[Development of a Series of Neutralizing Nanobodies against SARS-CoV-2 Spike Protein].

Countering the spread of new respiratory infections and reducing the damage they cause to society requires efficient strategies for rapidly developing of targeted therapeutics, such as monoclonal antibodies. Nanobodies, defined as variable fragments of heavy-chain camelid antibodies, have a set of characteristics that make them particularly convenient for this purpose. The speed at which the SARS-CoV-2 pandemic spread confirmed that the key factor in the development of therapeutics is obtaining highly effective blocking agents as soon as possible, as well as the diversity of epitopes to which these agents bind. We have optimized the selection process of blocking nanobodies from the genetic material of camelids and obtained a panel of nanobody structures with affinity to Spike protein in the lower nanomolar and picomolar ranges and with high binding specificity. The subset of nanobodies that demonstrate the ability to block the interaction between the Spike protein and the cellular ACE2 receptor was selected in experiments in vitro and in vivo. It has been established that the epitopes bound by the nanobodies are located in the RBD domain of the Spike protein and have little overlap. The diversity of binding regions may allow a mixture of nanobodies to retain potential therapeutic efficacy towards new Spike protein variants. Furthermore, the structural features of nanobodies, particularly their compact size and high stability, indicate the possibility of their utilization in the form of aerosols.

Engineered Removal of PD-1 From the Surface of CD19 CAR-T Cells Results in Increased Activation and Diminished Survival.

CAR-T cell therapy is the most advanced way to treat therapy resistant hematologic cancers, in particular B cell lymphomas and leukemias, with high efficiency. Donor T cells equipped ex vivo with chimeric receptor recognize target tumor cells and kill them using lytic granules. CAR-T cells that recognize CD19 marker of B cells (CD19 CAR-T) are considered the gold standard of CAR-T therapy and are approved by FDA. But in some cases, CD19 CAR-T cell therapy fails due to immune suppressive microenvironment. It is shown that tumor cells upregulate expression of PD-L1 surface molecule that binds and increases level and signal provided by PD-1 receptor on the surface of therapeutic CAR-T cells. Induction of this negative signaling results in functional impairment of cytotoxic program in CAR-T cells. Multiple attempts were made to block PD-1 signaling by reducing binding or surface level of PD-1 in CAR-T cells by various means. In this study we co-expressed CD19-CAR with PD-1-specific VHH domain of anti-PD-1 nanobody to block PD-1/PD-L1 signaling in CD19 CAR-T cells. Unexpectedly, despite increased activation of CAR-T cells with low level of PD-1, these T cells had reduced survival and diminished cytotoxicity. Functional impairment caused by disrupted PD-1 signaling was accompanied by faster maturation and upregulation of exhaustion marker TIGIT in CAR-T cells. We conclude that PD-1 in addition to its direct negative effect on CAR-induced signaling is required for attenuation of strong stimulation leading to cell death and functional exhaustion. These observations suggest that PD-1 downregulation should not be considered as the way to improve the quality of therapeutic CAR-T cells.

[A Modified Lentivirus-Based Reporter for Magnetic Separation of Cancer Stem Cells].

Cancer stem cells (CSCs) are the most malignant subpopulation of tumor cells that possess a tumorigenic potential and resistantance to chemotherapy. These properties make CSCs a promising target for the development of targeted antitumor therapy which is especially in demand in highly aggressive cancers. However, the correct identification of cancer cells with stem properties remains a challenge. A newly developed lentivirus-based reporter SORE6 allows to directly identify CSCs by measuring gene expression of the embryonic stem cell factors SOX2 and OCT4. In the current study the reporter was modified to enable isolation of SOX2^(+)/OCT4^(+) cells by immunomagnetic separation and then was used to transduce HCC1806 and MDA-MB-453 triple-negative breast cancer (TNBC) cell lines. To validate the modified reporter, SOX2^(+)/OCT4^(+) populations were isolated and analyzed for the content of NANOG, a key transcription factor of pluropotency which expression is regulated by SOX2/OCT4. The percentage of SOX2^(+)/OCT4^(+) cells was assessed for each cell line. An increased content of NANOG protein was found in isolated SOX2^(+)/OCT4^(+) cell fractions indicating that the modified reporter is suitable for further studying the CSC subset.

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.

[CD47 receptor as a primary target for cancer therapy].

Recently, a number of new highly efficient antibody-based anticancer therapeutics have emerged. These receptor-binding antibodies have beneficial toxicity profiles associated with relatively mild side effects. Therefore, the search for novel surface proteins that are present on cancer cells and play important metabolic or defensive roles has intensified. Additionally, the therapeutic stimulation of patient's immune system in order to aim its components, specifically, phagocytes and cytotoxic T-lymphocytes, at tumor cells is gaining traction. This review is focused on the CD47 surface receptor, a ubiquitously expressed molecule, which could nevertheless serve as a therapeutic target due to its ability to simultaneously stimulate both natural and adaptive immune response.

[Role of PDLIM4 and c-Src in Breast Cancer Progression].

High heterogeneity is characteristic of oncology diseases, often complicating the choice of optimal anticancer treatment. One cancer type may combine tumors differing in histogenesis, genetic lesions, and mechanism of cell transformation. Differences in the mechanism of cell malignant transformation result in specifics of cancer cell metabolism and sensitivity to various agents, including anticancer treatments. Hence, the molecular subtype of a tumor is essential to know for choosing the optimal therapeutic strategy. The review considers the role actin-associated proteins and tyrosine kinases, in particular, PDLIM4 and Src kinase, play in the formation of pathological signaling pathways.

Flavoprotein miniSOG Cytotoxisity Can Be Induced By Bioluminescence Resonance Energy Transfer.

In this study, we investigated the possibility of phototoxic flavoprotein miniSOG (photosensitizer) excitation in cancer cells by bioluminescence occurring when luciferase NanoLuc oxidizes its substrate, furimazine. We have shown that the phototoxic flavoprotein miniSOG expressed in eukaryotic cells in fusion with NanoLuc luciferase is activated in the presence of its substrate, furimazine. Upon such condition, miniSOG possesses photoinduced cytotoxicity and causes a 48% cell death level in a stably transfected cell line.

[Oncotoxic proteins in cancer therapy: mechanisms of action].

Cancer therapeutics based on protein biomolecules that exhibit selective toxic of inhibiting effects towards tumor cells without affecting normal tissue, are gaining extensive attention in cancer research. This heterogenous group of proteins consists of several subgroups, among them, are engineered cancer antigen-specific antibodies that suppress tumor growth by blocking proliferation-inducing receptors, or by direct action of a covalently attached toxin. Another subgroup of anticancer proteins that also represents promising potential therapeutic agents is oncotoxic proteins that can selectively trigger proapoptotic signaling in cancer cells. The oncotoxic proteins target such commonly disturbed processes in tumor calls as enhanced cell proliferation, altered cell-cycle control, deficient apoptotic response, inhibited mitochondrial respiration and activated glycolysis. The introduction of oncotoxic proteins to the clinic might substantially widen and upgrade modern arsenal of anticancer therapeutics.

[Protein complementation as tool for studying protein-protein interactions in living cells].

Association and degradation of protein complexes play essential role in a majority of normal and pathologic processes, which take place in living cell. Studying the underlying mechanisms of those interactions would give deeper understanding of specific causes of disease progression and would allow developing new therapeutic strategies. The majority of technical approaches currently used for detecting protein association include in vitro protein extraction and purification, whereas more relevant results require methods that can be used in vivo. One of a few approaches for in vivo protein association detection is based on reporter protein fragment complementation. Reporter systems based on protein complementation rely on reconstitution of reporter protein fluorescent or enzymatic activity which occurs upon reassociation of protein fragments and could be measured by colorimetry, luminometry or fluorimetry. Protein complementation is widely used to develop reporter systems for analysis of protein interactions, for functional dissection of signal transduction pathways and for performing high-throughput screenings to discover new protein interaction partners. Currently developed approaches that utilize protein fragment complementation have possibilities that extend far beyond simple detection of interaction in a pair of proteins.

[Organization and regulation of nucleocytoplasmic transport].

Separation of processes of DNA replication and transcription from protein synthesis, which occurs in eukaryotic cells, allows more precise control over these processes. Selective exchange of macromolecules between these two compartments is mediated by proteins of nuclear pore complex (NPC). Receptor proteins of karyopherin family interact with NPC components and transfer their cargos between nucleus and cytoplasm. Nucleocytoplasmic transport pathways are regulated on multiple levels by modulating the expression or function of single cargoes, transport receptors, or the transport channel. These levels of regulation have increasingly broad effects on transport pathways, and affect a wide range of processes, from gene expression to development and differentiation.

Organization and regulation of nucleocytoplasmic transport.

Separation of DNA replication and transcription, which occur in the nucleus, from protein synthesis, which occurs in the cytoplasm, allows a more precise regulation of these processes. Selective exchange of macromolecules between the two compartments is mediated by proteins of the nuclear pore complex (NPC). Receptor proteins of the karyopherin family interact with NPC components and transfer their cargos between the nucleus and cytoplasm. Nucleocytoplasmic transport pathways are regulated at multiple levels by modulating the expression or function of individual cargoes, transport receptors, or the transport channel. The regulatory levels have increasingly broad effects on the transport pathways and affect a wide range of processes from gene expression to development and differentiation.

[A lentivirus vector based assay system for quantitative detection of intracellular translocations of recombinant proteins].

An enzymatic assay system is described that allows quantitative localization within different cellular structures of recombinant proteins. The system is based on alpha-complementation of beta-galactosidase. The large omega-fragment of beta-galactosidase is expressed in predefined cellular structures with the aid of attached protein localization signals. The obtained reporter cell lines are used for the introduction of a second construct that expresses a protein of study fused with a shorter alpha-fragment of beta-galactosidase. Physical proximity of the two recombinant proteins carrying beta-galactosidase fragments results in reconstitution of an active enzyme, and the activity can be measured in a plate reader. The recombinant constructs are based on lentiviral vectors, which allows rapid and efficient introduction of recombinant proteins into cells by infection with stocks of lentiviral particles. The efficiency of the system is demonstrated with transcriptional factor FOXO3A, which is shuttling between cytoplasm and nuclei in model colon carcinoma cell line RKO.

[RNA interference: biology and perspectives of application in biomedicine and biotechnology].

RNA interference (RNAi) is among the most particular mechanisms of gene expression regulation. Besides, small interfering RNAs are significant players in cell defence either from viral infection or retrotransposons. Medical utilization of RNAi gives a handful of ways to cure viral and oncological illnesses. RNA interference, also, represents a useful tool for research, because it allows quick production of monogene functional knockouts. In this review we describe the most recent conceptions about RNAi mechanisms and actual approaches for it's usage.