Sergey M. Deyev

Design of multivalent complexes using the barnase·barstar module

The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185HER2-ECD 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185HER-2-ECD antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185HER-2-ECD-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.

Protein-assisted self-assembly of multifunctional nanoparticles

A bioengineering method for self-assembly of multifunctional superstructures with in-advance programmable properties has been proposed. The method employs two unique proteins, barnase and barstar, to rapidly join the structural components together directly in water solutions. The properties of the superstructures can be designed on demand by linking different agents of various sizes and chemical nature, designated for specific goals. As a proof of concept, colloidally stable trifunctional structures have been assembled by binding together magnetic particles, quantum dots, and antibodies using barnase and barstar. The assembly has demonstrated that the bonds between these proteins are strong enough to hold macroscopic (5 nm–3 μm) particles together. Specific interaction of such superstructures with cancer cells resulted in fluorescent labeling of the cells and their responsiveness to magnetic field. The method can be used to join inorganic moieties, organic particles, and single biomolecules for synergistic use in different applications such as biosensors, photonics, and nanomedicine.

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

Background RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells. Methodology/Principal Findings In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC50) ranging from 0.2 to 13 µM and to leukemia cell lines with IC50 values ranging from 2.4 to 82 µM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC50 = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3. Conclusions/Significance These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.

Biocomputing based on particle disassembly

Nanoparticles with biocomputing capabilities could potentially be used to create sophisticated robotic devices with a variety of biomedical applications, including intelligent sensors and theranostic agents. DNA/RNA-based computing techniques have already been developed that can offer a complete set of Boolean logic functions and have been used, for example, to analyse cells and deliver molecular payloads. However, the computing potential of particle-based systems remains relatively unexplored. Here, we show that almost any type of nanoparticle or microparticle can be transformed into autonomous biocomputing structures that are capable of implementing a functionally complete set of Boolean logic gates (YES, NOT, AND and OR) and binding to a target as result of a computation. The logic-gating functionality is incorporated into self-assembled particle/biomolecule interfaces (demonstrated here with proteins) and the logic gating is achieved through input-induced disassembly of the structures. To illustrate the capabilities of the approach, we show that the structures can be used for logic-gated cell targeting and advanced immunoassays.

Feasibility study of the optical imaging of a breast cancer lesion labeled with upconversion nanoparticle biocomplexes

Abstract. Innovative luminescent nanomaterials, termed upconversion nanoparticles (UCNPs), have demonstrated considerable promise as molecular probes for high-contrast optical imaging in cells and small animals. The feasibility study of optical diagnostics in humans is reported here based on experimental and theoretical modeling of optical imaging of an UCNP-labeled breast cancer lesion. UCNPs synthesized in-house were surface-capped with an amphiphilic polymer to achieve good colloidal stability in aqueous buffer solutions. The scFv4D5 mini-antibodies were grafted onto the UCNPs via a high-affinity molecular linker barstar:barnase (Bs:Bn) to allow their specific binding to the human epidermal growth factor receptor HER2/neu, which is overexpressed in human breast adenocarcinoma cells SK-BR-3. UCNP-Bs:Bn-scFv4D5 biocomplexes exhibited high-specific immobilization on the SK-BR-3 cells with the optical contrast as high as 10:1 benchmarked against a negative control cell line. Breast cancer optical diagnostics was experimentally modeled by means of epi-luminescence imaging of a monolayer of the UCNP-labeled SK-BR-3 cells buried under a breast tissue mimicking optical phantom. The experimental results were analyzed theoretically and projected to in vivo detection of early-stage breast cancer. The model predicts that the UCNP-assisted cancer detection is feasible up to 4 mm in tissue depth, showing considerable potential for diagnostic and image-guided surgery applications.

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.

Man-made antibodies and immunoconjugates with desired properties: function optimization using structural engineering

The review outlines progress and problems in the design of non-natural antibodies for clinical applications over the past 10–15 years. The modular structure of natural antibodies and approaches to its targeted modifications and combination with other structural elements and effector molecules are considered. The review covers modern methods for immunoglobulin engineering and promising strategies for the creation and applications of monoclonal antibodies, their derivatives and analogues, including abzymes and scaffolds, oriented to the use in the diagnosis and targeted therapy of cancer and other socially significant diseases. The bibliography includes 225 references.

ERBB oncogene proteins as targets for monoclonal antibodies.

General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

Cytotoxicity and non-specific cellular uptake of bare and surface-modified upconversion nanoparticles in human skin cells

The cytotoxicity and non-specific cellular uptake of the most popular composition of upconversion nanoparticle (UCNP), NaYF4:Yb3+:Er3+, is reported using normal human skin cells, including dermal fibroblasts and immortalized human epidermal linear keratinocytes (HaCaT). A new hydrophilization reaction of as-synthesized UCNPs based on tetramethylammonium hydroxide (TMAH) enabled evaluation of the intrinsic cytotoxicity of bare UCNPs. The cytotoxicity effects of the UCNP surface-coating and polystyrene host were investigated over the concentration range 62.5–125 μg/mL with 24-h incubation, using a MTT test and optical microscopy. The fibroblast viability was not compromised by UCNPs, whereas the viability of keratinocytes varied from 52% ± 4% to 100% ± 10% than the control group, depending on the surface modification. Bare UCNPs reduced the keratinocyte viability to 76% ± 3%, while exhibiting profound non-specific cellular uptake. Hydrophilic poly(D,L-lactide)- and poly(maleic anhydride-alt-1-octadecene)-coated UCNPs were found to be least cytotoxic among the polymer-coated UCNPs, and were readily internalized by human skin cells. Polystyrene microbeads impregnated with UCNPs remained nontoxic. Surprisingly, no correlation was found between UCNP cytotoxicity and the internalization level in cells, although the latter ranged broadly from 0.03% to 59%, benchmarked against 100% uptake level of TMAH-UCNPs.