Nanoantibody specifically binding muc1 protein, method of muc1 protein detection by nanoantibodies

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.

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

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The technical field

The present invention relates to the field of molecular immunology, biotechnology and medicine, in particular the production and use of specific single-domain antibodies (nanoentity) for recognition (detection) and binding protein MUC1 person.

The prior art of the present invention

One of the most promising tumor markers Mucin-1 (MUC1) is a heavily glycosylated protein present in the norm in many types of human cells may be identified as associated with the cell surface membrane, as well as the indirect or intracellular protein). The expression of this protein was significantly increased in the majority of malignant tumors of epithelial origin. Increased expression of MUC1 in tumor and depolarization distribution of the antigen in the cells are regarded as an adverse factor for the disease. Attach special importance to the existence of altered forms of mucin, the expression of which is the consequence of disturbances in the processes of glycosylation molecules in malignant cells. High content Sekretareva forms of MUC1 in the bloodstream of cancer patients with malignant tumors of epithelial origin directly correlates with the prevalence of the tumor process and turn retnam the prognosis of the disease. Suggest that Mucins are involved in the processes of intercellular adhesion, including the processes of interaction of tumor cells with cells of the immune system. Thus, MUC1 has traditionally been seen as a potentially effective target for immunotherapy.

Mucin-1 is the major protein of 1232 amino acids, which includes 42 repeating sequence of 20 amino acids. Moreover, the number of repetitions can vary and is one of the reasons for the high polymorphism of the gene MUC1. The area between amino acids 1097-1098 is autolysosome. As a result of hydrolysis of this connection is formed by two subunits, which after proteolysis form with each other noncovalent heterodimer. Extracellular alpha subunit of mucin-1 (fragment 24-1097) contains adhesive repetitions, which is associated with SAM and is involved in cell migration and metastasis of tumor cells. Alpha-subunit of mucin-1 has properties of cell adhesion, and protein can act as an adhesive and anti-adhesive. Provides a protective layer of epithelial cells against the damaging bacteria and enzymes. Cytoplasmic beta subunit of mucin-1 (fragment 1098-1255) contains a large number of binding sites with regulatory proteins and determines the part of mucin-1 in multiple signaling pathways Beta-subunit binds to SRC, CTNNB1 and proteins ERB group. Beta-subunit plays a role in signal transmission by the cell, which is mediated by protein phosphorylation and protein-protein interactions. The mucin-1 modulates signaling through ERK, SRC, and NF-κ. In activated T-cells has an effect on Ras/MAPK signaling pathway. Enhances the growth of tumors. Cells with more than 80% of carcinomas are characterized by high expression of mucin-1, changes in the pattern of glycosylation and loss of polarization. [Carcinoma is a type of malignant tumor developing from cells of the epithelial tissue of various organs (skin, mucous membranes, and many internal organs)].

The high level of glycosylation of MUC1 and the high level of its expression leads to the fact that it forms on the surface of cancer cells layer that prevents typically hydrophobic, chemotherapeutic drugs. On the other hand, mutiney layer holds numerous growth factors from the surrounding cell environment, which leads to their concentration on the surface of cells and accelerate the growth of cancer cells.

It is obvious that MUC1 is one of the most important factors stimulating metastasis of tumors, which is a consequence of its total stimulating action on the processes of increasing the mobility and invasiveness of the cells, and the process of angiogenesis. Numerous studied the I recent years has convincingly demonstrated, that MUC1 protein is an important diagnostic marker and an important target for developing new therapeutics.

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. In principle, potentially it is possible to obtain antibodies specific to virtually any extracellular or associated with the cell surface of the target.

Antibodies that bind to the mucin 1, can inhibit the development of tumors, restoring intercellular interaction, disrupting the major tumor-associated MUC1, thereby preventing metastasis and counteract immune suppression. Some anti-MUC1 antibodies may contribute to the destruction of tumor cells by antibody-dependent cytotoxicity (using cells of the immune system) (von Mensdorff-Pouilly et al. Natural and Induced Humoral Responses to MUC1. Cancers 2011, 3: 3073-3103. Natural and induced humoral responses to MUC1).

Previously have repeatedly attempted to obtain antibodies that bind to the MUC1 protein. The closest technical solution, which is the basis of the present invention, one can cite the following example.

Known single-domain antibody for mucinosa antigen-1 (application WO No. 2011099684, publ. 18.08.2011, Single domain antibody for Much antigen 1). Here announced the receipt of a special single-domain antibody, binding protein MUC1 on the surface of tumor cells, in order to identify (diagnose) of these cells, and to use the obtained antibodies as targets (MUC1) specific anticancer drug.

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:

1. Single-domain antibody were obtained on the basis of the sequence of the heavy chain of mouse immunoglobulin by GE is but engineering variation strictly defined amino acid positions in the hypervariable complementarity determining areas (CDR1: 24-30, in CDR2: 31-37, CDR3: 38-44). Thus, the method of obtaining antibodies was time-consuming and inefficient, as the resulting antibody

a) not passed an important stage of obtaining high-affinity antibodies is their affinity maturation in vivo

b) usually, this taken out of context traditional monoclonal antibodies protein sequence is poorly soluble and prone to aggregation.

2. Structural features of variable antigen-binding domain of traditional antibodies (unlike nanoentities) make impossible the creation of special paratopes able to learn some hidden epitopes, such as in cavities, crevices small size in the structure of proteins.

3. The resulting antibodies are likely to be relatively poorly soluble, so as usual this taken out of context traditional monoclonal antibodies protein sequence is poorly soluble and prone to aggregation.

Thus, in the prior art there is a need to develop new antibodies (antigen-binding molecules), devoid of the above disadvantages and specifically recognizing MUC1 protein.

The present invention was the creation of new specific antibodies able to bind MUC1 protein. These new specific antibodies must have the following advantages compared to the prototype(single domain antibody, derived heavy chain classic immunoglobulin mouse): production and storage should be more economical, efficient, and relatively simple; they receive should include the stage of immunization and affinity maturation in vivo, which should contribute to higher affinity and significantly improve the efficiency of selection specified antibodies, they should be well soluble, they must have new structural features, in principle allowing them to learn some "hidden" for normal antibody epitopes.

The problem is solved due to the fact that created nanoparticle, specific binding protein mucin 1 (MUC1) of a person and having the amino acid sequence of SEQ ID NO: 2. In this way the detection of MUC1 protein in biological fluids of a person includes bringing into contact a specified biological fluid with the stated nanoentities and detecting binding of this nanoparticle with MUC1 protein.

The detection of MUC1 protein with nanoparticle perform both in free form in biological fluids, and on the surface of cells, for example tumor, where the protein overexpression.

The basis of the present invention are not derived single-domain classical bivalent antibodies, which are considered as FR the type and small, in the original norm existing in the body of the animal (camel) 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 antibodies, they acquire a number of new positive qualities of practical importance. There are effective ways to get (and selection) of such antibodies against various (including netcommunity) mucin antigens, and thanks to its low immunogenicity of nanoparticle can be used to treat infections caused by pathogens of this group.

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 (NANOBODY), and "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 of cartilaginous fishes). These specific antibodies consist of a dimer of only one shortened (excluding the first constant region SN) heavy chain immunoglobulin 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 segment (define 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., L. Wyns Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains, TIBS 2001; 26, 230-235. Recognition of antigens by single-domain fragments of antibodies: superfluous luxury of paired domains). In this structure, all three hypervariable area 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 related to NGF what influence 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, at least - in the CDR2 and CDR3).

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 (see review: Tillib SV Camel nanoparticle is an effective tool for research, diagnostics and therapy. Molecular biology, 2011; 45(1), 77-85), is the following.

1. A highly efficient method of generating and breeding nanoentities.

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

3. 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, to the which can not be recognized significantly larger conventional antibodies.

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

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

6. 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, Nature 1993; 363: 446-448. - Existing in nature antibodies without light chains). Technical implementation of the selection of single-domain nanoentities, which is genetically engineered derivatives of the antigen-recognizing domains of the single-stranded antibodies the 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 of phage display is a 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 NI. 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 Curr Opin Struct Biol., 2007; 17:481-7. - Phage display for design and analysis of interactions of protein domains).

One particularly important application of this technology is the generation of specific recombinant antibodies to various antigens (Hoogenboom MR. Selecting and screening recombinant antibody libraries. Nat Biotechnol., 2005; 23:1105-16. Selection and analysis of libraries of recombinant antibodies. 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 sequence is th variable regions of the heavy and light chains of immunoglobulins, United a 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 changes. 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 specification is the durability 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-specific 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 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 can communicate both with the same epitope of the target antigen and 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 products (Conrath KE, Lauwereys M, Wyns L, Muyldermans S. Camel single-domain antibodies as modular building units in bispecific and left-hand drive vehicles antibody constructs. - Camel single-domain antibodies as modular building units in bispecific and bivalent structures of antibodies. J Biol Chem., 2001, Mar 9; 276 (10): 7346-50; Zhang J, Tanha J, Hirama T, Khieu NH, To R, Tong-Sevinc H, Stone E, Brisson JR, MacKenzie CR. Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents. Pentameric the Oia single-domain antibodies from phage libraries: a new strategy for rapid receipt reagents antibodies with high avidity; J Mol Biol. 2004, Jan 2; 335 (1): 49-56; Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De Baetselier P, Muyldermans S, Revets H. Efficient cancer therapy with a nanobody-based conjugate. Effective cancer therapy conjugates on the basis of nantel; Cancer Res., 2004 Apr 15; 64 (8): 2853-7; Baral TN, Magez S, Stij'lemans B, Conrath K, Vanhollebeke B, Pays E, Muyldermans S, De Baetselier P. Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor., Nat. Med., 2006, May; 12 (5): 580-4. Experimental therapy of African trypanosome using human trypanosomiasis factor conjugated to notelem.; Coppieters K, Dreier T, Silence, Haard HD, Lauwereys M, Casteels P, Beirnaert E, Jonckheere H, Wiele CV, Staelens L, Hostens J, Revets H, Remaut E, Elewaut D, Rottiers P. Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis., Arthritis Rheum., 2006, Jun; 54 (6): 1856-66. - Rich anti-TNFalpha VHH proteins isolated from camelids, demonstrate a high affinity to the inflamed joints in a murine model of collagen-induced arthritis); multimerization with the introduction of additional amino acid sequences of the interacting protein domains, such as lacinova the zippers (Harbury W., T. Zhang, Kirn P.S., et al. A switch between two-, three - and four-stranded coiled coils in GCN4 leucine zipper mutants. - D between two-, three - and chetyrekhstoechnymi spiral structures in mutants SSM - "latinboy lightning"; Science, 1993, 262:1401-1407; Shirashi So, Suzuyama k., Okamoto, H. et al. Increased cytotoxicity of soluble Fas ligand by fusing isoleucine zipper motif. - Reinforced cytotoxic the industry soluble Fas-ligand due to its connection with theme of "isoleucinol lightning"; Biochem. Biophys. Res. Communic. 2004, 322: 197-202; ChenchikA., Seth William page, A., Komarov, A., Natarajan V. Reagents and methods for producing secreted bioactive peptides, 2010, the application for the grant of U.S. patent No. 20100305002, Reagents and methods for the production of bioactive secreted peptides; or sequences of small proteins that form stable complexes (Deyev SM, Waibel R, Lebedenko EN, Schubiger AP, Pluckthun A. Design of multivalent complexes using the barnase*barstar module. Nat Biotechnol., 2003, 21(12):1486-92. - Design of multivalent complexes, using the module barnase-barstar).

It was also shown (Vincke S., Loris R, Saerens d, et al.; J. Biol. Chem., 2009. V., 284, No. 5. 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 obtaining nanoparticle linking antigenic epitope of MUC1 protein, includes the following principal stages:

1) induction of the formation of specific antibodies as a result of immunization representative of the family of camelids (in particular, two-humped camel) mixed substance prepared on the basis of native mucin isolated from human milk, as well as tumor mucin;

2) cloning of the sequences of the antigen-binding domains of nanoentities - specific single-chain antibodies, synthesized in the lymphocytes of the immunized animal, 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, MUC1 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 MUC1-containing antigenic material used mixed substance, prepared on the basis of native mucin isolated from plasma membranes of the fat globules of milk, as well as tumor mucin (g is mogenet tissue tumors of the breast, and the drug culture cells epidermoid cancer of the hypopharynx, Ner, in which there is increased expression of surface mucin).

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 temperatures 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 nano is NITEL or chimeras, in which in addition to nanoparticle is another protein with desired properties.

Although mainly for illustrative purposes, the author of the 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, is evident in this technical field.

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 l of culture).

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

3. High economic profitability. The author of this invented the I comes from the fact, which, as is well 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 can communicate both with the same epitope of the target antigen and 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 additionally introduced the e 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 author of the present invention understands under 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 above-mentioned adaptations or "formatting" and their variants. 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, for example 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 have places is in the regions of the polypeptide, which 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 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 is within 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, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, 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 (indicated in the list of sequences as CDR) the others but very similar in properties, 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 delegated, substituted, inserted or added. The 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 the CSOs 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 nanoparticle declared in accordance with the present invention and falling under the scope of the present invention.

Obtain functionally active nanoparticle anti-MUC1, which can be used for specific binding and detection of protein MUC1 person as being in a free state, and overexpression on the surface of tumor cells, as shown in examples 3 and 4.

Brief description of drawings

Figure 1 is the nucleotide sequence of cDNA encoding a received a single domain nanoparticle aMUC1 (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 anti-MUC1 MUC1 protein.

Figure 2 presents the results of the enzyme immunoassay recognition selected for single-Nanojuncetea anti-MUC1 Immo is risovannogo in the hole immunological die MUC1 protein, isolated from breast milk.

Figure 3 presents the results of the enzyme immunoassay recognition selected for single-Nanojuncetea anti-MUC1 immobilized in the wells of immune cells die Ner containing on the surface overexpression MUC1 protein.

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 (first injection) or incomplete (if other injections) 's adjuvant adjuvant. The second injection was performed 3 weeks after the first, then at intervals of two weeks spent another four immunization.

As MUC1-containing antigenic material used mixed substance, prepared on the basis of native mucin isolated from plasma membranes of the fat globules of milk, as well as tumor mucin (the homogenate tissue tumor, breast cancer, and the drug culture cells epidermoid cancer of the hypopharynx, Ner, in which there is increased expression of surface mucin). Antigenic material was obtained from the Department of Prof. Jakubowski R.I. (of Moscow them. PageRank). Native MUC1 were isolated by the method of immunoaffinity chromatography Enrichment on sepharose 4B, conjugated with MCA IC (monoclonal antibodies against mucin), partially purified by the method of ion exchange chromatography of plasma of breast milk. The concentration of mucin in the solution was estimated by the optical absorption of the solution, measured against a reference standard buffer solution, taking 1 oted absorption 10,000 U/ml of antigen. During three consecutive selections on immunoaffinity sorbent immobilized on sepharose 4B monoclonal antibodies IK gained 6 ml highly purified preparation of native antigen MUC1 from skim milk by the method described above. The optical absorption of the solution is reduced to 0,2 UNED (2000 U/ml). The specific activity of the antigen-tested method for inhibition ELISA using ICA IC. The resulting solution of the antigen was frozen and stored at -20°C.

The homogenate tissue tumors of the mammary gland was obtained by grinding the tissue in a mechanical homogenizer. Tissue samples of the tumor in 5 patients with clinical diagnosis of breast cancer obtained during surgery. Immediately after receipt of each tissue sample maximum freed from fat, crushed scissors and frozen at -20°C with the addition of physiological saline containing 0.01% thimerosal (1 ml of solution is 1 g of tissue). All manipulations were performed using sterile disposable plastic tableware and/or pre-disinfection metal tools. After dialing the desired number of material samples were thawed, pooled and subjected to additional grinding using a metal sieve and mechanical homogenizer, with the addition of saline solution (1/1 by volume). The resulting homogenates were centrifuged at 3000 rpm for deposition of large pieces of tissue and separation of tissue fat. Adosados selected, Liquefiable and kept at -80°C. the Obtained 3.5 ml of the clarified homogenate. The presence of MUC1 confirmed by ELISA for the inhibition of binding of MCA IC with immobilized antigen.

Cell culture epidermoid cancer of the larynx person Ner received from the Bank of the crops research Institute of Virology. Dijanoveckog, RAMS, Moscow, Russia. Cells were cultured in 25 cm2-bottles on the environment, Needle-MEM with the addition of 2 mm L-glutamine and 8% fetal calf serum, at 37°C in humidified atmosphere containing 5% carbon dioxide. The passage of the cells was carried out twice a week. Grown in several stages in the culture vials with approximately 150 million cell culture epidermoid cancer of the hypopharynx, Ner, were divided into 5 equal parts (according to the of nhcci) and frozen with 10% DMSO.

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 U/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 800g. 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(dT)-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 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 M-MuLV (Fermentas) and primers onnro(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 in the NcoI sites (>PST) and NotI 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 phage M13KO7 (New England Biolabs, USA).

Example 2

Selection of single-domain nanoentities, specifically recognizing MUC1

Selection of single-domain nanoentities was performed by the method of phage display using, primarily, the same as for immunization, preparation of native mucin MUC1 isolated from breast milk. MUC1 (10 μg in 100 μl of 1 well) 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 nanoparticle in the composition of the surface of phage protein pill) was repeated, usually three consecutive times. All manipulations were performed as described in publications (Tillib S.V., Ivanova TI, Vasiliev L.A. 2010. Fingerprinty analysis of breeding nanoentities method of phage display using two variants of phage-helpers. Acta Naturae 2010; 2 (3): 100-108; Hamers-Casterman S., Atarhouc So, Muyldermans S. et al. Nature 1993; 363: 446-448.; V.K. Nguyen, DesmyterA., 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 (HinfI, MspI, RsaI). The cDNA sequence of a single domain of nanoparticle anti-MUC1 (SEQ ID NO:1) was determined (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 anti-MUC1 MUC1 protein.

Products Manantial

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. Antimicrob Agents Chemother. 2001; 45:2807-12 - Beta-lactamase inhibitors derived from single-domain fragments of antibodies induced in camelids), allowing the second joining With the end of a single domain of nanoparticle (His) 6-epitope (immediately following the ON-epitope). Due to the presence of N-end expressed sequence signal peptide (peIB) accumulating recombinant protein (a single domain nanoparticle) accumulates 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. a Single domain nanoparticle was isolated from periplasmatic extracts using affinity chromatography on Ni-NTA-agarose using the system for cleaning QIAExpressionist (QIAGEN, USA).

Example 3

Detection of protein MUC1 person using the selected nanoparticle anti-MUC1

The ability nanoparticle anti-MUC1 contact immobilized in the wells of immunological dies protein MUC1 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 a secondary antibody TO the tag present at the C-end audited anoint the body anti-MUC1, 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, bovine serum albumin protein or ovalbumin chicken) did not contain the antigen and then were blocked and processionary in parallel with the experimental cells (antigen).

Figure 2 presents the result of the immunoassay, from which it follows that nanoparticle anti-MUC1 specifically binds to the immobilized protein MUC1 person. Nanoparticle took in four serial dilutions (1:2), starting with a concentration of 100 ng/ml and ending with a concentration of 12.5 ng/ml Clear, specific signal was obtained for nanoentities at a concentration of 25 ng/ml, which corresponds to the antibody with good affinity. The value of the column in the drawing corresponds to the relative values of the absorption at a wavelength of 405 nm, quantitatively reflect the efficiency of binding of nanoparticle anti-MUC1.

Thus, nanoparticle anti-MUC1 can specifically and efficiently to recognize (detect) protein MUC1 person.

PR is measures 4

Detection of MUC1 protein, expressed on the surface of immobilized cell line Ner, using selected nanoparticle anti-MUC1

The recognition by nanoparticulate isolated protein MUC1 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 anti-MUC1 for the detection of MUC1, overexpression on the surface of tumor cells, was performed enzyme-linked immunosorbent assay for cell culture epidermoid cancer of the larynx person Ner. Cell culture Ner received from the Bank of the crops research Institute of Virology. Dijanoveckog, RAMS, Moscow, Russia. Cells were cultured in 25 cm2-bottles on the environment, Needle-MEM with the addition of 2 mm L-glutamine and 8% fetal calf serum, at 37°C in humidified atmosphere containing 5% carbon dioxide. The passage of the cells was carried out twice a week. As a negative control used cells Chinese hamster ovary Cho, which were cultured in DMEM (Dulbecco''s modified Eagle's medium)containing 10% fetal calf serum. 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% buffered solution formalize the IDA 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 anti-MUC1 in four dilutions (factor 2), starting with 100 ng/ml and ending with a concentration of 12.5 ng/ml.

As secondary antibodies FOR-tag (present at the s-end audited nanoparticle anti-MUC1) 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 cells SNO) were blocked and processionary in parallel with the experimental cells.

Figure 3 presents the results described immunoassay recognition by nanoentities anti-MUC1 immobilized in the wells of immune cells die Ner containing on the surface overexpression MUC1 protein. It is seen that similar to the detection of an isolated MUC1 protein nanoparticle anti-MUC1 and in this case works effectively in a concentration of 25 ng/ml Specificity the civil recognition of cells Ner, most likely due to the enhanced expression in these cells MUC1 protein, which is located on the surface of these cells. In contrast, in control cells Cho MUC1 protein is almost not expressed, and this corresponds to a background value of optical density in the appropriate cells.

Thus, nanoparticle anti-MUC1 can specifically and efficiently to recognize (detect) protein MUC1 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 MUC1 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 contributes to higher affinity and significantly improve the efficiency of selection specified nanoentities, they are well soluble, have new structural features that allow them to learn some "hidden" for normal antibody epitopes. On the basis of the generated nanoentities will be new in the options for more efficient targeting of protein MUC1 and impact on associated biological processes and diseases.

1. Nanoparticle, specific binding protein mucin 1 (MUC1) of a person and having the amino acid sequence of SEQ ID NO: 2.

2. The method of detection of MUC1 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 with the MUC1 protein.

3. The method according to claim 2, characterized in that the detection of MUC1 protein with nanoparticle according to claim 1 carried out in a free form in biological fluids, and on the surface of cells, for example tumor, where the protein overexpression.



 

Same patents:

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.

EFFECT: preparing the high-quality diagnosticum.

4 cl, 2 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: method is implemented by detecting a marker in herparinised venous blood by incubating the blood with a mycobacterial antigen material (a test sample) and with normal saline (a reference); thereafter the blood is examined to measure CD45+ lymphocytes of the fluoroisothiocyanate-labelled markers; the fluorescence of the labelled CD45+ lymphocytes in the test sample in relation to the reference enables to consider that the individual has been infected with the very species of mycobacteria with the antigen material has been used for the blood incubation in a test tube.

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.

EFFECT: method involving the enzyme immunoassay enables increasing the effectiveness of health-improving measures, reducing the length of health-improvement in livestock farms with the negative brucellosis situation, dropping the disease incidence.

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.

EFFECT: using the method enables higher accuracy of the preclinical dysthyroidism prediction and enables applying the well-timed obstetric approach.

3 ex

FIELD: medicine.

SUBSTANCE: epidural electric stimulation of the spinal cord in the patients suffering a complicated superior cervical spine injury is preceded by preparing blood serum samples to be tested by enzyme immunoassay. A value of recombinant human ciliary neurotrophic factor CNTF is derived and accepted as a reference - Pref. Similarly, on the 7th day from the beginning of the epidural electric stimulation of the spinal cord, a value of CNTF - Pcur is determined and compared to the reference. If observing Pref.<Pcur., higher activation of the functional status of spinal motoneurons is stated.

EFFECT: invention provides more objective assessment combined with improved sensitivity and specificity of the declared method.

1 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.

EFFECT: invention can find application in treating, preventing and diagnosing the diseases and disorders associated with excessive and uncontrolled complement activation, particularly alternative pathway of complement.

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).

EFFECT: invention provides the successful treatment of rheumatoid arthritis ensured by the selective delivery of biologically active compounds conjugated on the antibody to the disease locations.

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: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to expression constructs, and may be used for immunoglobulin expression. An expression vector contains one open reading frame (sORF) insert which contains a first sequence of nucleic acid coding a first polypeptide; a first intermediate sequence of nucleic acid coding a first protein cleavage site containing an autoprocessing element with an intein segment providing proteolytic sORF polypeptide cleavage between the first polypeptide and the intein segment and the second polypeptide, but not ligation of said first polypeptide with said second polypeptide; and a second sequence of nucleic acid coding the second polypeptide. The expression vector is able to express a mammalian polypeptide coding sORF and cleaved in said first protein cleavage site in a host cell; consisting of the first polypeptide - an immunoglobulin heavy chain, and the second polypeptide - an immunoglobulin light chain able to be assembled into a multimer.

EFFECT: invention provides functional antibody production with 'correct' setup and assembly.

40 cl, 9 dwg, 57 tbl, 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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