G. M. Proshkina

Genetically encoded immunophotosensitizer 4D5scFv-miniSOG is a highly selective agent for targeted photokilling of tumor cells in vitro

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.

Recombinant targeted toxin based on HER2-specific DARPin possesses a strong selective cytotoxic effect in vitro and a potent antitumor activity in vivo.

DARPins fused with other proteins are promising non-immunoglobulin scaffolds for specific binding to target cells. In this study HER2-specific DARPin (DARPin_9-29) was used as a tumor-targeting moiety for the delivery of a cytotoxic agent - the fragment of Pseudomonas aeruginosa exotoxin A. It was determined that DARPin-PE40 possesses a considerable cytotoxic activity and induces apoptosis in HER2-positive cells. Cytotoxic effect of DARPin-PE40 strongly correlates with the HER2 expression level. The effect of intravenous administration of DARPin-PE40 was tested in the xenograft model of breast cancer. It was shown that treatment of animals with DARPin-PE40 caused strong and prolonged suppression of xenograft tumor growth.

Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu

Here we propose a simple and reliable approach for detection of the tumor marker HER2/neu using the targeting fluorescent hybrid protein DARPin-mCherry. As a targeting module, we used DARPin9-29, which is a member of a novel class of non-immunoglobulin targeting proteins that can highly selectively recognize the extracellular domain of the epidermal growth factor receptor HER2/neu. The red fluorescent protein mCherry was used as the detecting module. The hybrid protein DARPin-mCherry was prepared with high yield in a bacterial expression system and purified in one step by affinity chromatography. The purified protein is not prone to aggregation. The specificity of DARPin-mCherry binding with the HER2/neu tumor marker was demonstrated using confocal microscopy, flow cytofluorimetry, and surface plasmon resonance. The dissociation constant of the DARPin-mCherry protein complex with the HER2/neu receptor determined by surface plasmon resonance was calculated to be 4.5 nM. These characteristics of the hybrid protein DARPin-mCherry suggest it as a promising agent for immunofluorescent assay and an attractive alternative to antibodies and their fragments labeled with fluorescent dyes that are now used for this purpose.

Synthesis, Characterization, and Selective Delivery of DARPin–Gold Nanoparticle Conjugates to Cancer Cells

We demonstrate that the designed ankyrin repeat protein (DARPin)_9-29, which specifically targets human epidermal growth factor receptor 2 (HER 2), binds tightly to gold nanoparticles (GNPs). Binding of the protein strongly increases the colloidal stability of the particles. The results of experimental analysis and molecular dynamics simulations show that approximately 35 DARPin_9-29 molecules are bound to the surface of a 5 nm GNP and that the binding does not involve the receptor-binding domain of the protein. The confocal fluorescent microscopy studies show that the DARPin-coated GNP conjugate specifically interacts with the surface of human cancer cells overexpressing epidermal growth factor receptor 2 (HER2) and enters the cells by endocytosis. The high stability under physiological conditions and high affinity to the receptors overexpressed by cancer cells make conjugates of plasmonic gold nanostructures with DARPin molecules promising candidates for cancer therapy.

Bifunctional Toxin DARP-LoPE Based on the Her2-Specific Innovative Module of a Non-Immunoglobulin Scaffold as a Promising Agent for Theranostics

We have generated and characterized HER2-specific targeted toxin based on the low-immunogenic variant of Pseudomonas exotoxin A (LoPE), in which most of the human immunodominant B-cell epitopes have been inactivated. Nonimmunoglobulin DARPin-based HER2-specific protein was used as a targeting module for toxin delivery to the cellular target. Using confocal microscopy, it has been found that both domains in this hybrid toxin retained their functionality, i.e., the specific interaction with HER2 receptor, as well as the internalization and effective transport to ER typical of the wild-type Pseudomonas exotoxin A. The HER2-dependent cytotoxic effect correlated with receptor expression level at the cell surface, as shown in vitro using cell lines with different levels of HER2 expression. Due to the very high selective cytotoxicity against HER2-positive human tumor cells, as well as expected low immunogenicity, we believe that this new targeted toxin may be promising for future in vivo studies as a therapeutic agent for HER2-positive tumors.

Mechanism of the cytotoxic action of immunophototoxin 4D5scFV-miniSOG on HER2/neu-positive cancer cells

16 Photodynamic therapy (PDT) is a modern method of influence on cancer cells, in which a photosensitizer (PS) that is nontoxic in unexcited state and harmless visible light are used [1]. When switched to the excited state by illumination with light of a certain wavelength, the PS can participate in photochemical reactions [2] and produce reactive oxygen species (ROS), such as singlet oxygen 1 О 2 , as well as free radicals OH, HO 2 , and. ROS produced in photochemical reactions during PDT trigger a cascade of biochemical events that lead to one of three types of cell death (apoptosis, necrosis, or autophagy). Unfortunately, the majority of PSs currently used in medical practice for the treatment of cancer have a significant drawback such as the nonselective accumuu lation in healthy tissues, leading to the development of allergic reactions or skin photosensitization by sunn light. To overcome this problem, a new PDT direction— targeting photodynamic therapy (TPT)—is rapidly developing [3], the goal of which is the development of the macromolecular conjugates that use the specific affinity of an address module to surface tumor markers (recognition of ligand by receptor or antigen by antii body) for targeting at certain cells. It was shown that in vivo chemical conjugates of PSs with monoclonal antibodies or their fragments have advantages comm pared to the free PSs [4–6]. However, the chemical conjugation of PSs with antibodies is always associated with a number of difficulties and does not always allow obtaining products with a constant composition. The O • – 2 genetically encoded PSs, which are used in electron microscopy and optogenetics, are devoid of this draww back [7, 8]. Currently, they are also used for selective photoactivatable destruction of tumor cells [9, 10]. Previously, we described and completely characterr ized the genetically encoded immunophototoxin that is able to highly selectively recognize the HER2/neuu positive human cancer cells [10]. Photoactivatable flaa voprotein miniSOG [7], capable of generating ROS when illuminated with blue light [7, 11, 12], was taken as a phototoxic module. The 4D5scFv miniantibody, which is able to highly selectively recognize the HER2/neu tumor marker, was used as an address modd ule. In in vitro experiments with the breast adenocarcii noma cells, the immunophototoxin 4D5scFvvminiSOG showed a high specificity with respect to the HER2/neuuoverexpressing cancer cells and effectively reduced their viability under irradiation with blue light. The IC 50 value was found to be …

Flavoprotein miniSOG BRET-induced cytotoxicity depends on its intracellular localization

It is proposed to use the bioluminescent resonance energy transfer to solve the problem of creating the internal light sources in photodynamic therapy of cancer. Energy donor in the developed system is the oxidized form of the luciferase NanoLuc substrate furimamide, and acceptor is the phototoxic fluorescent protein miniSOG. It is shown that, in the proposed system, the photoinduced cytotoxicity of flavoprotein miniSOG in vitro depends on the intracellular localization, and the cytotoxic effect is 48% for the cytoplasmic localization of the fusion protein, 65% for the mitochondrial localization, and 69% for the membrane localization. The obtained data indicate that, for maximization of the photodynamic effect in vivo, it is appropriate to use the NanoLuc–miniSOG fusion protein in the membrane localization.

A Highly Specific Substrate for NanoLUC Luciferase Furimazine Is Toxic in vitro and in vivo

The bioluminescent platform based on a genetically engineered luciferase NanoLuc and its synthetic substrate furimazine is widely used in modern molecular biology for optical imaging and therapy of deep tissue tumors. Taking into account the perspectives of using this system in molecular biology research, a detailed study of the toxicity of furimazine in vitro and in vivo is of great interest. In this work, the cytotoxicity of furimazine in vitro was studied using four different cell lines. Systemic toxicity of furimazine in vivo was investigated under conditions of prolonged administration of the substrate in animals. It has been found that seven-day intravenous injection of the substrate in animals causes hydropic dystrophy of liver and necrosis of hepatocytes. Splitting of the dose into several injections reduces hepatotoxicity of furimazine.

The Cause of ErbB2 Receptor Resistance to Downregulation

ErbB2/HER2 is a tyrosine kinase receptor belonging to the family of epidermal growth factor receptors (EGFRs); it is overexpressed in 25–30% of human breast cancer cases and has a number of structural and functional differences from other receptors of this family. Typically, the activation of tyrosine kinase receptors, i.e., formation of their homo- or heterodimers, and the subsequent signal transmission into the cell occurs when the ligand is bound to them. After dimers are formed, the internalization of a complex takes place, which plays a key role in the regulation of receptor activity. Unlike other receptors of the family, ErbB2 does not have natural ligands, but is the preferred partner for the formation of heterodimers with other members of the ErbB family. ErbB2 is also resistant to internalization and degradation. Thus, staying for a long time at the cell membrane after activation, ErbB2 continues to transmit regulatory signals to the cell nucleus. Although mechanisms ensuring the ErbB2 resistance to downregulation are not fully understood, a significant pool of experimental data suggests that such key points as interaction with Hsp90 chaperon, the ability to suppress the formation of clathrin pits, the ability to quickly return to the membrane from early endosomes, and interaction with the calcium pump PMCA2, allow ErbB2 receptor to avoid internalization.

Bifunctional Recombinant Protein Agent Based on Pseudomonas Exotoxin A Fragment for Targeted Therapy of HER2-Positive Tumors

Four variants of bifunctional HER2-specific recombinant proteins (anti-HER2-toxins) were created composed of HER2-specific scFv antibody (4D5scFv) or HER2-specific DARPin (D29) as targeting module and Pseudomonas exotoxin A fragment (PE40) as toxic module, and distinguished by the way of bacterial expression (cytoplasmic or periplasmic). Physicochemical, immunochemical, and functional properties of the created recombinant proteins as well as their expression yields were analyzed and compared with the option of the most promising one, D29-PE40, for further implementation as an agent for targeted therapy of HER2-overexpressing tumors.