High-Affinity Single-Domain Antibodies for Analyzing Human Apo- and Holo-Transferrin.

A highly efficient technology for generating new monoclonal single-domain recombinant antibodies (nanobodies) was used to obtain a panel of nanobodies recognizing human apo- and/or holo-transferrin. This article is devoted to the primary analysis of the properties of two different variants of the new nanobodies obtained by us, as well as to the demonstration of the unique potential of their application for diagnostic studies. The simultaneous use of immunosorbents based on these nanobodies apparently makes it possible to detect changes in the relative abundance of apo- and holo-transferrin in human biological fluids. Such changes could potentially be indicative of an increased risk or degree of development of pathological processes, such as malignant neoplasms in humans.

[Single-Domain Antibodies Used to Pretreat the Human Urinary Proteome in Cancer Biomarker Testing].

A number of single-domain antibodies (nanobodies) obtained previously to major marker blood proteins were tested as tools to preprocess urine samples from patients with bladder cancer. Nanobody-based tools demonstrated unique possibilities for noninvasive diagnostic studies along with other conventional methods, such as electrophoresis and, in prospect, mass spectrometric analysis. A testing of 22 samples from bladder cancer patients showed that the development of bladder cancer is accompanied by an increase in the urine contents of major blood proteins, including those known as potential bladder cancer biomarkers. New nanobody-based immunosorbents allow both specific enrichment and specific removal of particular antigenic proteins and subproteomes associated with them from a biological fluid. The isolation of immune complexes from the urine of a particular patient is of particular interest. An initial study of the complexes showed not only increased contents of IgA and IgG at advanced stages of the disease, but also many other components, which provide potential biomarkers of the pathological process in a particular patient. It is intended to use the approaches proposed in this work in a future larger-scale study of urine samples from patients with bladder cancer at different stages of the disease in order to identify new promising biomarkers of bladder cancer.

Generation and Evaluation of Bispecific Anti-TNF Antibodies Based on Single-Chain VHH Domains.

Systemic cytokine inhibition may be an effective therapeutic strategy for several autoimmune diseases. However, recent studies suggest that tissue or cell type-specific targeting of certain cytokines, including TNF, may have distinct advantages and show fewer side effects. Here we describe protocols for generating and testing bispecific cytokine inhibitors using variable domain of single-chain antibodies from Camelidae (VHH) with a focus on cell-specific TNF inhibitors.

[Prospective Applications of Single-Domain Antibodies in Biomedicine].

Methods that utilize highly specific antibodies, anti-idiotypic antibodies, various recombinant molecules with antibody properties and immunocorrection and immunoprophylaxis with the help of vaccines are in demand and are intensely developed in the field of biomedicine. Techniques to generate specific single-domain recombinant antibodies (nanobodies) and their derivatives have raised great expectations in the past years. The review considers the recent literature data on the use of nanobodies in basic research, diagnosis, and design of new immunotherapeutic agents. Special sections focus on the prospects of using nanobodies as targeted molecules of microbiota components, the use of anti-idiotypic nanobodies, and a search for promising targets for early diagnosis based on nanobodies.

[Differentially Expressed Long Noncoding RNAs in the Promoter Region of the fork head Gene in Drosophila melanogaster Detected by Northern Blot Hybridization].

It is known that long (200-300 nucleotides and longer) non-protein-coding RNAs (ncRNAs) tissue-specifically expressed from the regulatory regions of developmental genes can regulate the transcription of the mRNA of these genes. In this study, an attempt is made to identify differentially expressed ncRNAs in the extended promoter region of the fork head (fkh) gene of the fruit fly Drosophila melanogaster. We investigated four preparations of the total RNA: from embryos, from adult flies (separately from females and males), and from the S2 cell line of cultured Drosophila cells. In the total RNA preparations from embryos and adult flies, the levels of fkh expression differed substantially, whereas in S2 cells its expression is not detected at all (shown in this work). We perform classical Northern blot analysis of gel-separated RNAs hybridized to a series of radioactively labeled DNA fragments corresponding to the adjacent and partially overlapping regions of the promoter region of the fkh gene. Several previously unknown differentially expressed ncRNAs are detected, including those in the regions overlapping with the previously detected regulatory elements (TRE1 and salivary gland enhancer sgE) and the transcription start site of the fkh gene. The collected data complement and clarify the results of the previously conducted RNA-seq experiments, in particular, in terms of the length of the detected RNAs. These results may serve as a foundation for further studies of the mechanisms of tissue-specific regulation of the fkh gene expression.

[A Method for the Parallel and Sequential Generation of Single-Domain Antibodies for the Proteomic Analysis of Human Blood Plasma].

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.

[Single-domain adapted antibodies against Chlamydia trachomatis, preserving the development of chlamidic infection in vitro]. / Odnodomennye adaptirovannye antitela protiv Chlamydia trachomatis, podavliaiushchie razvitie khlamidiinoi infektsii v usloviiakh in vitro.

The technology for the generating of single-domain recombinant monoclonal antibodies (nanoantibodies) based on the immunization of a camel, cloning of induced sequences encoding single-domain antigen-recognizing fragments of non-canonical camel antibodies, as well as functional selection of clones of nanoantibodies by the phage display method, was used to obtain new effective tools for more efficient diagnostics of Chlamydia infection and to develop new approaches for effective therapy. Two promising nanoantibodies were obtained. They showed effective binding to extracellular and intracellular forms of C. trachomatis, and also had activity that inhibited the development of chlamydial infection in vitro.

Construction of a pIX-modified Adenovirus Vector Able to Effectively Bind to Nanoantibodies for Targeting.

Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to "nanoantibody-modified RPAN" binding.

Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk.

Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated KD varied from 0.43nM to 3.7nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins.

Experimental models of arthritis in which pathogenesis is dependent on TNF expression.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by joint damage as well as systemic manifestations. The exact cause of RA is not known. Both genetic and environmental factors are believed to contribute to the development of this disease. Increased expression of tumor necrosis factor (TNF) has been implicated in the pathogenesis of RA. Currently, the use of anti-TNF drugs is one of the most effective strategies for the treatment of RA, although therapeutic response is not observed in all patients. Furthermore, due to non-redundant protective functions of TNF, systemic anti-TNF therapy is often associated with unwanted side effects such as increased frequency of infectious diseases. Development of experimental models of arthritis in mice is necessary for studies on the mechanisms of pathogenesis of this disease and can be useful for comparative evaluation of various anti-TNF drugs. Here we provide an overview of the field and present our own data with two experimental models of autoimmune arthritis - collagen-induced arthritis and antibody-induced arthritis in C57Bl/6 and BALB/c mice, as well as in tnf-humanized mice generated on C57Bl/6 background. We show that TNF-deficient mice are resistant to the development of collagen-induced arthritis, and the use of anti-TNF therapy significantly reduces the disease symptoms. We also generated and evaluated a fluorescent detector of TNF overexpression in vivo. Overall, we have developed an experimental platform for studying the mechanisms of action of existing and newly developed anti-TNF drugs for the treatment of rheumatoid arthritis.

Molecular analysis of heavy chain-only antibodies of Camelus bactrianus.

In this work, IgG content and structures of antigen-binding domains and hinge regions of different IgG subtypes of Camelus bactrianus were analyzed in detail for the first time. Our data demonstrate that C. bactrianus contains a very large amount of heavy chain-only antibodies that can be used as a source of VHH domain-containing molecules. Despite some minor sequence differences identified in this study, C. bactrianus VHH domains possess principally the same unique features as those of C. dromedarius and the llama. These features are important for developing an efficient phage display-based antibody selection technology. We conclude that C. bactrianus is a very suitable animal to raise an immune response that serves as a source to identify antigen-specific VHHs selected after phage display.

Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor A(165).

Nanoantibodies (single-domain antibodies, nanobodies) derived from noncanonical single-chain immunoglobulins provide an attractive tool for in vitro and in vivo diagnostics as well as for development of targeted drugs for clinical use. Nanoantibodies against several clinically important targets have been developed and are actively investigated. However, no development of nanoantibodies against vascular endothelial growth factor VEGF-A(165) has been reported. We describe here the generation of nanoantibodies derived from single-chain Bactrian camel immunoglobulins directed against VEGF-A(165). We demonstrate that these nanoantibodies are suitable for enzyme-linked immunoassay to quantify human VEGF-A(165) as well as for blocking its activity. Our results provide a basis for diagnostic kit development for quantification of VEGF-A(165), which emerges as a biomarker useful in various pathological conditions. In addition, the nanoantibodies might be used for development of therapeutic molecules targeting VEGF-A(165)-dependent pathological neoangiogenesis.

["Camel nanoantibody" is an efficient tool for research, diagnostics and therapy].

This short review provides an introduction to the rapidly developing field of generation and utilization of "camel nanoantibodies" (or "nanobodies"). The term "nanoantibody" or "nanobody" was given to single-domain variable fragments of special type of antibodies that naturally exist (in addition to classical types of antibodies) in blood of Camelidae family animals and in some chondrichthyan fishes. The existence of very efficient technology of nanobody generation and some very useful characteristic features promise a big potential for their use in immunobiotechnology and medicine.

Effective Genetic Expression of Nanoantibodies by Recombinant Adenoviral Vector in vitro.

The present study is devoted to the feasibility of expressing the single-domain mini-antibody (nanoantibody) selected from the library of sequences of the variable domains of special single-stranded antibodies derived from an immunized camel, a gene of which was introduced into eukaryotic cells within a recombinant adenoviral vector. A vector bearing the gene of a single-domain nanoantibody was obtained using the AdEasy Adenoviral Vector System (Stratagene). This method of delivering the nanoantibody gene facilitates efficient expression of this gene and functional activity of the nanoantibody. The results obtained can be used to produce passive immunizing tools against pathogens or new-generation immunobiological antitoxic medication.

Fingerprint-like Analysis of "Nanoantibody" Selection by Phage Display Using Two Helper Phage Variants.

This paper discusses the selection of mini-antibody (nanoantibody, nanobody® or single domain antibody) sequences of desired specificity by phage display-based method using a generated library of antigen-binding domains of special heavy-chain only antibodies (single-stranded antibodies) of immunized camel. A comprehensive comparison of the efficiency of parallel selection procedures was performed by using the traditional (M13KO7) and modified (with N-terminal deletion in the surface gIII protein) helper phages. These two methods are partly complementary, and by using them in parallel one can significantly improve the selection efficiency. Parallel restriction analysis (fingerprinting) of PCR-amplified cloned sequences coding for mini-antibodies (HMR-analysis) is proposed for identifying individual clones, as a replacement to sequencing (to a certain extent). Using this method, unique data were collected on the selection of mini-antibody variants with the required specificity at various stages of a multi-stage selection procedure. It has been shown that different sequences coding for mini-antibodies are selected in different ways, and that, if this feature is not taken into account, some mini-antibody variants may be lost.

Tumor Necrosis Factor and the consequences of its ablation in vivo.

Although TNF has been discovered due to anti-tumor activity, its physiological functions are different. Current knowledge places TNF downstream of many receptors of innate immunity, implying its primary role in host defense and inflammation. When overproduced systemically or locally, TNF may exert deleterious effects on the organism. Anti-TNF therapy is highly efficient in several autoimmune and inflammatory diseases. However, due to TNF unique beneficial functions in immune system, such therapy cannot be entirely free of adverse effects. We review the current status of the field with the focus on drugs and strategies used for TNF ablation in vivo.

Camelid immunoglobulins and nanobody technology.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.