Bismuth Nanoparticles Increase Effectiveness of Proton Therapy of Ehrlich Carcinoma.

We studied the antitumor activity of the combined use of local proton irradiation in two modes (10 and 31 Gy) with preliminary intra-tumoral injection of two types of bismuth nanoparticles differing in surface coating: coated with the amphiphilic molecule Pluronic-F127 or Silane-PEG (5 kDa)-COOH polymer. Nanoparticles were used in doses of 0.75 and 1.5 mg/mouse. In two independent series on experimental tumor model (solid Ehrlich carcinoma), bismuth nanoparticles of both modifications injected directly into the tumor enhanced the antitumor effects of proton therapy. Moreover, the radiosensitizing effect of bismuth nanoparticles administered via this route increased with the increasing the doses of nanoparticles and the doses of radiation exposure. In our opinion, these promising data obtained for the first time extend the possibilities of treating malignant neoplasms.

Immunoliposomes As a Promising Antiviral Agent against SARS-CoV-2.

According to the World Health Organization, as of January 3, 2020 to September 13, 2023, there were approximately 23 million confirmed cases of COVID-19 reported in the Russian Federation, about 400 thousand of which were fatal. Considering the high rate of mutation of the RNA-containing virus genome, which inevitably leads to the emergence of new infectious strains (Eris and Pyrola), the search for medicinal antiviral agents remains an urgent task. Moreover, taking into account the actively mutating receptor-binding domain, this task requires fundamentally new solutions. This study proposes a candidate immunoliposomal drug that targets the S protein of SARS-CoV-2 by the monoclonal neutralizing antibody P4A1 and ensures the penetration of a highly active ribonuclease into the virus-infected cell, which degrades, among cellular RNA, viral RNA too. We demonstrate a more than 40-fold increase in the neutralizing activity of the developed drug compared to the free monoclonal neutralizing antibody.

Subpopulation Heterogeneity of NK Cells during the Genetic Modification for Subsequent Use in Targeted Therapy.

Obtaining genetically engineered NK cells is a developing area of immunotherapy. In this work, we analyzed the subset heterogeneity of NK cells subjected to retroviral transduction, taking into account the content of adaptive NK cell progenitors. It was shown that subsets KIR2DL2/DL3+, as well as CD57-KIR2DL2/DL3+NKG2C+, can be modified with greater efficiency than the corresponding subsets that do not carry the KIR2DL2/DL3 and NKG2C markers. After genetic modification, the CD57-KIR2DL2/DL3+NKG2C+ cells began to express CD57 de novo, acquiring the adaptive NK cell phenotype.

Specific Cytotoxicity of Targeted 177Lu and 212Pb-Based Radiopharmaceuticals.

Two radiopharmaceutical preparations were developed on the basis of artificial targeted polypeptide ZHER2 specific to HER2/neu tumor marker and radionuclides 177Lu (ZHER2-HSA-chelator-177Lu) or 212Pb (ZHER2-HSA-chelator-212Pb). The objective was to evaluate in vitro the cytotoxic activity of the targeted radiopharmaceuticals using two cultured human breast cancer cell lines with different expression of HER2/neu: SK-BR3 (high expression of HER2/neu) and MCF-7 (low expression of HER2/neu). It was shown that the cytotoxic effect of both preparations was significantly higher against the SK-BR-3 cells. The cytotoxicity correlated with the incubation period (it was higher after 72 h than after 24 h) and was significantly more pronounced in comparison with activity of radionuclide salts without a specific ligand. In vivo preclinical study of these pharmaceuticals seems to be very promising in animals with xenografted tumors showing high expression of HER2/neu marker.

Upconversion Nanoparticles Decorated with Polysialic Acid for Solid Tumors Visualization In Vivo.

Upconversion nanoparticles (UCNPs) are a promising nanoplatform for bioreagent formation for in vivo imaging, which emit UV and blue light under the action of near-infrared radiation, providing deep tissue penetration and maintaining a high signal-to-noise ratio. In the case of solid tumor visualization, the UCNP surface functionalization is required to ensure a long circulation time, biocompatibility, and non-toxicity. The effective UCNP accumulation in the solid tumors is determined by the disturbed architecture of the vascular network and lymphatic drainage. This work demonstrates an approach to the UCNP biofunctionalization with endogenous polysialic acid for in vivo bioreagent formation. Bioreagents possess a low level of nonspecific protein adsorption and macrophage uptake, which allow the prolongation of the circulation time in the bloodstream up to 3 h. This leads to an intense photoluminescent signal in the tumor.

Involvement of Actin Filaments in the Cytotoxic Effect of GD2-Specific Antibodies.

Induction of direct cell death is one of the mechanisms of the antitumor effect of GD2-specific antibodies used for the therapy of high-risk neuroblastoma. The mechanisms of the cytotoxic signal triggered by antibody binding to GD2 ganglioside on the surface of the tumor cell remain insufficiently studied. Using inhibitor analysis we demonstrated that actin microfilaments are involved in the cell death induced by GD2-specific antibodies. Specifically, a strong antagonistic influence of cytochalasin D on the cytotoxic effect induced by GD2-specific antibodies was demonstrated in GD2+ tumor cell lines, which was expressed in at least 20% increase in cell survival and a significant decrease of the fraction of cells with fragmented DNA.

Stably Fluorescent Cell Line of Human Ovarian Epithelial Cancer Cells SK-OV-3ip-red.

Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.

Study of Fibronectin Type III-Like Domains Role in Activation of gp130 Receptor.

Chimeric gp130 receptors were produced to study the role of three fibronectin type III-like domains in activation of gp130 receptor machinery. The ligand-induced dimerization of gp130 was sufficient to trigger STAT3 signaling pathway. These findings can be used as the basis in designing novel therapeutic gp130 inhibitors.

[Cancer gene therapy with plasmid expressing anti-HER2/neu-toxin].

The HER2/neu receptor is over-expressed on the surface of many types of cancer cells, and is widely used for tar- geted delivery of anticancer drugs. We have created geneti- cally engineered construct expressing the PE40 fragment of Pseudomonas toxin bound with the DARPin molecule which recognizes the HER2/neu receptor with high specificity. The construct destroyed transfected tumor cells in vitro. Intra-tumor injections of the construct complexed with polyethyleneimine led to growth retardation of D2F2/E2 tumors in mice. These results suggest a possibility of using this approach to develop new anticancer drugs.

[Immunocytochemical visualization of P185(HER2) receptor by antibody fused with dibarnase and conjugate of barstar with colloidal gold].

We studied the localization of transmembrane receptor P185(HER2) in SKOV-3 and BT-474 cancer cells by fluorescence, confocal and electron immunomicroscopy. P185(HER2) is a marker of breast and ovarian tumors, it is considered as a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, first, to study the distribution of P185(HER2) in cancer cells, secondly, to remove P185(HER2) from the cell surface and, thirdly, to eliminate target cells. In this work for visualization of P185HER2 We prOposed immunocytotoxic system, consisting of the monoclonal miniantibody 4D5 scFv to extracellular P185E domain fused with two molecules of barnase (ribonuclease from Bacillus amyloliquefaciens) and of its specific inhibitor barstar. Fluorescence microscopy has showed that the module 4D5 scFv-dibarnase:barstar efficiently identified P185(HER2) on the surface of cancer cells. It was revealed by confocal microscopy that interaction with 4D5 scFv-dibarnase lead to internalization of P185(HER2). The localization of P185(HER) in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185(HER) distributed on the cell surface unequally with preferential localization on protrusions or close to their bases and in contacts between protrusions and cell membrane. At 37 degrees C, P185(HER2) internalized through coated pits and vesicles and concentrated in the endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in cells BT-474.

[Genetically encoded photoimmunosensitizer].

Photosensitizer-antibody conjugates are successfully used for targeted elimination of cancer cells bearing specific membrane markers. This method is known as photoimmunotherapy. However, chemical conjugation of photosensitizer and antibody poses a number of complications such as low reproducibility, aggregation and unconjugated photosensitizer impurities. Here we report a fully genetically encoded photoimmunosensitizer, consisting of an anti-HER2/neu miniantibody 4D5scFv and a phototoxic fluorescent protein KillerRed. Both domains in this photoimmunosensitizer retained their functional qualities - high affinity for HER2/neu antigen and phototoxicity respectively. 4D5scFv-KillerRed fusion protein showed high specificity for HER2/neu-over-expressing cells and effectively lowered their viability upon illumination.

[Differences in the functional activity of human neutrophilic granulocytes in their interactions with semiconductor quantum dots].

The uptake of quantum dots (QD-5 nm particles of CdSe/ZnS-mercaptoacetic acid) by human neutrophilic granulocytes was studied using the methods of scanning laser and scanning probe microscopy. The results show that the neutrophilic granulocytes may be subdivided into three subpopulations: (1) the cells with no uptake of QD (10.0 +/- 2.0%); (2) cells that accumulate QD in their volume (28.0 +/- 1.9%), and (3) cells, surrounded by a halo of QD (59.0 +/- 2.2%). The dispersion of these characteristics may suggest the differences in neutrophilic granulocyte plasma membrane permeability.

[Antibody engineering: barnase-barstar module as a molecular constructor].

Today, antibody engineering for clinical applications is a rapidly progressing field of science and a big business. The basic functions of an antibody can be spatially differentiated and attributed to various structural domains of a molecule. Therefore, each of them may be an object for engineering with the aim of using a definite antibody function. In this sense, the potential of antibodies is unique. In this article, recent achievements and current problems of antibody engineering are briefly reviewed. The main attention is focused on a molecular constructor that allows for obtaining, with the help of a versatile barnase-barstar module, mono- and multiva-lent miniantibodies and their derivatives with outlined properties.

[Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells].

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG1 or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.

Dynamic spin label study of the barstar-barnase complex.

The dynamic spin label method was used to study protein-protein interactions in the model complex of the enzyme barnase (Bn) with its inhibitor barstar. The C40A mutant of barstar (Bs) containing a single cysteine residue was modified with two different spin labels varying in length and structure of a flexible linker. Each spin label was selectively bound to the Cys82 residue, located near the Bn-Bs contact site. The formation of the stable protein complex between Bn and spin labeled Bs was accompanied by a substantial restriction of spin label mobility, indicated by remarkable changes in the registered EPR spectra. Order parameter, S, as an estimate of rapid reorientation of spin label relative to protein molecule, was sharply increasing approaching 1. However, the rotational correlation time tau for spin-labeled Bs and its complex with Bn in solution corresponded precisely to their molecular weights. These data indicate that both Bs and its complex with Bn are rigid protein entities. Spin labels attached to Bs in close proximity to an interface of interaction with Bn, regardless of its structure, undergo significant restriction of mobility by the environment of the contact site of the two proteins. The results show that this approach can be used to investigate fusion proteins containing Bn or Bs.

[Antiviral activity of Bacillus intermedius RNAase in guinea-pigs and rabbits infected with outdoor rabies virus].

Single injection of Bacillus intermedius RNAase in a dose of 5 mg/kg could protect 40 and 50-70% of the outdoor rabies virus-preinfected guinea-pigs and rabbits, respectively. In the control group there were 100 and 75-100% deaths of the RNAase-untreated guinea-pigs and rabbits, respectively. Animal protection was observed only when RNAase was injected into the site of viral administration. The intramuscular injection of RNAase, other than the site of viral administration failed to protect the infected animals. The efficacy (75%) of RNAase injected into the rabbits was similar 1 and 24 hours after animal infection.

[The antiviral activity of RNAse Bacillus intermedius in experiments with mice preinfected with street rabies virus]. / Protivovirusnaia aktivnost' RNKazy Bacillus intermedius v éksperimentakh na myshakh linii CBA, predvaritel'no zarazhennykh ulichnym virusom beshenstva.

RNAse Bacillus intermedius, when administered once and according to 11 repeated experiments, protected the preliminarily infected CBA mice with street rabies virus (protection of 40-67%; p < 0.01-0.001). A reliable protection of Animals was registered only when RNAse was administered intramuscularly at the virus introduction spot; it was not effective, when the bacterial RNAse was injected in the brain, vein, under the skin or in muscles of a non-infected extremity. Neither did it produce any suppressive effect on the vaccinal antirabic immunity.

[Biosynthesis of mini-antibodies to human ferritin in plant and bacterial producers]. / Biosintez mini-antitel k ferritinu cheloveka v rastitel'nykh i bakterial'nykh produtsentakh.

A recombinant scFv antibody against human spleen ferritin was expressed as a barstar-fused protein in Escherichia coli and in Nicotiana tabacum plants and suspension cell cultures. As demonstrated by immunoblotting with antibarstar antibodies, direction of the recombinant protein to the endomembrane system of plant cells ensured its stability and solubility. Production of the recombinant protein did not differ between parental transgenic plants and their first-generation progeny. Fusion with barstar allowed not only immunochemical detection of the recombinant scFv antibody, but also their purification from the plant material by affinity chromatography with barnase-His6 immobilized on a metal-affinity carrier.

[Thermostability of alkaline phosphatase of Meithermus ruber bacteria: cloning of the gene, expression in Escherichia coli cells and biochemical characterization of the recombinant protein]. / Termostabil'naia shchelochnaia fosfataza bakterii Meiothermus ruber: klonirovanie gena, ékspressiia v kletkakh Escherichia coli i biokhimicheskaia kharakteristika rekombinantnogo belka.

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60-65 degrees C and pH 11.0 and had K(m) = 0.055 mM as estimated with p-nitrophenyl phosphate (pNPP). The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60 degrees C and being completely inactivated in 2 h at 80 degrees C. In substrate specificity assays, the highest enzymic activity was observed with pNPP and dATP. Vanadate, inorganic phosphate, and SDS inhibited M. ruber alkaline phosphatase, while thiol-reducing agents had virtually no effect. The enzymic activity strongly depended on exogenous Mg2+ and declined in the presence of EDTA.