties has been proposed. The method employs two unique proteins, barnase and barstar, to rapidly join the structural components together directly in water solutions. The properties of the superstructures can be designed on demand by linking different agents of various sizes and chemical nature, designated for specific goals. As a proof of concept, colloidally stable trifunctional structures have been assembled by binding together magnetic particles, quantum dots, and antibodies using barnase and barstar. The assembly has demonstrated that the bonds between these proteins are strong enough to hold macroscopic (5 nm-3 μm) particles together. Specific interaction of such superstructures with cancer cells resulted in fluorescent labeling of the cells and their responsiveness to magnetic field. The method can be used to join inorganic moieties, organic particles, and single biomolecules for synergistic use in different applications such as biosensors, photonics, and nanomedicine.superstructures | cancer cells | targeting | fusion proteins | magnetic nanoparticles R ecently, nanoparticles have become attractive objects for life science applications, in particular, in such rapidly growing areas as express diagnostics and advanced medical treatment. Encapsulation of nanoparticles with drug molecules (1, 2) or attaching them to viruses, bacteria, etc. are of special interest. Time-controlled release of the absorbed drugs would be advantageous for treatment of many diseases, e.g. diabetes, because of a decreased number of injections compared to that of molecular insulin. Furthermore, fluorescent or colored particles such as quantum dots (QD) (3), nanodiamonds (4), and gold nanoparticles (5) can be used for diagnostics as markers that provide visual information about the distribution of labeled agents in tissues and blood. Magnetic particles (MP) (6) can be also used as efficient labels for MRI diagnostics and can be precisely quantified even inside a living organism by an external induction probe (7,8). At present, MP are widely studied for hyperthermia of tumors by heating in an AC magnetic field and for targeted delivery of drugs by magnetic field gradients, to avoid systemic intoxication of the organism (9, 10). Specific immunological targeting of nanoparticles by antibodies against pathogenic cells is another noteworthy application. Not only does it allow marking tumors for accurate dissection, but it also enhances drug delivery to the target cells.The above-mentioned functional aspects of nanoparticles are brought into play in many life science applications. In certain cases, however, it would be beneficial to use multifunctional structures (11-13), which consist of several types of particles. Extensive studies were devoted to the synthesis of hybrid complexes of magnetic particles and different fluorophores (quantum dots or conventional chemical dyes) to allow visual MP tracking. Most approaches (14-16) emp...

Tumor-targeted delivery of cytotoxins presents considerable advantages over their passive transport. Chemical conjugation of cytotoxic module to antibody is limited due to insufficient reproducibility of synthesis, and recombinant immunotoxins are aimed to overcome this disadvantage. We obtained genetically encoded immunophotosensitizer 4D5scFv-miniSOG and evaluated its photocytotoxic effect in vitro. A single-chain variable fragment (scFv) of humanized 4D5 antibody was used as a targeting vehicle for selective recognition of the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) overexpressed in many human carcinomas. As a phototoxic module we used a recently described photoactivated fluorescent flavoprotein miniSOG. We found that recombinant protein 4D5scFv-miniSOG exerts a highly specific photo-induced cytotoxic effect on HER2/neu-positive human breast adenocarcinoma SK-BR-3 cells (IC50= 160 nM). We demonstrated that the 4D5scFv-miniSOG specifically binds to HER2-positive cells and internalizes via receptor-mediated endocytosis. Co-treatment of breast cancer cells with 4D5scFv-miniSOG and Taxol or junction opener protein JO-1 produced remarkable additive effects.

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Nanoparticle surface engineering can change its chemical identity to enable surface coupling with functional biomolecules. However, common surface coupling methods such as physical adsorption or chemical conjugation often suffer from the low coupling yield, poorly controllable orientation of biomolecules, and steric hindrance during target binding. These issues limit the application scope of nanostructures for theranostics and personalized medicine. To address these shortfalls, we developed a rapid and versatile method of nanoparticle biomodification. The method is based on a SiO-binding peptide that binds to the nanoparticle surface and a protein adaptor system, Barnase*Barstar protein pair, serving as a "molecular glue" between the peptide and the attached biomolecule. The biomodification procedure shortens to several minutes, preserves the orientation and functions of biomolecules, and enables control over the number and ratio of attached molecules. The capabilities of the proposed biomodification platform were demonstrated by coupling different types of nanoparticles with DARPin9.29 and 4D5scFv-molecules that recognize the human epidermal growth factor receptor 2 (HER2/neu) oncomarker-and by subsequent highly selective immunotargeting of the modified nanoparticles to different HER2/neu-overexpressing cancer cells in one-step or two-step (by pretargeting with HER2/neu-recognizing molecule) modes. The method preserved the biological activity of the DARPin9.29 molecules attached to a nanoparticle, whereas the state-of-the-art carbodiimide 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/ N-hydroxysulfosuccinimide method of conjugation led to a complete loss of the functional activity of the DARPin9.29 nanoparticle-protein complex. Moreover, the method allowed surface design of nanoparticles that selectively interacted with antigens in complex biological fluids, such as whole blood. The demonstrated capabilities show this method to be a promising alternative to commonly used chemical conjugation techniques in nanobiotechnology, theranostics, and clinical applications.