Nanoantibody specifically binding cea protein, method of its usage for detection of this protein

FIELD: biotechnologies.

SUBSTANCE: single-domain antibody (nanoantibody) is proposed, which specifically binds a carcino-embrional antigen (CEA) of a human being and characterised by a full amino acid sequence. Also the method is considered to detect a CEA protein in biological fluids and tissues of a human being with usage of a nanoantibody according to the invention.

EFFECT: invention may find further application in diagnostics and therapy of cancer.

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The technical field of the present invention

The present invention relates to the field of molecular immunology, biotechnology and medicine.

Prior art

The carcinoembryonic antigen (CEA Russian-language publications) or Carcinoembryonic antigen (CEA in English-language publications), is a glycoprotein with a molecular weight of about 180-200 kDa, produced by the gastrointestinal tract and pancreas of the embryo and secreted into the circulation system. In adults, the CEA is determined by tumors in the pleural fluid in the effusion in the ascites, the secrets of the gastrointestinal tract and urine. CEA is the most widely used marker of cancer of the gastrointestinal tract. Although CEA is primarily associated with rectal cancer, the increased level can also be caused by malignant tumors of the breast, lung, stomach, ovary and other organs. It was shown that in the serum of patients with various types of carcinomas frequently detected elevated (compared with the sera of healthy individuals) levels of CEA (above 2.5 ng/ml). Increasing the concentration of CEA can cause and some benign processes, such as pneumonia, cirrhosis of the liver or benign neoplasms. In the group of heavy smokers, the level of CEA in serum exceeds nor the actual values for healthy people. Quantitative determination of CEA is used for mass screening for early diagnosis of tumors. In clinical practice, research level of CEA are used mainly for the diagnosis of recurrent colorectal cancer and colon cancer after surgery, as well as for monitoring patients with diagnosed malignancies with elevated levels of CEA.

Protein CEA was first identified in 1965 in extracts of tissues of human colon (Gold and Freedman, 1965. The Journal of experimental medicine, 121: 439-462). Since that time, and especially in recent years, substantial progress has been made in the study of this and similar proteins. Today, CEA-like proteins known to be involved in the processes of cell adhesion (binding of cells to extracellular matrix and other cells), merge into the family SEASON (CEA-related cell adhesion molecules) and the immunoglobulin superfamily of cell adhesion molecules (IgCAM). In humans, the proteins of this family are encoded 29 genes. These genes encode proteins, referred to as SEASON, SEASON - SEASON, SEASON 16, SEASON 18 - SEASON and PSG1-PSG11 (11 glycoproteins, the expression of which is associated with pregnancy). All proteins of this family belong to the superfamily of immunoglobulins (Ig) and consist of one variable (similar to the variable domain of the Ig)and varying number constant (the same constant domains Ig) domains. SEASON-molecules normally associated with the cellular membrane transmembrane domain or anchored with the participation of glycosyl phosphatidylinositol modification. Protein function is SEASON-family varies widely: they function as adhesion molecules, tumor suppressors and regulators of the activity of dendritic cells and lymphocyte receptors of bacteria. Actually the above-mentioned protein CEA corresponds in the new nomenclature squirrel SEASON. Study of the expression level of this protein as a tumor marker in tumors of epithelial origin and today remains one of the most common diagnostic tests. (Horst, A., Wagener SSEA-related CAMs. / CEA-like cell adhesion molecules. (2004) In: Behrens J, Nelson WJ, editors. Cell Adhesion. Nelson Lab. pp.283-341. K. Kuespert, Pils, S., C.R. Hauck CEACAMs: their role in discrimination and pathophysiology. / SEASON proteins: their role in physiology and pathophysiology. Curr. Opin. In Cell Biol. 2006, 18, 565-571).

Protein CEA/CEACAM5 is the product of the gene SEASON and consists of 7 extracellular domains: 34-amino acid sequence of the leader peptide, followed by a 108-amino acid N-terminal site that is similar to the variable domain of the Ig, followed by three dual-domain 178-amino acid stretch that is similar to the constant C2 domain of Ig and named, respectively, A1 and B1, A2 and B2, A3 and B3. At the end of this protein is a 27-amino acid stretch that includes glycotope Idil-inititally end, which polupronicaema the cell membrane and provides a binding protein on the cell surface (Berinstein, 2002, J.Clin.Oncol., v.20, 2197-2207).

In recent years increasing attention in the creation of new diagnostic and therapeutic products for the detection and treatment of many diseases on drugs based on antibodies, usually monoclonal classical antibodies. The antibody is able to specifically bind to a specific antigen/protein as free or in complex with other molecules, and protein, overexpression and localized on the surface of cells of a particular type. Such binding may block important biological process, cascade, and to stimulate the immune system of the patient to attack cells that have contacted the antibody. The antibody can also be a means for the specific delivery of other therapeutic molecules, compounds or radioisotopes. Finally, you can use di - and poly-functional and multi-component structures on the basis of the obtained antibodies, or derivatives thereof. Previously have repeatedly carried out to obtain antibodies specifically bind to the protein CEA. The closest technical solution, which is the basis of the present invention, one can cite the following example.

Application WO2011023787 (Affinity-maturated humanzed anti CEA monoclonal antibodies - The last process of affinity maturation humanized anti-CEA monoclonal antibodies, date of publication - 03.03.2011). This patent describes obtaining humanized monoclonal antibodies, have undergone a process of affinity maturation in vitro by introducing new mutations in hypervariable sites and subsequent selection of the most high-affinity variants of antibodies using the phage display technique. Antibodies were obtained to the epitope of CEA-specific B3 domain which is specific for CEA protein associated with the cell surface. The resulting antibodies can be used for the detection of membrane-bound CEA, and therapeutic effects on tumor cells on which surface overexpression CEA.

This technical solution is the closest to the claimed composition of the active substance and how it is used is selected by the authors of the present invention for the prototype.

The disadvantages of the prototype are:

- Relatively expensive production of antibodies, the complexity of the maintenance and storage of a producer, very high demands on the quality of reagents and culture conditions.

The relatively large size of antibodies, which leads to reduced permeability of fabrics.

- Structural features impose Ogre is a restraint to recognize some hidden epitopes, are, for example, grooves, slits small size in the structure of proteins.

- Limited and the relative complexity of genetic engineering manipulation, adaptation for specific tasks, the complexity of creating multivalent and multifunctional derivatives of the claimed antibodies.

Thus, in the prior art there is a need to develop new antibody - antigen-binding molecules devoid of the above disadvantages and specifically recognizing the protein of CEA.

The present invention is the creation of new antibodies capable of effectively binding protein CEA, obtaining these antibodies, production and storage which should be economical. New antibodies should be effective and relatively simple; their size should be several times smaller than the classical antibodies.

The technical problem is solved due to the fact that the obtained nanoparticle, specific binding protein CEA person and having the amino acid sequence of SEQ ID NO: 2. In this way the detection of CEA protein in biological fluids of a person includes bringing into contact a specified biological fluid with nanoentities according to claim 1 and detecting binding of nanoparticle according to claim 1 with CEA protein. Moreover, the detection of CEA protein using nanoparticle perform both in free form in biological fluids and in which ernesti cells of different types, for example, a tumor, where the protein overexpression.

The basis of the present invention are not classical bivalent antibodies, which are considered as a prototype, and small single-domain of nanoparticle, which have several advantages compared to classical monoclonal antibodies for practical use in treatment of diseases. Because nanoparticle (molecular weight of about 12-15 kDa) on the order of smaller traditional classical antibodies, they acquire a number of new positive qualities of practical importance. Nanoparticle may contain novel structural features, in principle allowing them to learn some "hidden" for normal antibody epitopes. There are effective methods of obtaining and selection of such antibodies against various, including netcommunity antigens.

Full equivalent of the term "single domain of nanoparticle" for the purposes of the present invention is subsumed into General use designation "nanotesla"entered the firm ABLYNX, as well as "single-domain mini-antibody".

Recombinant single domain of nanoparticle is obtained on the basis of a special non-canonical single-chain antibodies existing in the norm along with the classical antibodies in animals of the family of Camelids and in some species the cartilaginous fishes. These specific antibodies consist of a dimer of only one shortened without first constant region SN heavy chain immunoglobulin and fully functional in the absence of the light chain of the immunoglobulin. For the specific recognition and binding of the antigen when it is necessary and sufficient for only one variable domain (VHH, "nanoparticle", "nanobody" or a single domain nanoparticle) of this antibody. Organization variable domains (VHH) of the non-canonical antibodies substantially similar to that of the variable domains (VH) classical antibodies (human VH domains of antibodies of the IgG3 subclass are particularly pronounced homology to the VH and VHH camelids). In both cases, V-domains consist of four conservative frame sections (FR, "framework regions")surrounding the three hypervariable area defining complementarity, CDR, from a "complementarity determining regions". In both cases, the domains form the typical V-domain immunoglobulin spatial structure of the two beta-layers (sheets), one of four amino acid chains, and the second of the five (Padlan E.A. X-Ray crystallography of antibodies. Adv. Protein Chem. 1996; 49: 57-133. Muyldermans, S., Cambillau C, Wyns L. Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Recognition of antigens by single-domain fragments of antibodies: superfluous luxury of paired domains. TIBS 2001; 26: 230-235). In this structure, all three hypervariable the x plot are clustered on one side of the V-domain, where they are involved in antigen recognition and are located in the loops connecting the beta-structure. However, there are important differences relating to the operation of VHH in the format of a single domain. So, hypervariable sites CDR1 and CDR3 significantly increased in the case of VHH. Often in hypervariable sites VHH detected cysteine residues, and are present in two areas (mostly in CDR1 and CDR3, rarely in CDR2 and CDR3). In the study of crystal structures VHH was shown that these cysteine residues form a disulfide bond, which leads to an additional stabilization of the structure of loops of a given antigen. The most explicit and reproducible hallmark VHH are four substitutions of hydrophobic amino acid residues on the hydrophilic second frame section (Val37Phe, Gly44Glu, Leu45Arg, Trp47Gly, numbered according to Kabat). This framed area in the case of the VH domain is highly conserved, enriched in hydrophobic amino acid residues and are particularly important for the formation of ties with variable domain VL light chain. VHH domain in this respect is very different: the replacement of hydrophobic amino acids on the hydrophilic make it impossible for the Association VHH and VL. These replacements also explain the high solubility VHH, nanoparticle, when it is obtained in the form of a recombinant protein (Tillib ST. "Camel nanoparticle" is an effective tool for research, diagnosis and therapy. Molecular biology 2011; 45(1): 77-85).

Compared with the traditional and pure recombinant antibodies camel nanoparticle have a number of advantages, suggesting a great potential for their future use in various research and in the creation of new biotechnological devices, as well as for clinical purposes for the diagnosis and treatment of diseases.

Characteristics of single-domain nanoentities that define a great potential for their use for a variety of practical applications in immunobiotechnology, are the following (see review: Tillib ST. "Camel nanoparticle" is an effective tool for research, diagnostics and therapy. Molecular biology 2011; 45(1): 77-85).

- A highly efficient method of generating and breeding nanoentities.

- Small size, ~2×4 nm, 13-15 kDa (enhanced permeability of the cell).

- Structural features (the ability to form unusual for classical antibody paratope that allows to communicate with the recesses and the active centers of proteins); can be used to identify "hidden" epitopes or epitopes, which can not be recognized significantly larger conventional antibodies.

High expression yield, efficiency developments in large quantities. Usually n is nontitle develop in periplasm bacteria E.coli (1-10 mg from 1 liter of culture). The possibility of their effective practices in yeast, plants and mammalian cells.

- Easy various genetic manipulations, adaptation for specific tasks, the ability to create multivalent and multifunctional derivatives.

Low immunogenicity; the opportunity cost to "humanize" the antibody without appreciable loss of their specific activity.

The possibility of obtaining recombinant single domain of nanoentities with a given specificity is determined by the existence of the representatives of the family Camelidae functional and has a sufficiently wide range of recognition of the non-canonical antibodies. Non-canonical antibodies consist of a dimer of only one shortened heavy chain of immunoglobulin (without light chains), the specificity of recognition of which is determined by only one variable domain (Hamers-Casterman C, Atarhouch T, Muyldermans S, et al. Naturally occurring antibodies devoid of light chains. - Existing in nature antibodies without light chains. Nature 1993; 363:446-448). Technical implementation of the selection of single-domain nanoentities, which is genetically engineered derivatives of the antigen-recognition domain of single-chain antibodies camel, based on a highly efficient procedure for the selection of antigen-binding polypeptide exposed on the particle surface of filamentous phage "phage display".

Method f the new display is very effective and widely used technology for functional screening of large recombinant libraries of DNA sequences encoding peptides and proteins with desired properties and expressed in the composition of the surface protein of filamentous phage (Brissette R & Goldstein N1. The use of phage display peptide libraries for basic and translational research. - Using peptide phage-display libraries for basic research and research translation. Methods Mol Biol. 2007; 383:203-13; Sidhu SS & Koide S. Phage display for engineering and analyzing protein interaction interfaces. - Phage display for design and analysis of interactions of protein domains. Curr Opin Struct Biol. 2007; 17:481-7). One particularly important application of this technology is the generation of specific recombinant antibodies to various antigens (Hoogenboom HR. Selecting and screening recombinant antibody libraries. Selection and analysis of libraries of recombinant antibodies. Nat Biotechnol. 2005; 23:1105-16). Usually, instead of a great many molecules of classical antibodies for display on the surface of phage use a hybrid recombinant single-chain proteins, which represents a random combination of the cloned sequences of the variable regions of the heavy and light chains of immunoglobulins, United short serine/glycine-rich linker sequence. Such a chimeric molecule, if the correct combination of domains, are able to maintain the specificity of the original immunoglobulin, despite entered compared to the native antibody molecule changed the Yami. One of the problems with conventional recombinant technology is the need to work with very large libraries of recombinant antibodies, which should be represented all possible combinations of two random variable regions (heavy and light chains of immunoglobulins), United linker

sequence. Aside from the issue of representation here is clear and the problem of formation of the correct relative conformation of the two domains, and the problem of the solubility of the individual variable domains, which often have a tendency to aggregation. These problems may be avoided by using a single domain of nanoentities, as almost every clone variable domain of single-chain antibodies will in this case have a certain antigen-binding specificity, corresponding to one of the antibody immunized animal, and can now be selected from a relatively small library of such domains.

Single domain of nanoparticle with a given specificity or their derivatives can be used as classical antibodies, in various applications, including, but not limited to, detection of antigens (for both research and diagnostic purposes), by blocking the activity of a protein antigen, the specific the th delivery by binding to the desired antigen molecules, conjugated with the antibody.

Also a single domain of nanoparticle can be a source modules (blocks) more complex multimodal drugs.

It has been shown (Vincke, Loris R, Saerens d, et al. // J. Biol. Chem. 2009.V. 284. No. 5. P. 3273-3284)that you can "humanize" these camel nanoparticle without appreciable loss of their specific activity, having a small number of point substitutions of amino acids. This opens up the potential widespread use of nanoentities as a means of passive immunization for prevention of various infectious diseases (J. Wesolowski, Alzogaray V., Reyelt J. et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. -Single-domain antibodies: a promising experimental and therapeutic tools in the field of infection and immunity. Med. Environ. Immunol. 2009; 198, 157-174.).

The disclosure of the present invention

The method of producing nanoentities linking antigenic epitopes of the protein CEA includes the following principal stages:

1) induction of the formation of specific antibodies in the immunization representative of the family of Camelids (in particular, two-humped camel) ofinnocence protein CEA person;

2) cloning of the sequences of the antigen-binding domains (nanoentity) special single-chain antibodies, synthesized in the lymphocytes of the immunized alive the spas, in fahmida vector (used in the subsequent procedure of phage display), obtaining specifically enriched library sequences encoding nanoparticle;

3) selection of the method of phage display (function selection of phage particles bearing surface expressed by nanoparticle, and inside - DNA encoding this Nanojuncetea) clones of nanoentities, communicating with a given antigen, CEA protein, obtained from libraries nanoentities;

4) conducting perchlorovinyl and genetically engineered modifications of coding sequences selected nanoentities for their efficient solutions in the bacterial expression system and subsequent effective treatment (as producer of the active substance used E. coli).

As antigen for immunization and selection of used ofinnocence protein CEA person.

The method of obtaining a single domain of nanoentities described in examples 1 and 2.

Nanoparticle (molecular weight of about 12-15 kDa) on the order of smaller traditional antibodies and are fully functional antigen-binding units, with a number of new positive qualities of practical significance. Nanoparticle is a single-domain protein that is highly soluble and has a high stability in a wide range of the e temperature and pH). This avoids problems with solubility and proper folding of antibody molecules by products prokaryotic cells and leads to significantly lower costs of production compared to traditional methods of obtaining therapeutic monoclonal antibodies in eukaryotic expression systems. Considerably easier as well as for storing and transporting antibodies compared with significantly less stable traditional antibodies. Due to a smaller size nanoparticle have a greater ability to penetrate tissue. Finally, nanoparticle make it easy to carry out genetic engineering manipulations for the purpose of subsequent products bespecifically of nanoentities or chimeras, which in addition to nanoparticle is another protein with desired properties.

Although mainly for illustrative purposes, the authors present invention focuses on the production of antibodies using E. coli, the average expert in the art it will be obvious that under the scope of the present invention fall and other types of systems implementing the present invention not expressly stated herein. For example, as a eukaryotic producer can be used yeast culture or any other system, the obvious is the art.

The choice of the ways of implementation to obtain nanoentities with the claimed properties of prokaryotic expression system, in accordance with one of preferred embodiments of the present invention due to the following factors:

1) High expression yield, efficiency developments in large quantities due to the expression of nanoentities in periplasm bacteria E.coli (1-10 mg from 1 liter of culture).

2) Simplicity of various genetic manipulations, adaptation for specific tasks, the ability to create multivalent and multifunctional derivatives.

3) High economic profitability. The author of the present invention proceeds from the fact that, as

it is known to the average expert in the art, the primary source of the received sequence of single-domain nanoentities can then be adapted or "rich" in a different way for future practical use.

Thus, a single domain of nanoparticle can be a source modules (blocks) more complex multimodal drugs. It is possible to unite in one multivalent derived two, three or more monovalent primary single domain of nanoentities. These merged into one single domain design of nanoparticle may contact as the same epitope of the target antigen, and with its different epitopes or even with different antigens on target. It is also possible combo unite into one single domain design of nanoentities and other molecules or drugs with obtaining multifunctional drugs; multimerization with the introduction of additional amino acid sequences of the interacting protein domains, such as lacinova the zippers or small sequences of proteins that form stable complexes. For modulating properties of the preparation of single-domain nanoparticle, for example, to increase the lifetime or improved method of purification, the resulting compounds may be introduced an additional amino acid sequence. Average expert in the art it will be obvious that such modifications and other options antibodies underlying the present invention fall under the scope of the present invention, as are the structural and functional variants of a single domain of nanoentities. Thus, the authors of the present invention is meant by the term "single domain of nanoparticle" as the primary source derived, "minimum" amino acid sequences of single-domain nanoentities and their modifications resulting from the aforementioned adaptation or "formatting" and their vari the options. The term "variant antibodies" for the purposes of the present invention means a polypeptide that contains the changes in amino acid sequence, i.e. deletions, insertions, additions or substitutions of amino acids, provided that it retains the desired activity of the protein, e.g., at least 10% of the activity of the original single-domain of nanoparticle. A number of changes in the variant protein is dependent on the position or the type of amino acid residue in the three-dimensional structure of the protein. The amount of change may be, for example, from 1 to 30, more preferably from 1 to 15 and most preferably from 1 to 5 changes in the sequence of the original single-domain of nanoparticle. These changes can occur in regions of the polypeptide that are not critical to its function. This is possible due to the fact that some amino acids have a high homology with each other, and therefore the tertiary structure or activity of the protein is not violated by such a change. Therefore, as a variant protein can be a protein that is characterized by a homology of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, relative to the amino acid sequence of the original single-domain of nanoparticle while maintaining active the activity of the polypeptide. Homology between amino acid sequences can be established using well-known methods, for example using sequence alignment computer program BLAST 2.0, which calculates three parameters: the account, the identity and similarity.

Substitution, deletion, insertion, addition or substitution of one or several amino acid residues will represent a conservative mutation or conservative mutations, provided that the activity of the protein is maintained. An example of a conservative mutation(s) is a conservative substitution(s). "Conservative amino acid substitution" is a substitution in which the amino acid residue is substituted with amino acid residue having a similar side chain. In this field of technology is defined family of amino acids with similar side chains. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, series, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, the'alene, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). As hypervariable regions of nanoentities determine their specific interaction with the antigen, it is homologous replacement of amino acids in these areas can lead to several different sequences nanoentities, which have identical or similar properties. Thus, the average expert in the art it will be obvious that under the scope of the present invention are subject not only the application sequence nanoentities, but also those that can be obtained by substitutions of amino acids in the hypervariable sites (listed in the sequence listing as CDR1, CDR2 and CDR3, complementarity determining antibodies plots), but very similar in properties of amino acids (conservative substitutions).

DNA fragments that encode essentially the same functional polypeptide can be obtained, for example, by modifying the nucleotide sequence of the DNA fragment encoding the original single domain nanoparticle, for example, using the method of site-directed mutagenesis, so that one or more amino acid residues at a specific site will be deleterows, substituted, inserted or added. DNA fragments modified, as described above, can be obtained using traditional processing methods with the aim of obtaining mutations.

DNA fragments that encode essentially the same functional polypeptide original single domain of nanoparticle, can be obtained by ekspressirovali DNA fragments with mutations described above, and determine the activity of the expressed product.

Substitution, deletion, insertion or addition of nucleotides described above, also include mutations that occur in nature and, for example, due to variability.

The single domain polypeptide of nanoentities according to the present invention can be encoded by a large variety of molecules of nucleic acid, which is the result of well-known in the art, the phenomenon of degeneracy of the genetic code. The essence of the phenomenon is that any amino acids (except tryptophan and methionine), which is part of the natural peptides, can be encoded by more than one triplet nucleotide codon. Any of these degenerate molecules encoding nucleic acids may be part of cassettes expressing antibodies declared in accordance with the present invention, and falling under the scope of the present invention.

Obtain functionally active nanoentities ACEA-1, specifically linking the boe is OK CEA, described in examples 1 and 2.

The possibility of using the obtained nanoentities ACEA-1 for the detection of CEA protein in a free state, and in the case where it is associated with the surface of the cells where it is expressed in increased amounts in biological fluids of a person shown in the examples 3, 4.

Brief description of figures

Figure 1 presents the nucleotide sequence of cDNA encoding a received a single domain nanoparticle ACEA-1 (SEQ ID NO: 1). From it was derived the corresponding amino acid sequence nanoparticle (SEQ ID NO: 2). In this sequence are underlined, respectively (from left to right, from N - to C-end) hypervariable sites CDR1, CDR2 and CDR3, which are decisive for the specific recognition by nanoentities ACEA-1 protein CEA. Arrows indicate the positions of amino acid residues, which is characteristic for the variable domains of specific single-chain antibodies (different from the residues in the variable domains of the heavy chains of classical antibodies). In CDR1 and CDR3 parts of the ACEA-1 are identified cysteine residues, it is common in nanortalik and stabilizing nanoparticle, forming additional C-C bond between the first and third hypervariable sites.

Figure 2 presents electrophoregram polyacrylamide gel gained is passed through selected cloned the coding sequence and then purified nanoentities ACEA-1. Right - track (M) with marker proteins of known size (in kDa).

Figure 3 presents the results of the enzyme immunoassay recognition selected for single-Nanojuncetea ACEA-1 immobilized in the wells of immunological die ofinnocence protein CEA person. As control was used wells (No. 1) with the immobilized protein bovine serum albumin. The intensity of the signal reflects the efficiency of binding of nanoparticle ACEA-1.

Figure 4 presents the results of enzyme-linked immunosorbent assay (ELISA) detection of protein CEA expressed on the cell surface lines A and Lim1215 (or control CEA-negative cell line NEC), using selected nanoparticle ACEA-1 (100ng/ml). The top shows the cell with the corresponding three types of immobilized cells after conducting ELISA. The optical density at a wavelength of 405 nm is shown on the graph, represents the efficiency of binding of nanoparticle.

The embodiments of the present invention

Example 1

Obtaining a library of single-chain variable domains of antibodies

Immunization

Bactrian camel Camelus bactrianus consistently were immunized 5 times by subcutaneous administration of antigenic material mixed with an equal volume of complete (when the first injection) or incomplete (if the OST is lnyh injections) 's adjuvant adjuvant. As the antigen used protein CEA person, purified using immunoaffinity chromatography from the blood of a patient diagnosed with metastatic liver carcinoma. This protein (no R224) was purchased from the company "Hema Medica" (Russia). Took about 0.26 mg protein for each stage of the injection. The second injection (stage immunization) was performed 3 weeks after the first, then at intervals of two weeks spent three immunization. Blood samples (150 ml) was performed 5 days after the last injection. To prevent clotting of blood was added 50 ml of a standard phosphate-saline (PBS)containing heparin (100 units/ml) and EDTA (3 mm).

The blood was diluted 2 times with a solution of PBS containing 1 mm EDTA. 35 ml of diluted blood was layered on the step special medium (Histopaque-1077, Sigma) with a density of 1.077 g/ml volume of 15 ml and perform the centrifugation for 20 min at HD. Mononuclear cells (lymphocytes and monocytes) was collected from the interphase zone plasma/Histopaque, then washed with a solution of PBS containing 1 mm EDTA.

Total RNA from b-lymphocytes were isolated using TRIzol reagent (Invitrogen). Then on a column of oligo(ATA)-cellulose from the total RNA was purified poly(A)-containing RNA. The concentration of RNA was determined using biophotometer (Eppendorf) and checked the quality of the selected RNA with POM is by electrophoresis in 1.5%agarose gel with formaldehyde.

The reverse transcription reaction was performed according to standard Protocol (Sambrook et al., 1989) using reverse transcriptase H-M-MuLV and primer oligo(dt)15as seed.

The products of reverse transcription were used as matrix in the two-stage polymerase chain reaction and the resulting amplification products were cloned sites Ncol(Pstl) and Not! in formigny vector as described previously (Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006). The selection process was also similar to that in the mentioned works. It was based on the method of phage display, in which the phage-assistant used the bacteriophage MC (New England Biolabs, USA).

Example 2

Selection of single-domain nanoentities, specifically recognizing CEA.

Selection of single-domain nanoentities was performed by the method of phage display using the same, as for immunization, drug protein CEA man who was immobilized on the bottom of the holes 96-well ELISA plate. Used polystyrene immunological die with high sorption MICROLON 600 (Greiner Bio-One). To block used 1% BSA (Sigma-Aldrich, USA) and/or 1% nonfat milk (Bio-Rad, USA) in PBS. The procedure of selection and subsequent amplification of selected phage particles containing the gene of single-domain nanoparticle inside, and expressed a single domain on antitelo - in the composition of the surface of phage protein pIII) was repeated, usually three consecutive times. All manipulations were performed as described in the publications (Tillib ST., Ivanova TI, Vasiliev L.A. 2010. Fingerprinty analysis selection "Manantial" method of phage display using two variants of phage-helpers. Acta Naturae 2010; 2 (3): 100-108; Hamers-Casterman C, Atarhouch T., Muyldermans, S. et al. Nature 1993; 363: 446-448; V.K. Nguyen, A. Desmyter, Muyldermans S. Adv. Immunol. 2001; 79: 261-296; Saerens, D., Kinne j, Bosmans E., U. Wernery, Muyldermans s, Conrath K. J Biol Chem. 2004; 279: 51965-51972; Rothbauer, U., K. Zolghadr, Tillib, S., et al. Nature Methods 2006; 3: 887-889).

Sequences of clones selected nanoentities were grouped according to the similarity of their fingerprints obtained by electrophoretic separation of the hydrolysis products of the amplified sequences of single-domain nanoentities three parallel customease restriction endonucleases (Hinfl, Mspl, Rsal). The cDNA sequence of a single domain of nanoparticle aMTS1 (SEQ ID NO: 1) was determined (see figure 1). From it was derived the corresponding amino acid sequence nanoparticle (SEQ ID NO: 2). In this sequence are underlined, respectively (from left to right, from N - to C-end) hypervariable sites CDR1, CDR2 and CDR3, which are decisive for the specific recognition by nanoentities ACEA-1 protein CEA. Arrows indicate the positions of amino acid residues, which is what I characteristic for the variable domains of specific single-chain antibodies (different from the residues in the variable domains of the heavy chains of classical antibodies). In CDR1 and CDR3 parts of the ACEA-1 are identified cysteine residues, it is common in nanortalik and, apparently, stabilizing Naantali, forming additional C-C bond between the first and third hypervariable sites.

Products nanoentities

The cDNA sequence selected nanoentities was periglomerular in the expression plasmid vector is a modified vector pHEN6 (Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, Kinne J, Wyns L, Muyldermans S. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. - Beta-lactamase inhibitors derived from single-domain fragments of antibodies induced in Camelids. Antimicrob Agents Chemother. 2001; 45:2807-12), allowing connection to the C-end of the single domain nano-antibodies (His)6-apicola (immediately following the ON-epitope). Due to the presence of N-end expressed sequence signal peptide (peIB) accumulating recombinant protein (single domain nano-antibody) is stored in periplasm bacteria, which enables him to select the method of osmotic shock, do not actually destroying bacterial cells. Production of single-domain nanoentities was performed in E. coli (strain BL21). Expression was induced by adding 1 mm indolyl-beta-D-galactopyranoside and the cells were incubated with vigorous stirring for 7 hours at 37°C or overnight at 29°C. Onedomain the s nanoparticle was isolated from periplasmatic extracts using affinity chromatography on Ni-NTA-agarose using the system for cleaning QIAExpressionist (QIAGEN, USA).

Figure 2 presents electrophoregram polyacrylamide gel developed (with the help of selected cloned the coding sequence) and then purified nanoentities ACEA-1.

Example 3

Detection of protein CEA person using the selected nanoparticle ACEA-1.

The ability nanoparticle ACEA-1 contact immobilized in the wells of immunological dies protein CEA person was tested using the standard Protocol method enzyme immunoassay. As control was used wells with immobilized protein a chicken ovalbumin or bovine serum albumin (non-specific proteins).

As secondary antibodies FOR-tag (present at the s-end audited nanoparticle ACEA-1) used conjugated with horseradish peroxidase anti-monoclonal antibodies (CHGT-45P-Z, ICL, Inc., USA). The activity of horseradish peroxidase was determined using the chromogenic substrate ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonate).

The optical density was measured at a wavelength of 405 nm using a tablet fluorimetry. Control wells (with immobilized nonspecific protein, protein-chicken ovalbumin or bovine serum albumin) did not contain the antigen and then were blocked and processionary in parallel with the experimental cells (from what Tienam).

Figure 3 presents the result of the immunoassay, from which it follows that nanoparticle ACEA-1 (100ng/ml - in hole # 2 and 10 ng/ml - in hole # 3) specifically binds to immobilized in the wells 2 and 3 protein CEA person. As control was used wells (No. 1) with the immobilized protein bovine serum albumin. The intensity of the signal (the magnitude of the absorption at a wavelength of 405 nm) reflects the efficiency of binding of nanoparticle ACEA-1.

Example 4

Detection of protein CEA expressed on the cell surface lines A and Lim1215, using selected nanoparticle ACEA-1.

The recognition by nanoparticulate isolated protein CEA does not mean that a recognizable epitope of this protein will also be available, when this protein is localized on the cell surface. To test the possibility of using the obtained nanoparticle ACEA-1 for the detection of CEA, overexpression on the surface of tumor cells, was performed enzyme-linked immunosorbent assay for cell culture lines A (cell carcinoma of the lung) and Lim1215 cells of colon cancer). As a negative control used cell lines NEC (originating from cells of the embryonic human kidney), in which according to literature data CEA protein is not detected. Cells were obtained from the Bank of the crops research Institute of Virology. DIE Nevskogo, RAMS, Moscow.

Cells were grown in the wells of 96-hole tablet before reaching 80% of confluently, then 3 times washed with a solution of a standard phosphate-saline (PBS) and fixed in 3.7% solution of buffered formaldehyde for 10 minutes, fixation was stopped by adding a solution of glycine to a concentration of 125 mm, fixed cells were washed 3 times with PBS solution. Fixed cells were blocked in a solution of 1% BSA, 1×PBS for 2 hours, then washed 1×PBS was added to the solution (1×PBS, 0.1% BSA) with nanoentities ACEA-1 at a concentration of 100 ng/ml.

As secondary antibodies FOR-tag (present at the s-end audited nanoparticle ACEA-1) used conjugated with horseradish peroxidase anti-monoclonal antibodies (CHGT-45P-Z, ICL, Inc., USA). The activity of horseradish peroxidase was determined using the chromogenic substrate ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonate).

The optical density was measured at a wavelength of 405 nm using a tablet fluorimetry. Control wells (with immobilized cell line NEC) were blocked and processionary in parallel with the experimental cells.

Figure 4 presents the results described immunoassay recognition by nanoentities ACEA-1 immobilized in the wells of immune cells die lines A and Lim1215, with whom on the surface containing a series of overexpression protein CEA, in the absence of such recognition in the wells with the control CEA-negative cell line NECK. It is seen that, like the detection of isolated protein CEA, nanoparticle ACEA-1 and in this case works effectively at a concentration of 100 ng/ml Specific recognition of cells A and Lim1215 is associated with enhanced expression in these cells protein CEA, which is located on the surface of these cells. In contrast, in control cells NC protein CEA almost not expressed, and this corresponds to a background value of optical density in the appropriate cells.

Thus, nanoparticle ACEA-1 can specifically and efficiently to recognize (detect) protein CEA person, expressed on the surface of tumor cells.

Examples with illustrations prove the fulfillment of this invention tasks. Created a new special nanoparticle able to bind CEA protein. These new special nanoparticle have the following advantages compared to the prototype (single domain antibody, which is derived from the heavy chain of the classical immunoglobulin mouse): their synthesis, production and storage more economical, efficient and relatively simple; receiving includes a step of immunization and affinity maturation in vivo, which usually promotes higher affinnative significantly improve the efficiency of selection specified nanoentities, they are well soluble, have new structural features that allow them to potentially find some "hidden" for normal antibody epitopes. On the basis of the established nanoentities will be new opportunities for more efficient targeting of protein CEA and the impact on associated biological processes and diseases.

Thus, the objective of the present invention, is solved.

1. Nanoparticle, specific binding protein CEA person and having the amino acid sequence of SEQ ID NO: 2.

2. The method of detection of CEA protein in biological fluids of a person, comprising bringing into contact a specified biological fluid with nanoentities according to claim 1 and detecting binding of nanoparticle according to claim 1 protein CEA.

2. The method according to claim 2, characterized in that the detection of protein CEA using nanoparticle perform both in free form in biological fluids, and on the surface of cells of various types, such as tumor, where the protein overexpression.



 

Same patents:

FIELD: biotechnologies.

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EFFECT: invention may find further application in diagnostics and therapy of cancer.

3 cl, 3 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely endocrinology, and concerns predicting the clinical effectiveness in diabetic polyneuropathy in the patients suffering type 2 diabetes and dyslipidemia. That is ensured by evaluating the intensity of diabetic polyneuropathy manifestations taking into account neuropathic dysfunctional score, evaluating triglycerides and glycolised blood hemoglobin before the treatment; the derived data are used to specify a therapeutic approach followed by further estimation of a probability of a successful correction of diabetic polyneuropathy by formula P=111+e(0,991,78Z), wherein e is the basis of hyperbolic logarithm e=2.72; Z is a regression coefficient calculated by formula 7=4.56-0.05*"ДлСД"-0.02*IntDPN-0.05*TG-0.53*HbAlc+0.14TherApp; if the value P is 0.7 or more, the successful correction of DPN is predicted in the patients, and the value P less than 0.7 requires the therapeutic approach to be changed.

EFFECT: method provides the improved clinical effectiveness ensured by the individual therapeutic approach based on the lipid and carbohydrate metabolism of a specific patient.

2 ex

FIELD: medicine.

SUBSTANCE: diagnosticum comprises human lymphocytes to identify donor-specific major histocompatibility complex antigen antibodies during the post-transplant period, characterised by the fact that the diagnosticum comprises lymphocytes obtained from the multiorgan donor's spleen; a method for preparing a diagnosticum, predicting a rejection reactions following the transplantation from multiorgan donors, including a reaction to identify the donor-specific major histocompatibility complex antigen antibodies, characterised by the fact that the above diagnosticum is used. The diagnosticum contains T lymphocytes bearing the class I major histocompatibility complex antigens, and B lymphocytes bearing the class II major histocompatibility complex antigens in ratio suitable to provide an adequate antigen-antibody reaction in studying the donor-specific class I and II HLA antibodies in recipient's serum for a long transplantation period for humoral rejection diagnosis and evaluation of the immunosuppression therapy effectiveness.

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4 cl, 2 ex, 2 tbl

FIELD: medicine.

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EFFECT: method enables supporting for the fact of the mycobacterial infection, identifying the infection agent species, assessing the vaccination effectiveness; the method can be implemented for a relatively short time.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses using the monoclonal antibodies (MCAs) produced by the hybridomas B/4H1 and 4H7 and directed to variable determinants as a part of influenza B virus proteins. The hybridoma B/4H1 and 4H7 strains are deposited in the Russian Collection of Vertebral Cell Cultures (RKKK P) under Nos. 719 D and 720 D, respectively. The two current influenza B viral evolution lines are identified with using the MCAs B/4H1 and MCAs 4H7 directed to variable determinants of Victorian-type and Yamagata-type influenza B hemagglutinin, respectively.

EFFECT: using the prepared MCA kit enables identifying the newly recovered influenza B viruses Besides, the MCAs under the invention may be used to study the antigenic structure and variability of this agent, to identify them as belonging to a specific evolution line; the MCAs may be also used to construct the diagnostic test systems.

3 cl, 12 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention describes a chemically modified peptide that is a fragment of human protein S100β consisting of a sequence of 15 amino acids corresponding to the amino acids in position 18-32 of the amino acid sequence of protein S100β. The peptide is covalently bound to a spacer (Ahx) consisting of 2-aminoheptane acid or a similar compound, and covalently bound to the spacer by a biotin molecule (Bio). The peptide is described by general formula: A-Tyr1-Ser2-Gly3-Arg4-Glu5-Gly6-Asp7-Lys8-His9-Lys10-Leu11-Lys12-Lys13-Ser14-Glu15-E, wherein A represents Bio-Ahx, Bio-Acp, Bio or 0, and B represents 0 or -NH2. Bio means biotin; Ahx means a residue of 2-aminoheptane acid; Acp means a residue of 6-aminocapronic acid, 0 means the absence of any amino acid. A diagnostic technique and a method for prediction of melanoma provides measuring blood natural antibodies (nAB) to the peptide according to the invention with the stages of melanoma diagnosed by the antibody concentration as compared to the reference. The clinical outcome and therapeutic effectiveness in a craniocereberal injury are predicted by measuring the nAB to the peptide according to the invention. If observing the blood nAB level in the patients lower as compared to the standard reference, the favourable clinical outcome is diagnosed, and the therapy is stated to be effective.

EFFECT: invention may be used effectively as instant diagnosing of the disturbed blood-brain barrier and monitoring of the clinical effectiveness in CCIs and some cancers.

3 cl, 9 dwg, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: technique according to the invention involves the two-stage immobilisation of S and R brucellosis antigens on a tray by a non-specifically adsorbed mouse's monoclonal antibodies 2H2 and 2H8 in wells of the immunological tray (the first stage), specific binding S and R brucellosis antigens (the second stage) thereto, introduction and incubation of the analysed material (blood serum or milk) combined with introduction of mixed mouse's monoclonal antibodies 2H2 and 2H8 marked by horseradish peroxidase. The brucellosis agent antibodies are detected by being competitive with the antigen in binding on the well surfaces of the tray between the antibodies of the analysed sampled and fragment-marked monoclonal antibodies. A decreased level of the enzymatic signal in the analysed sample as compared with the negative reference testifies to contamination of the animal. Introducing the analysed sample into the two wells of the tray with S and R brucellosis antigens enables differentiation thereof for antibody specificity to any given form of the disease agent.

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3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: ultrasound-assisted transcutaneous targeted needle liver-biopsy is used in the patients with acute destructive pancreatitis; that is followed by the immunohistological analysis of the biopsy micropreparations in parallel sections 5 mcm thick marked with monoclonal antibodies: CD 79a for identifying B-Lm and CD 68 for identifying MF. That is followed by optical microscopy at magnification x400 with morphometry of B-Lm periportal regions and MF lobe parenchyma; the results are further processed by automated image analysis systems. A relative area taken by the analysed cell elements in the micropreparation is determined by formula (A). If the value A for the B-Lm periportal regions falls within the range of 0.4 to 1.14, while the value A for the MF lobe parenchyma falls within the range of 0.89 to 2.95, a pre-infectious phase of acute destructive pancreatitis is stated. If the value A for the B-Lm periportal regions falls within the range of 1.41 to 4.98, while the value A for the MF lobe parenchyma falls within the range of 4.34 to 9.52, a phase of suppurative septic complications of acute destructive pancreatitis is stated.

EFFECT: using the method provides the well-timed correction of the therapeutic actions; the method is attended by a lower risk of the needle biopsy; it possess high accuracy and simplicity of implementation.

2 tbl, 2 ex, 6 dwg

FIELD: medicine.

SUBSTANCE: total pre-therapeutic immunoglobulin E is measured in blood serum of the patients suffering the aggravated chronic obstructive pulmonary diseases (COPD), and if observing the upper normal limits to be overridden, the high efficacy of the systemic glucocorticoid therapy is predicted.

EFFECT: using the declared method enables predicting the efficacy of the systemic glucocorticoid therapy.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: according to the method, the amniotic fluid substance P is determined at the beginning of the stage I of labour or antepartum amniorrhea using ELISA. If the substance P value is equal to 2.9 ng/ml and higher, dysthyroidism is predicted.

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3 ex

FIELD: biotechnologies.

SUBSTANCE: single-domain antibody (nanoantibody) is proposed, which specificaly binds a mucin 1 protein (MUC1) of a human being and characterised by a full amino acid sequence. Also the method is considered to detect a MUC1 protein in biological fluids of a human being with usage of a nanoantibody according to the invention.

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3 cl, 3 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention discloses a bispecific antibody or its functional fragment, specifically binding with IL-4 and IL-13, which contain variable domains of light and heavy chains with established amino acid sequence. The invention also includes use of antibodies or its functional fragments within a pharmaceutical composition for treatment of diseases or disturbances mediated by IL-4 and/or IL-13, including allergic diseases, asthma, cancer. The invention discloses a molecule of nucleic acid, which codes a bispecific antibody or its fragment, an expression vector and a master cell for production of a bispecific antibody and its functional fragment.

EFFECT: invention makes it possible to produce and use new inhibitors of cytokines, preserving stability in process of production and use in vivo.

17 cl, 2 dwg, 8 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is an anti-factor D antibody and an antigen-binding fragment thereof, as well as a pharmaceutical composition, a commercial product and methods for treating the complement-binding diseases. What is considered is a polynucleotide, an expression vector, a host cell and a method for preparing the antibody according to the invention or the antigen-binding fragment thereof.

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70 cl, 5 ex, 3 tbl, 13 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. There are presented versions of recovered monoclonal antibodies and an antigen-binding portion thereof specific to human IP-10. There are described: an immunoconjugate, bispecific molecule thereof, as well as versions of a composition of the antibody, immunoconjugate or bispecific molecule - for treating autoimmune and inflammatory diseases. There are also disclosed: a coding nucleic acid, an expression vector thereof and a host cell carrying this vector to produce the antibody. What is described is using the antibody or antigen-binding portion thereof for preparing a medicine for treating: either a viral or bacterial infection entailing undesired IP-10 activity, or autoimmune and inflammatory diseases caused by undesired IP-10 activity. What is presented is a hybridoma producing the antibody, derived from a transgenic mouse splenocyte cross-linked to an immortalised cell.

EFFECT: use of the invention provides the novel antibodies that can be find application in medicine to treat a variety of diseases associated with IP-10 activity.

22 cl, 30 dwg, 9 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a medicine for treating a patient with rheumatoid arthritis on the basis of an antibody or a fragment thereof that bind the Extra Domain-A (ED-A) isoform of fibronectin. The antibody or fragment thereof contain the domain VH comprising a frame and a number of complementary-determining regions HCDR 1 -3, and the domain VL comprising a frame and a number of complementary-determining regions LCDR 1-3 with the antibody or fragment thereof being conjugated with IL-10. The invention discloses a medicine on the basis of the above antibody or fragment thereof for delivering a molecule conjugated with the antibody or fragment thereof wherein the molecule represents IL-10 to a newly formed vascular tree of the rheumatoid arthritis regions in a patient. There are disclosed method of treating rheumatoid arthritis with using the medicines under the invention, method of delivering IL-10 conjugated with the antibody or fragment thereof to the newly formed vascular tree of the rheumatoid arthritis regions in the patient, as well as a conjugate of the antibody or fragment thereof binding ED-A of fibronectin to interleukine-10. The antibody or fragment thereof may be represented by a diabody, contain one-chain Fv, present a small immunoprotein (SIP).

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35 cl, 8 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: what is presented is a single-domain nanoantibody aMTS1 specifically binding the human and mice proteins S100A4/Mts1 with the characterised amino acid and nucleotide sequences, as well as using it for the purpose of detection of the protein S100A4/Mts1 in human and mice biological fluids.

EFFECT: invention can find further application in therapy of the S100-mediated diseases.

2 cl, 4 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: protein complex is proposed with improved activity of long-term action and biostability, containing a physiologically active polypeptide, an Fc-domain of immunoglobulin and a non-peptidyl polymer, having three functional ends. The method is disclosed to produce a protein complex containing a physiologically active polypeptide, an Fc-domain of immunoglobulin and a non-peptidyl polymer, having three functional ends A pharmaceutical composition is proposed, which contains the effective quantity of the specified protein complex, having the improved resistance in-vivo of the physiologically active polypeptide.

EFFECT: invention makes it possible to produce a protein complex with improved activity of long-term action and biostability.

24 cl, 3 dwg, 4 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: there are presented versions of the peptide (A) or (B) with the amino acid sequence of SEQ ID NO: 1 or 2 respectively presented in this description. The peptide has activity to induce a cytotoxic T-cell when an antigen-presenting cell carrying HLA-A2 (A*0201) presents it. There are described the versions of the peptide antibodies prepared by immunisation by the proper peptide. There are presented: an agent, methods for inducing: a cytotoxic (killer) T-cell, an antigen-presenting cell, CDH3 expressing cancer immunity; as well as a method of treating CDH3 expressing cancer on the basis of the peptide. What is presented is an isolated cytotoxic T-cell induced by said method on the basis of the peptide. There are described: the antigen-presenting cell and exosome presenting the complex containing the peptide and HLA-A2 (A*0201).

EFFECT: higher effectiveness of the use of the invention in treating CDH3 expressing cancer.

15 cl, 5 dwg, 2 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are studied: a method of decreasing the load and of reducing the number of plaques in the layer of retinal ganglion cells, of reducing the total amount of soluble amyloid-beta in the layer of retinal ganglion cells and of maintaining or reducing intraocular pressure in a subject, as well as a method of preventing, treating and relieving the symptoms of ophthalmic diseases associated with amyloid-beta related pathological disorders or changes in tissues of the visual system, a diagnostic technique for such disease and predisposition thereto, and a method of monitoring the minimal residual ophthalmic disease associated with the amyloid-beta related pathological disorders or changes, involving administering the composition containing the anti-amyloid-beta antibody to the patient. The present invention can find further application in a therapy of the ophthalmic diseases.

EFFECT: what is presented is a pharmaceutical composition for treating the ophthalmic diseases containing the anti-amyloid-beta antibodies.

31 cl, 3 ex, 13 tbl, 20 dwg

FIELD: medicine.

SUBSTANCE: what is presented is a method for differentiation of osteoarthritis, rheumatoid arthritis, and non-pathological states in a sample, including the measurement of the concentration of the peptide of a C1 protein 2 chain of the cartilage intermediate layer (CILP-2). The high level of the C1 protein 2 chain CILP-2 compared with rheumatoid arthritis and the normal state indicates that the patient has osteoarthritis. What is also presented is a peptide for differentiation of osteoarthritis and rheumatoid arthritis and non-pathological states, comprising SEQ ID NO:1. There are presented antibodies immunoreactive to the mentioned peptide, and a kit containing the antibodies and the analysis regulation.

EFFECT: effective agents and methods for differentiation of osteoarthritis, rheumatoid arthritis and non-pathological states by the increase of the concentration of the C1 protein 2 chain of the cartilage intermediate layer in the sample.

7 cl, 2 dwg, 4 ex

FIELD: biotechnology, medicine, proteins.

SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.

EFFECT: valuable medicinal properties of polypeptide.

19 cl, 33 dwg, 1 ex

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