Ekaterina N. Lebedenko

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.

Self-Assembling Complexes of Quantum Dots and scFv Antibodies for Cancer Cell Targeting and Imaging

Semiconductor quantum dots represent a novel class of fluorophores with unique physical and chemical properties which could enable a remarkable broadening of the current applications of fluorescent imaging and optical diagnostics. Complexes of quantum dots and antibodies are promising visualising agents for fluorescent detection of selective biomarkers overexpressed in tumor tissues. Here we describe the construction of self-assembling fluorescent complexes of quantum dots and anti-HER1 or anti-HER2/neu scFv antibodies and their interactions with cultured tumor cells. A binding strategy based on a very specific non-covalent interaction between two proteins, barnase and barstar, was used to connect quantum dots and the targeting antibodies. Such a strategy allows combining the targeting and visualization functions simply by varying the corresponding modules of the fluorescent complex.

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

Passive and active targeting of quantum dots for whole‐body fluorescence imaging of breast cancer xenografts

Far-red and near-infrared fluorescent quantum dots (QDs) have become advancing contrast agents for efficient whole-body tumor imaging. In this study, we investigated the possibility of the vital fluorescence imaging of tumor using two contrast agents on the basis of QDs: bioinert QDs coated with polyethyleneglycol and QDs bound with anti-HER2/neu scFv antibodies. HER2/neu-positive breast cancer tumor xenografts in nude mice were used as a model. It was shown that both bioinert and tumor-targeted QD probes can be successfully applied for visualization of the tumor using in vivo imaging method, but fluorescent signal of QD-4D5scFv in tumors was considerably stronger than that of QD-PEG.

A high-level prokaryotic expression system: synthesis of human interleukin 1α and its receptor antagonist

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

Imaging of human ovarian cancer SKOV-3 cells by quantum dot bioconjugates.

41 Development of molecular approaches to treat ment of oncological diseases determines the necessity of development ultrasensitive and highly precise methods for rapid visualization of specific markers of tumor cells. The main role of such cellular molecular markers, which are determined immediately in tumor tissue, is that, by characterizing the molecular profile of each certain tumor, they may help to determine the immune status of the tumor and select individual ther apy course. Creation of fluorescent constructs able to specifically bind to such markers is a promising approach to early molecular diagnostic of cancer. Semiconductor nanocrystals (quantum dots) are a new class of fluorophores widely used in various bio logical studies. In comparison to organic dyes and flu orescent proteins, which are conventionally used in fluorescence diagnostics, quantum dots have a num ber of advantages, such as a high quantum yield, inten sity, high molecular extinction coefficient, broad absorption spectrum, high resistance to photobleach ing, and exceptional resistance to photodegradation and chemical degradation. Generalized results of studies performed in recent years showed that quan tum dots linked to various guiding agents specific for tumor markers are promising tools for fluorescence diagnostics and determination of immune status of tumors and visualization of peripheral metastases (for review, see [1]). Targeted delivery of quantum dots is successfully ensured using full length monoclonal antibodies linked to nanocrystals directly through various cross linking agents or using a three stage labeling system based on the biotin–streptavidin affinity pair (primary antibodies, biotinylated secondary antibodies, and conjugates of quantum dots with streptavidin). How ever, in some cases, the use of constructs based on the antigen binding fragments of antibodies is more promising. In contrast to full length antibodies, these small fragments contain no constant domain; this has no effect on their guiding properties but reduces the possibility of occurrence of side effects caused by interaction of constant domains with immune cell receptors and complement proteins. An example of such constructs are single chain Fv fragments (scFv antibodies)—recombinant polypep tides in which variable domains of the light and heavy immunoglobulin chains are linked through a flexible linker. Such small (25–30 kDa) antibodies rapidly reach target cells, effectively recognize the antigen, and are easily withdrawn from the body [2]. In this study, we described a construct based on flu orescent CdSe/CdS nanocrystals and genetic engi neering recombinant miniantibodies, intended for detecting cells overexpressing the HER2/neu onco marker. The HER2/neu protein belonging to the fam ily of epidermal growth factors is an important marker in diagnosing cancer diseases. Its overexpression on the surface of ovarian, cervical, bladder, colonic, stomach, esophageal, and breast tumor cell correlates with unfavorable prognosis of disease development and increased resistance to chemotherapy. For this reason, early detected of cells overexpressing HER2/neu is of great clinical importance [3].

SYNTHESIS AND EXPRESSION OF SOME GENES FOR HUMAN CYTOKINES

Abstract Intronless genes for human mature interleukin 1a (IL1a) and its receptor antagonist (IL1ra) have been synthesized and efficiently expressed in a specially devised bacterial plasmid vector as part of a versatile two-cistron prokaryotic expression system.

Construction of a Translation Enhancer-Containing Vector for Gene Expression in a Prokaryotic Two-Cistron System

Abstract A prokaryotic two-cistron system for gene expression, based on the pGEM1 plasmid and containing a translation enhancer in the coding part of the first cistron, has been constructed. The gene to be expressed can be inserted into the vector by means of a PCR-mediated approach using type IIS restriction endonucleases (SDL method). Efficiency of the system is exemplified by the expression of a synthetic gene encoding human interleukin lα.