Department of immunology

All publications (show selected)

Ram Petrov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. , office.
  • Phone: +7(495)954-32-76
  • E-mail: petrov@pran.ru

Key factors contributing to the green-to-red fluorescent protein transformation were identified

Laboratory of biomolecular modeling,  Group of in silico analysis of membrane proteins structure,  Laboratory of molecular theranostics

Through the examples of two highly homologous fluorescent proteins from Zoanthus sp. (zoanGFP and zoan2RFP), amino acid residues participating in the transformation of a protein with the green fluorescence (GFP) into the red fluorescent protein (RFP) were explored. As the result of zoanGFP mutagenesis, internal amino acid residues (a.a.r.) became identical to those of zoan2RFP. However, this mutant underwent only partial transformation into the red form. To elucidate the extra factors that might affect red chromophore biosynthesis, we used comparative molecular dynamics simulations of zoan2RFP and zoanGFPmut. As the result, additional a.a.r. were discovered on the surface of the protein that might influence both the arrangement and flexibility of the chromophore-surrounding a.a.r. Site-directed mutagenesis of these external a.a.r. confirmed the crucial role of these residues in red chromophore biosynthesis.

A method has been developed for the production of human NK cell clones using IL-2 and K562-mbIL21 feeder cells expressing membrane-bound IL-21

Laboratory of cell interactions

1. Using different models of clone cultivation, it was revealed that the cultivation conditions (the frequency of restimulation using feeder cells) affect the phenotype, functional characteristics, expansion level, and clone life expectancy, which can reach 14 weeks.
2. The CD57 marker can completely disappear from the cell surface of CD57-positive NK cells when cultured under stimulation conditions of IL-2 / K562-mbIL21.
3. Expression of the NKG2A receptor can occur de novo in the progeny of initially NKG2A-negative NK cells.

Gold nanostructures for biomedical applications.

Laboratory of molecular immunology

A biosensor-Fourier transducer based on periodic gold nanostructures was created and studied for the first time, which allows achieving ultrahigh sensitivity of the analysis of compounds (10-15 g/ml) in biological matrix. The developed methodology will allow solving the problems of highly sensitive analysis of target compounds in complex matrices, including hormones and other bioregulators acting in very low concentrations (doping control), highly toxic substances (biotoxins), pathogens (for biosafety problems, antibioterrorism protection). For the first time in the world, gold nanorods coated with a tumor-specific addressing module DARPin were obtained, which find tumor cells of a corresponding molecular profile and suppress their growth when irradiated with infrared light in the “Biotissue Transparency Window” (IC50 3.4 nM).

Publications

  1. Proshkina G, Deyev S, Ryabova A, Tavanti F, Menziani MC, Cohen R, Katrivas L, Kotlyar A (2019). DARPin_9-29-Targeted Mini Gold Nanorods Specifically Eliminate HER2-Overexpressing Cancer Cells. ACS Appl Mater Interfaces 11 (38), 34645–34651
  2. Kabashin AV, Kravets VG, Wu F, Imaizumi S, Shipunova VO, Deyev SM, Grigorenko AN (2019). Phase-Responsive Fourier Nanotransducers for Probing 2D Materials and Functional Interfaces. Adv Funct Mater 29 (26),

Radioactive (90Y) upconversion nanoparticles conjugated with recombinant targeted toxin for synergistic nanotheranostics of cancer

Group of oncanotechnology,  Laboratory of molecular immunology

We report combined therapy using upconversion nanoparticles (UCNP) coupled to two therapeutic agents: beta-emitting radionuclide yttrium-90 (90Y) fractionally substituting yttrium in UCNP, and a fragment of the exotoxin A derived from Pseudomonas aeruginosa genetically fused with a targeting designed ankyrin repeat protein (DARPin) specific to HER2 receptors. The resultant hybrid complex UCNP-R-T was tested using human breast adenocarcinoma cells SK-BR-3 overexpressing HER2 receptors and immunodeficient mice, bearing HER2-positive xenograft tumors. The photophysical properties of UCNPs enabled background-free imaging of the UCNP-R-T distribution in cells and animals. Specific binding and uptake of UCNP complexes in SK-BR-3 cells was observed,with separate 90Y- and PE40-induced cytotoxic effects characterized by IC50 140 μg/mL (UCNP-R) and 5.2 μg/mL (UCNP-T), respectively. When both therapeutic agents were combined into UCNP-R-T, the synergetic effect increased markedly, ∼2200-fold, resulting in IC50 = 0.0024 μg/mL. The combined therapy with UCNP-R-T was demonstrated in vivo. PNAS USA, 2018. In colaboration with Lobachevsky University.

Publications

  1. Shilova ON, Shilov ES, Lieber A, Deyev SM (2018). Disassembling a cancer puzzle: Cell junctions and plasma membrane as targets for anticancer therapy. J Control Release 286, 125–136
  2. Guryev EL, Volodina NO, Shilyagina NY, Gudkov SV, Balalaeva IV, Volovetskiy AB, Lyubeshkin AV, Sen AV, Ermilov SA, Vodeneev VA, Petrov RV, Zvyagin AV, Alferov ZI, Deyev SM (2018). Radioactive (90Y) upconversion nanoparticles conjugated with recombinant targeted toxin for synergistic nanotheranostics of cancer. Proc Natl Acad Sci U S A 115 (39), 9690–9695
  3. Sokolova EA, Vodeneev VA, Deyev SM, Balalaeva IV (2018). 3D in vitro models of tumors expressing EGFR family receptors: a potent tool for studying receptor biology and targeted drug development. Drug Discov Today 24 (1), 99–111
  4. Shipunova VO, Zelepukin IV, Stremovskiy OA, Nikitin MP, Care A, Sunna A, Zvyagin AV, Deyev SM (2018). Versatile Platform for Nanoparticle Surface Bioengineering Based on SiO2-Binding Peptide and Proteinaceous Barnase, Barstar Interface. ACS Appl Mater Interfaces 10 (20), 17437–17447

SUBPOPULATION OF HLA-DR-POSITIVE NK-CELLS CHARACTERIZED BY HIGH PROLIFERATIVE AND FUNCTIONAL ACTIVITY

Laboratory of cell interactions

In order to elaborate clones of cytotoxic lymphocytes for clinical application, a method has been developed for the expansion of NK cells using irradiated K562-mbIL21 cells expressing the membrane-bound form of IL-21. The distribution in the peripheral blood and the functional features of the HLA-DR+ NK cells, which are predominant in the populations of NK cells obtained using this method of stimulation, have been characterized.

Plasmonic gold nanoparticles for photothermal therapy of cancer

Laboratory of molecular immunology

The new agents for tumor theranostics with different mechanisms of action were constructed on the base of hybrid nanoparticles. The 5 nm gold nanoparticles conjugated with designed ankyrin repeat protein (DARPin), which specifically targets human epidermal growth factor receptor 2 (HER 2), are of the utmost interest.  The high stability under physiological conditions and high a ffinity to the receptors overexpressed by cancer cells make conjugates of plasmonic gold nanostructures with DARPin molecules promising candidates for cancer photothermal therapy. This work was supported by the Russian Science Foundation (project no.14-2400106). 

Publications

  1. Deyev S, Proshkina G, Ryabova A, Tavanti F, Menziani MC, Eidelshtein G, Avishai G, Kotlyar A (2017). Synthesis, Characterization, and Selective Delivery of DARPin-Gold Nanoparticle Conjugates to Cancer Cells. Bioconjug Chem 28 (10), 2569–2574
  2. Mironova KE, Khochenkov DA, Generalova AN, Rocheva VV, Sholina NV, Nechaev AV, Semchishen VA, Deyev SM, Zvyagin AV, Khaydukov EV (2017). Ultraviolet phototoxicity of upconversion nanoparticles illuminated with near-infrared light. Nanoscale 9 (39), 14921–14928
  3. Semenova G, Stepanova DS, Dubyk C, Handorf E, Deyev SM, Lazar AJ, Chernoff J (2017). Targeting group i p21-activated kinases to control malignant peripheral nerve sheath tumor growth and metastasis. Oncogene 36 (38), 5421–5431
  4. Sokolova E, Guryev E, Yudintsev A, Vodeneev V, Deyev S, Balalaeva I (2017). HER2-specific recombinant immunotoxin 4D5scFv-PE40 passes through retrograde trafficking route and forces cells to enter apoptosis. Oncotarget 8 (13), 22048–22058
  5. Liang L, Lu Y, Zhang R, Care A, Ortega TA, Deyev SM, Qian Y, Zvyagin AV (2017). Deep-penetrating photodynamic therapy with KillerRed mediated by upconversion nanoparticles. Acta Biomater 51, 461–470

Fluorogenic marker for instant live-cell membrane staining and imaging

Group of growth factors expression and differentiation,  Group of Molecular Physiology,  Laboratory of molecular theranostics

A new organic-compound-based fluorogenic marker has been created for live-cell membrane staining. Unlike current commercial cell markers, the obtained fluorogenic marker does not fluoresce in the aquatic environment, but acquires fluorescence immediately after being placed in a nonpolar medium, for example, in the cell membrane. This property allows one to instantly stain cells without further washing out the unbound dye. This marker can be applied in fluorescence microscopy for live-cell imaging and flow cytometry.

Publications

  1. Pakhomov AA, Deyev IE, Ratnikova NM, Chumakov SP, Mironiuk VB, Kononevich YN, Muzafarov AM, Martynov VI (2017). BODIPY-based dye for no-wash live-cell staining and imaging. Biotechniques 63 (2), 77–79

Ethanol-dependent expression of the NKG2D ligands MICA/B in human cell lines and leukocytes

Laboratory of cell interactions

Stress-induced molecules MICA and MICB are capable to regulate activity of cytotoxic lymphocytes through the interaction with receptor NKG2D, which substantially affects the functionality of cellular immunity. In cell line models ethanol caused different changes in surface expression of MICA/B, particularly it induced the translocation of intracellular proteins MICA/B to the cell surface and shedding of MICA (in soluble and microparticle-associated forms) from the plasma membrane. The observed results are not linked with cell death in cultures, taking place only under higher doses of ethanol. Ethanol at physiologically relevant concentrations (and higher) stimulated expression of MICA/B genes in different cell types. Presumably, changes in MICA/B expression, caused by ethanol, can affect functions of NKG2D-positive cytotoxic lymphocytes, modulating immune reactions at excessive alcohol consumption.

Retroviral gene transfer into primary human NK cells activated by IL-2 and K562 feeder cells expressing membrane-bound IL-21

Laboratory of cell interactions

Natural killer (NK) cells are capable of rapidly recognizing and efficiently killing tumor cells. This makes them a potentially promising agent for cancer immunotherapy. Additional genetic modifications of NK cells may further improve their anti-tumor efficacy. Numerous technical challenges associated with gene delivery into NK cells have significantly tempered this approach. We achieved efficient retroviral vector transduction of primary human NK cells that were stimulated by a combination of IL-2 and engineered K562 cells expressing membrane-bound IL-21. The activated NK cells were in less differentiated state and expressed NK cell activation receptors NKG2D, NKp30, CD16, and were highly HLA-DR-positive. This NK cell population was highly susceptible to the transduction by both GFP- and NGFR-expressing retroviral vectors. More mature CD57+ NK cell population was generally resistant to retroviral vector transduction because of poor response to the stimulation. Our findings may facilitate retroviral vector-mediated genetic engineering of human primary NK cells for future immunotherapies.

The localization of positively and negatively charged chitosan nanoparticles in tumor cells has been characterized

Laboratory of cell interactions

In collaboration with Centre of Bioengineering RAS, Moscow

 

Using in vitro culture of RAW264 cells and nanoparticles fabricated from positive and negative chitosan derivations we demonstrated penetration of the particles into tumor cells and their intracellular localization: in mitochondria for positively charged nanoparticles and in lysosomes for negatively charged nanoparticles. Accumulation of both types of the particles resulted in reducing mitochondrial membrane potential and in exocytosis of mitochondria and lysosomes from live tumor cells. Our results suggest that positively charged chitosan derivates may be used for development of nanopreparations for treatment of the diseases with mitochondrial dysfunctions, and negatively charged chitosan derivates – for anticancer preparations.

Zubareva A.A., Shcherbinina T.S., Varlamov V.P., Svirshchevskaya E.V. Intracellular Sorting of Differently Charged Chitosan Derivatives and Chitosan-Based Nanoparticles. NanoScale. 2015 Nanoscale, 2015, 7, 7942 - 7952.