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Proteins of the AID/APOBEC family are capable of cytidine deamination in nucleic acids forming uracil. These enzymes are involved in mRNA editing, protection against viruses, the introduction of point mutations into DNA during somatic hypermutation, and antibody isotype switching. Since these deaminases, especially AID, are potent mutagens, their expression, activity, and specificity are regulated by several intra-cellular mechanisms. In this review, we discuss the mechanisms of impaired expression and activation of AID/APOBEC proteins in human tumors and their role in carcinogenesis and tumor progression. Also, the diagnostic and potential therapeutic value of increased expression of AID/APOBEC in different types of tumors is analyzed. We assume that in the case of solid tumors, increased expression of endogenous deaminases can serve as a marker of response to immunotherapy since multiple point mutations in host DNA could lead to amino acid substitutions in tumor proteins and thereby increase the frequency of neoepitopes.

Monoclonal antibodies are the classical basis for targeted therapy, but the development of alternative binding proteins has made it possible to use non-immunoglobulin proteins as targeting modules. The advantages of DARPins, scaffold proteins based on ankyrin repeats, over antibodies are as follows: small size, stability over a wide range of temperatures and pH values, low aggregation tendency, and ease of production in heterologous expression systems. The differences in the structure of the paratope of DARPin and antibodies broaden the spectrum of target molecules, while the ease of creating hybrid fusion proteins allows one to obtain bispecific and multivalent constructs. In this article, we summarize recent data on the development of therapeutic and imaging compounds based on DARPins.

Photodynamic therapy (PDT) is a clinical, minimally invasive method for destroying cancer cells in the presence of a photosensitizer, oxygen, and a light source. The main obstacle for the PDT treatment of deep tumors is a strong reduction of the excitation light intensity as a result of its refraction, reflection, and absorption by biological tissues. Internal light sources based on bioluminescence resonance energy transfer can be a solution of this problem. Here we show that luciferase NanoLuc being expressed as a fusion protein with phototoxic flavoprotein miniSOG in cancer cells in the presence of furimazine (highly specific NanoLuc substrate) induces a photodynamic effect of miniSOG comparable with its LED-excited (Light Emitting Diode) phototoxicity. Luminescence systems based on furimazine and hybrid protein NanoLuc-miniSOG targeted to mitochondria or cellular membranes possess the similar energy transfer efficiencies and similar BRET-induced cytotoxic effects on cancer cells, though the mechanisms of BRET-induced cell death are different. As the main components of the proposed system for BRET-mediated PDT are genetically encoded (luciferase and phototoxic protein), this system can potentially be delivered to any site in the organism and thus may be considered as a promising approach for simultaneous delivery of light source and photosensitizer in deep-lying tumors and metastasis anywhere in the body.

It was found that the fluorescent properties of the phototoxic domain of miniSOG allow to assess the ability of toxins to bind to human breast adenocarcinoma cells SK-BR-3 and study the dynamics of their internalization. We established that the main cause of the decrease of the fluorescence intensity of the recombinant proteins 4D5scFv-miniSOG and DARPin-miniSOG during their internalization in the complex with the HER2 receptor is their shielding and absorption of the fluorescence of miniSOG by the cells fluorophores.

Despite an enhanced permeability and retention effect typical of many solid tumors, drug penetration is not always sufficient. Possible strategies for the drug delivery improvement are a modification of the tumor cell-to-cell junctions and usage of cell membrane permeabilization proteins. In this review we discuss epithelial cell junctions as targets for a combined anticancer therapy and propose new possible sources of such agents. We suggest considering viral and bacterial pathogens disrupting epithelial layers as plentiful sources of new therapeutic agents for increasing tumor permeability for other effector agents. We also observe the application of pore forming proteins and peptides of different origin for cytoplasmic delivery of anti-cancer agents and consider the main obstacles of their use 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.

Controlling background fluorescence remains an important challenge in flow cytometry, as autofluorescence can interfere with the detection of chromophores. Furthermore, experimental procedures can also affect cellular fluorescence in certain regions of the emission spectrum. In this work, the effects of fixation, permeabilization, and heating on cellular autofluorescence are analyzed in various spectral regions, along with the influence of trypan blue as a quenching dye for these treatments. The impact of these procedures on the staining of SK-BR-3 cells with a dim green fluorophore, a miniSOG (mini Singlet Oxygen Generator) flavoprotein in the form of the recombinant protein DARPin-miniSOG, is also evaluated. The data presented here indicate that fixation of certain types of cells leads to noticeable increase of the autofluorescence. Our results also suggest that trypan blue should be used as an autofluorescence quencher only with bright green emitters since it interferes with the fluorescent signal in a longer-wavelength region of the spectrum and as a result causes reduction of the signal from dim green fluorescent agents. © 2017 International Society for Advancement of Cytometry.

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.

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.

The protein photosensitizer miniSOG is a promising agent for photodynamic therapy. The genetically encoded phototoxins 4D5scFv-miniSOG and DARPin-miniSOG specifically bind to the HER2 receptor overexpressed on the surface of cancer cells and promote receptor-mediated internalization of HER2. We show that ingestion of proteins in a complex with the receptor reduces the fluorescent signal of the phototoxic module in endosomes. In order to clarify the mechanism of decrease in the fluorescence intensity of miniSOG-based proteins as they enter a cancer cell during internalization, we analyzed the influence of different factors, including low pH, proteolysis, cofactor reduction, and shielding, on changes in the fluorescence of photosensitizers. Shielding and absorption of miniSOG fluorescence by cell fluorophores, including cytochrome c, were found to contribute significantly to the changes in the fluorescent properties of miniSOG.

Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.

Development and functional characterization of novel, high-affinity protein compounds able to selectively kill human cancer cells is an urgent task of modern biomedical research. In this work, we studied the cytotoxicity of a recombinant phototoxic protein DARPin-miniSOG against the HER2-positive human breast adenocarcinoma cells. It was found that targeted phototoxin DARPin-miniSOG interacts specifically with HER2 receptor and causes the light-induced death of HER2-positive cells by the mechanism of necrosis. Irradiation of the cells in the presence of ascorbic acid eliminates the light-induced cytotoxicity of DARPin-miniSOG, which proves the prooxidant mechanism of phototoxin action.

The concept of targeted therapy implies the development of bifunctional agents complementing the therapeutic module with a targeting one. A promising target for the delivery of imaging and/or toxic modules is the HER2 (ErbB2) receptor. Earlier, we have functionally characterized the targeted photosensitizers 4D5scFv-miniSOG and DARPin-miniSOG, causing the death of HER2-overexpressing cells when irradiated with blue light. However, the cytotoxicity of targeted toxins 4D5scFv-miniSOG and DARPin-miniSOG (both having functionally active targeted and cytotoxic modules in recombinant proteins) against human breast adenocarcinoma cells differs 5 times. The study of the dynamics of internalization of 4D5scFv-miniSOG and DARPin-miniSOG proteins in the complex with HER2 in this work showed that the rate of internalization contributes most significantly to the toxicity of these photosensitizers, because it determines the duration of the presence of the phototoxin in the lipid bilayer of the cell membrane, where its damaging effect is maximum.

Cytotoxic effects of a new targeted phototoxin DARPin-miniSOG and mechanism of its action were investigated in vitro. It was determined that DARPin-miniSOG causes light-induced death of HER2/neu-positive cancer cells (IC50 0.8 μM). Treatment of the cells with DARPin-miniSOG in the presence of ascorbic acid eliminated the light-induced cytotoxic action of the protein. This observation suggests the involvement of oxidative stress in the mechanism of the phototoxin action. DNA fragmentation analysis, caspase-3 activity assay and PI-staining of HER2/neu-positive cancer cells treated with DARPin-miniSOG indicated that phototoxin induces necrotic cell death under blue light illumination. Co-localization analysis showed that DARPin-miniSOG accumulates mostly in endosomes and lysosomes.

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.

Design and evaluation of new high-affinity protein compounds that can selectively and efficiently destroy human cancer cells are a priority research area in biomedicine. In this study we report on the ability of the recombinant phototoxic protein DARPin-miniSOG to interact with breast adenacarcinoma human cells overexpressing the extracellular domain of human epidermal growth factor receptor 2 (HER2). It was found that the targeted phototoxin DARPin-miniSOG specifically binds to the HER2 with following internalization and slow recycling back to the cell membrane. An insight into the role of DARPin-miniSOG in HER2 internalization could contribute to the treatment of HER2-positive cancer using this phototoxic protein.