Modified ubiquitin proteins having specific binding activity for extradomain b of fibronectin

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to novel hetero-multimeric proteins obtained from modified ubiquitin, and can be used in medicine to treat or diagnose diseases associated with hyperprodution of the extradomain B of fibronectin (ED-B). The protein includes two monomeric ubiquitin links which are differently modified through substitutions of at least 6 amino acids in positions 4, 6, 8, 62, 63, 64, 65 and 66 of SEQ ID NO: 1. In the first monomer link the substitutions include: F4W, K6(H, W or F), Q62N, E64(K, R or H), S65(L, F or W), T66(S or P), and in the second monomer link: K6(T, N, S or Q), L8(Q, T, N or S), Q62(W or F), K63(S, T, N or Q), E64(N, S, T or Q), S65(F or W), T66(E or D).

EFFECT: invention enables to obtain a modified heterodimeric ubiquitin protein, capable of binding with ED-B with high affinity.

28 cl, 18 dwg, 3 tbl, 7 ex

 

AREA of TECHNOLOGY

The present invention relates to new heteropolymeric proteins capable of binding extradosed b of fibronectin (ED-B). In addition, the invention relates to a composite protein, incorporating heteropolymeric binding protein, combined with a pharmaceutically and/or diagnostically active component. The invention is further directed to a method of creating such heteropolymeric binding protein protein or compound and to pharmaceutical and/or diagnostic compositions containing the mentioned heteropolymer binding proteins. Additionally, the invention relates to libraries containing DNA encoding the aforementioned proteins.

In other embodiments, the invention is directed to polynucleotides that encode mentioned heteropolymeric binding protein or compound is a protein, vectors comprising the aforementioned polynucleotide, the host cell, incorporating the protein integral protein, vector and/or the polynucleotide. In one preferred embodiment, the implementation of those heteropolymeric binding protein protein or compound included in the medicament or diagnostic agent. Additionally, describes how to obtain the recombinant protein or protein composite, and the use of said proteins in the ways that health l�learning from.

The LEVEL of TECHNOLOGY

There is a growing demand for a binding molecule composed of amino acids that are not immunoglobulins. Although the present antibodies are the best studied class of binding molecules, there is still a need for new binding molecules to obtain ligands with high affinity and specificity, since the molecules of immunoglobulins have major shortcomings. Although they can easily get and they can be directed to almost any target, they have very complicated molecular structure. There is a continuing need in substitution of the antibody to smaller molecules, which were easy to work with. These alternative linking agents can be profitably used, for example, in medical diagnosis, prevention and treatment of diseases.

Proteins that have a relatively specific three-dimensional structure, usually called protein cages can be used as a starting material for constructing these alternative binding agents. These frames usually contain one or more areas that are affected by specific or random change in the sequence, and such a randomization sequence is often performed to obtain a library of proteins, which can �be selected for specific binding molecule. Molecules smaller than antibodies and with comparable or even more than a good affinity to the target antigen will be, as expected, better antibodies mean pharmacokinetic properties and immuno-hennoste.

In several previous approaches do use protein cages as source material for binding proteins. For example, in document WO 99/16873 describes the developed modified proteins of the family of lipocalin (so-called anticalin) exhibiting binding activity of some ligands. The structure of peptides of the family of lipocalin is modified by substitution of amino acids in their natural binding pocket, using the methods of genetic engineering. Like antibodies, these anticline can be used to

identification or linking molecular structures. Similar to antibodies, modify the structure of the flexible loop; these modifications give the ability to recognize ligands that are different from natural.

In document WO 01/04144 described the artificial creation of a binding domain on the surface of the protein in proteins with the structure of beta-Plast as those that lack the binding site. Through this newly created artificial binding domain can be obtained, for example, changes in γ-crystallin is a structural protein of the eye lens - which interact with ligands with high affinity and specificity. In contrast to the modification of binding sites, which are already present and formed from structures with a flexible loop that mentioned above for anticalins, these domains linking recreated on the surface of beta-layers. However, in document WO 01/04144 describes only the changes are relatively large proteins to create new binding properties. Because of their size, proteins according to WO 01/04144 can be modified at the level of genetic engineering only in ways that require some effort. In addition, in the proteins disclosed still, only a relatively small percentage of total amino acids is modified to maintain the overall structure of the protein. Therefore, only a relatively small surface region of the protein available for binding properties that did not exist previously. Moreover, WO 01/04144 reveals only the creation of properties of binding to γ-crystallin.

In document WO 04/106368 described the creation of artificial binding proteins on the basis of ubiquitin proteins. Ubiquitin is a small, Monomeric and cytosol-tion protein which is highly preserved in the sequence and is present in all known eukaryotic cells from protozoa to vertebrates organisms. In the body it plays a very important role in the regulation of the controlled degradation of cellular proteins. For �in proteins, destined for degradation are covalently associated with ubiquitinate or polyubiquitinated chains during their passage through the cascade of enzymes to selectively degrade because of this label. According to the latest results, ubiquitin or tagging of proteins with ubiquitin, respectively, also plays an important role in other cellular processes, such as the import of several proteins or gene regulation.

In addition to the elucidation of its physiological function, ubiquitin is the subject of research mainly because of its structural and protein-chemical properties. The polypeptide chain of ubiquitin consists of 76 amino acids, arranged in an extremely compact α/β structure (Vijay-Kumar (Vijay-Kumar), 1987): almost 87% of the polypeptide chain is involved in the formation of secondary structural elements by means of hydrogen bonds. Secondary structures are alpha 3,5-spiral coils, as well as antiparallel beta-layer, consisting of four strands. The characteristic arrangement of these elements is antiparallel beta-Plast open protein surface on the rear side which Packed the alpha helix, which lies vertically on its top is generally considered the so-called ubiquitin-like motif styling. Another structural feature is the pronounced hydrophobic region of the protein between�alpha-helix and beta-layer.

Because of its small size artificial preparation of ubiquitin can be carried out both by chemical synthesis or by biotechnological methods. Because of the favorable properties of stacking ubiquitin can be obtained by genetic engineering using microorganisms such as Escherichia coli, in relatively large quantities or in the cytosol or in the periplasmic space. Due to the oxidizing conditions prevailing in the periplasm, the latter strategy is usually reserved for the receipt of secretory proteins. For a simple and effective bacterial preparation of ubiquitin can be used as a partner in the Association obtained for other foreign proteins, which causes difficulties. By Association with the ubiquitin is possible to achieve high solubility and, through this, increased product yield.

Compared with antibodies or other alternative frameworks, artificial binding proteins based on ubiquitin proteins (also called Affilin®) have many advantages: small size, high stability, high affinity, high specificity, cost-effective microbial production and regulation of serum half-life. However, there is still a need for further development of these proteins in the sense of the new terapeuticas�x approaches with a high degree of affinity to specific targets. Although in document WO 05/05730 described in General terms the use of frameworks of ubiquitin to produce artificial binding proteins, there is not a proposed solution, how to modify ubiquitinate protein to get specific and high-affinity binding to ED-b of fibronectin.

In document WO 2008/022759 described recombinant binding proteins, and domain 3 (SH3) Src homology FYN kinase is used to obtain a new binding proteins. It is established that the target specificity can be developed through mutation RT-loop and/or Src-loops to develop protein therapies and/or diagnostics. As in lipocalin used as a framework, mutagenesis contain amino acid residues lie in a variable and flexible loop regions, simulating the principle underlying the binding functions of antibodies and/or antigen. This overall flexibility of the site of interaction, which antibodies bind an epitope that is mostly enthalpies ongoing process; this process, however, leads to an unfavourable entropic contribution loss of mobility upon binding of the flexible region, defining complementarity. In contrast, using ubiquitin as a scaffold, the authors of the present invention is not changed amino acid residues mainly in the flexible loop regions, and in rigid and not flexible beta threads obl�STI beta-layer or next to the beta thread. The advantage of selecting amino acid residues in not flexible and hard beta strands or near the beta-strands of ubiquitin in the areas of binding to ED-B is, inter alia, the following: it is believed that communicating partners already represent complementary geometry that is suitable for tight binding. Consequently, these interactions are complementary in shape, charge and hydrophilic/hydrophobic elements of the more rigid structures of the communicating partners. These interactions rigid body optimize the interface surface and provide a biological function.

Fibronectin (FN) are an important class of high molecular weight extracellular matrix picobello, abundantly expressed in healthy tissues and body fluids. Their main role is to facilitate in the adhesion of cells to a number of different extracellular matrices.

The presence of fibronectin on the surface of untransformed cells in culture, as well as their absence in the case of transformed cells led to the identification of fibronectin as an important adhesion proteins. They interact with numerous other different molecules, e.g., collagen, heparan sulfate-proteoglycans and fibrin and thus regulate the shape of cells and the creation of the cytoskeleton. In addition, they are responsible for the migration of cells differentiatio cells during embryogenesis. They also play an important role in the healing of wounds, in which they facilitate the migration of macrophages and other immune cells, and in the formation of blood clots, allowing adhesion of platelets to damaged areas of blood vessels.

Extradosed B (ED-B) of fibronectin is a small domain, which is administered by alternative splicing of the primary transcript RNA molecule fibronectin. This molecule is present or absent in molecules of fibronectin extracellular matrix and represents one of the most selective markers associated with angiogenesis and tissue remodeling, as it is abundantly expressed around new blood vessels, but cannot be detected in virtually all normal adult tissues (with the exception of the uterus and ovaries). It is known that ED-B is involved mainly in cancer. High levels of expression of ED-B were detected in the areas of primary lesions, and metastasis of many solid cancer sites in humans, including breast, non-small cell lung, colorectal, pancreatic, skin, hepatocellular, increased intracranial meningioma, glioblastoma (Menrad and Menssen (Menrad u. Menssen), 2005). In addition, ED-B may be associated with diagnostic agents and can advantageously be used as diagnostic tools. One example I�is its use in molecular imaging for example, atherosclerotic plaques and the detection of cancer, for example, by immunoscintigraphy cancer patients. Also many other possible ways of use in diagnosis.

Amino acid sequence of 91 amino acids of human extradata B (ED-B) of fibronectin is shown in SEQ ID NO: 2. For protein expression have to add the starting methionine. ED-B is abundantly present in mammals, for example, in rodents, cattle, primates, carnivores, etc. Examples of animals that have 100% sequence identity with ED-B man, are Rattus norvegicus, Bos taurus, Mus musculus, Equus caballus, Macaca mulatta, Canis lupus familiaris, and Pan troglodytes.

ED-B is specifically accumulates in neovascular structures and represents a molecular target for intervention in cancer. A number of antibodies or fragments of antibodies to the domain ED-B fibronectin is known in this area as a potential therapeutic agent for cancer and other indications (see, for example, WO 97/45544, WO 07/054120, WO 99/58570, WO 01/62800). Fragment ScFvL19 antibody single chain Fv (also referred to as L19) is specific to the domain ED-B of fibronectin and, as confirmed in experimental models of tumors and in patients with cancer, selectively focuses on the formation of new blood vessels in tumors. In addition, the conjugates comprising a�t-ED-B antibody or anti-ED-B antibody fragment with cytokine such as IL-12, IL-2, IL-10, IL-15, IL-24 and GM-CSF, have been described for drugs against target cells for the production of a medicament for inhibiting, in particular, cancer, angiogenesis is the growth of metastases (see, e.g., WO 06/119897, WO 07/128563, WO 01/62298). Selective targeting to the formation of new blood vessels in solid tumours with anti-ED-B antibodies or anti-ED-B fragments of antibodies, such as L19, conjugating with an appropriate effector function, such as cytotoxic or immune stimulating agent has been successful in animal experiments. For the treatment of pancreatic cancer compound proteins comprising a portion of interleukin-2 (IL-2) and part of the anti-ED-B antibodies were combined with a small molecule of gemcitabine (2'-deoxy-2 D'- diversityin) (see, for example, WO 07/115837).

The above documents from an existing prior art describe the use of different frames of proteins, including antibodies, to create new binding ED-B protein. Targeting ED-B with currently available compounds has certain disadvantages. More small molecules (such as heteropolymer binding ED-B proteins of the present invention on the basis of ubiquitin) with comparable or even higher affinity to the antigen ED-B is expected to have significant advantages over antibodies or other binding proteins.

Because the�ku cancer is one of the leading causes of death in the world, there is a growing need for improved agents for the treatment of cancer. Current chemotherapeutic agents and radiation treatment have poor selectivity, and most chemotherapeutic agents do not accumulate in the site of the tumour and, thus, may not achieve adequate levels in the tumor. There is a strong medical need for effective treatment of cancer.

Thus, the purpose of the present invention is to offer heteropolymer binding proteins on the basis of ubiquitin, is able to specifically communicate with very high affinity to the extracellular domain of fibronectin (ED-B). Another objective of the present invention is to identify and propose new binding proteins with a very high binding specificity to ED-B, for example, for use in the treatment of cancer. In addition, there must be a method to get mentioned heteropolymer binding molecule.

The above objective is solved by the subject matter of the attached independent claims of the claims. Preferred embodiments of the invention are included in the dependent claims and in the following description, examples and figures in the drawings.

Description of the INVENTION

More specifically, the inventors propose a protein, the method�th bind ED-B fibronectin person including modified heterodimeric ubiquitinate protein in which two Monomeric ubiquitin (ubiquitin link) related to the location of the "head to tail", and each monomer mentioned dimeric protein is differently modified by substitution of at least 6 amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO: 1, and wherein the said substitution include

(1) in the first Monomeric unit substitution, at least in amino acid positions 6, 8, 63, 64, 65 and 66; and

in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; optionally 2, or

(2) in the first Monomeric unit substitution, at least in amino acid positions 2, 4, 6, 62, 63, 64, 65 and 66; and

in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; optionally 2,

and, optionally, further modification, preferably a substitution of other amino acids, and referred to the modified Monomeric ubiquitinate link has amino acid identity with SEQ ID NO: 1 by at least one from the group of 80%, at least 83%, at least 85%, at least 83% and at least 90%, and over and above protein has specific binding affinity with the domain ED-B of fibronectin with kilodaltons =10-7-10-12M � exhibits a monovalent binding activity with extraluminal B (ED-B) of fibronectin.

In one preferred embodiment of implementation, the protein is recombinant.

In other embodiments, 7, 8, 9 or all of the amino acids in positions 2,4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO: 1 is modified in each monomer ubiquitination link. It should be understood that the present invention allows the combination of each of these variations in each Monomeric unit, i.e., in the first and second link. For example, the first monomer unit may include 6 modifications, when the second link includes 7 or 8 modifications, the first link may include 8 modifications and the second link 7 modifications etc. Each of these amino acids may be selected in the first and the second link, and then both links of combine. Preferred substitution is described below.

Definitions of important terms used in this application

The term "extradosed In fibronectin or shortly referred to as "ED-B" includes all proteins that show sequence identity with SEQ ID NO: 2 by at least 70%, optionally 75%, further optionally in 80%, 85%, 90%, 95%, 96% or 97% or more, or by 100% and which are set out above functionality ED-B.

The terms "protein, able to bind" or "spaziali protein" refer to ubiquitination protein comprising a domain of binding to ED-B, which is defined below. Any such linking Bel�to on the basis of ubiquitin may include additional protein domains, are not stasiewski domains, such as, for example, fluid multimerization, polypeptide tags, polypeptide linkers and/or non-protein molecules are polymers. Some examples of non-protein molecules polymers are gidroksiètilovyh starch, polyethylene glycol, polypropylene glycol or polyoxyalkylene.

Antibodies and fragments thereof are well known to the person skilled in the art. Binding protein of the invention is not an antibody or its fragment, such as Fab fragments or scFv. In addition, the binding domain of the invention does not include an immunoglobulin, as laid in antibodies.

In the present description of the invention, the terms "ligand" and "target" and "binding partner" are used interchangeably and can be mutually replaced. A ligand is any molecule that is able to communicate with the affinity defined here for heteropolymers modified ubiquitinated protein.

The term "ubiquitinate protein comprises ubiquitin in accordance with SEQ ID NO: 1 and its modifications according to the following definition. Ubiquitin highly retained in eukaryotic organisms. For example, all mammals,

studied to date, ubiquitin has an identical amino acid sequence. Especially preferred are molecules of ubiquitin from human�and, rodents, pigs and primates. In addition, you can use ubiquitin from any other eukarioticheskogo source. For example, ubiquitin yeast differs only in three amino acids from the sequence SEQ ID NO: 1. In General, ubiquitinate proteins covered referred to by the term "ubiquitinate protein" indicate amino acid identity of more than 70%, preferably more than 75% or more than 80%, more than 85%, more than 90%, more than 95%, more than 96% or until sequence identity of 97% with SEQ ID NO: 1.

The term "modified ubiquitinate protein" refers to modifications to ubiquitinated protein of any one of substitution, insertions or deletions of amino acids or combinations thereof, wherein the substitution are the most preferred modifications which may be supplemented with any one of the modifications described above. The number of modifications is strictly limited, as mentioned modified Monomeric ubiquitinate links have amino acid identity with SEQ ID NO: 1 at least one of the group of 80%, at least 83%, at least 85%", at least 83% and at least 90%. At most, the total number of substitutions in the Monomeric unit, therefore, is limited to 15 amino acids corresponding to 80% of amino acid identity. The total number of modified amino acids in heterodimeric the Molek�e of ubiquitin is 30 amino acids, corresponding to 20% of amino acid modifications on the basis heterodimeric protein. Amino acid identity dimeric modified ubiquitinated protein compared to non-modified dimeric ubiquitinates a protein with a basic monomer sequence SEQ ID NO: 1 is selected at least one from the group of 80%, at least 83%, at least 85%, at least 83% and at least 90%.

To determine the degree of sequence identity is derived of ubiquitin to the amino acid sequence of SEQ ID NO: 1 can be used, for example, the program SIM local similarities (Sawkin Huang and Webb Miller (Xiaoquin Huang and Webb Miller), "Advances in applied mathematics, Volume 12: 337-357,1991) or Clustal W. (Clustal, W.) (Thompson and others (Thompson et al.), Nucl Acids Res., 22(22): 4673-4680,1994.).

Preferably, the degree of identity to the sequence of the modified protein with SEQ ID NO: 1 is determined relative to the full sequence of SEQ ID NO: 1.

Heterodimers composite protein" or "heterodimeric protein" of the invention is considered to be a protein that consists of two differently modified Monomeric ubiquitin protein with two interacting binding domain areas together provides monovalent binding property (spaziali domain) for ED-B as a specific binding partner. Heteroge�EP is produced by connecting two Monomeric molecules of ubiquitin, both of these molecules are modified in different ways, as described herein.

The advantage of multimerization differently modified by monomers of ubiquitin to generate heteropolymeric binding proteins (here: heterodimerize proteins) with monovalent binding activity is to increase the total number of amino acid residues that can be modified to create a new property of high-affinity binding to ED-B. the Main advantage is that although modify even more amino acids, the chemical integrity of the protein is maintained without compromising the overall stability of the frame referred to the newly created protein binding to ED-B. the Total number of residues that can be modified to create a new binding site for ED-increases, because the modified residues may be divided into two Monomeric ubiquitin protein. The number of modifications may therefore be equal to two, corresponding to the number of modified Monomeric molecules of ubiquitin. The modular structure of the protein binding to ED-B on the basis of ubiquitin allows to increase the total number of modified amino acids, as mentioned modified amino acids are included in two Monomeric molecules of ubiquitin. This method provides identification of heterodimer�tion of molecules of ubiquitin, having one monovalent specificity for a single epitope) for ED-B.

Thus, the use of heterodimers that share a common binding site for binding partners, opens the possibility to introduce an increased number of modified residues that do not impact unduly on the chemical integrity of the protein end-binding molecules, since the cumulative number of these modified residues are distributed between two Monomeric units, which form a dimer. Mentioned heterodimerize modified ubiquitinate proteins, binding to ED-B, presented at the library of proteins.

"Monovalent" should be understood as the possibility that the two connecting areas created in the first and second Monomeric unit the modified dimeric ubiquitin, together bind ED-B synergistically and are United, i.e., both connecting areas work together to form a monovalent binding activity. If you take each separately connecting the first region and the second modified ubiquitin mentioned in heterodimeric the molecule, this region is obviously going to bind ED-B with much less efficiency and affinity than the dimeric molecule. Both connecting areas form a unique binding site that is formed as a contiguous region of amino acids n� surface heterodimeric modified ubiquitinated protein so mentioned modified ubiquitin is able to communicate much more effectively with ED-B than each Monomeric protein, taken separately. It is particularly important that, according to the present invention two Monomeric protein not linked after screening the strongest binding molecules of ubiquitin, but the screening process is performed in the presence heterodimeric of ubiquitin. After obtaining sequence information on the most strongly binding of ubiquitin molecules, these molecules can be obtained in any other way, for example, by chemical synthesis or methods of genetic engineering, for example, by linking already identified two of the ubiquitin monomer units together.

According to the invention, two differently modified by monomers of ubiquitin that bind to one ligand must be linked by connecting the "go-fishing-to-tail" with each other using, for example, genetic methods. These different modified combined monomers of ubiquitin bind monovalent and effective only if both "region binding domain" ("GSO") are acting together. "Region binding domain" is defined here as the area in the monomer of ubiquitin, which has a modified amino acid of at least 6 amino acids in positions 2,4, 6, 8, 62,3, 64, 65, 66, 68 SEQ ID NO: 1, which are involved in binding of the target.

Modified, and the associated monomers of ubiquitin, which form heteroge-dimensional protein bind to the same epitope related through a single coupling region. This adjacent area heteromer is formed by both determining the binding regions of the two modules, formed by two differently modified monomers of ubiquitin.

"The connection head-to-tail" must be understood as a compound of two proteins together by connecting them in the direction of the N-C-N-C-, depending on the number of links contained in the dimer. When this compound head-to-tail monomers of ubiquitin can be directly linked without any linker.

Alternatively, coupling of ubiquitin monomers can be performed by means of linkers, for example, the linker having at least the amino acid sequence GIG or having at least the amino acid sequence of SGGGG or any other linker, for example GIG, SGGGG, SGGGGIG, SGGGGSGGGGIG or SGGGGSGGGG. Known in the art and can be used and other linkers to the genetic connection of the two monomers of ubiquitin.

Modified ubiquitinate proteins of the invention constructed with new communicating sredstvami to ED-B as the target or the ligand (this expression

used here usamos�replaceable). The term "substitution" also includes chemical modifications of amino acids by, for example, substitution or addition of chemical groups or residues to an original amino acid. The amino acid substitution in at least one region with an open surface proteins, including amino acids located in at least one strand of the beta-layer region of the beta reservoir or facility to 3 amino acids near the thread of the beta reservoir is very important.

The amino acid substitution to create a new binding domain specific for ED-B, can be made according to the invention with any desired amino acid, i.e., to modify, to create a new binding property to the ED-B, it is not necessary to take care that the amino acids had any particular chemical property or side chain, respectively, which are similar to that of the replaced amino acids, so for this purpose you can use any desired amino acid.

The phase modification of the selected amino acids is performed, according to the invention, preferably by mutation at the genetic level by non-specific mutagenesis, i.e., non-specific substitution of selected amino acids. Preferably, the modification of ubiquitin perform the methods of genetic engineering to modify the DNA belonging to the corresponding protein. Preferred�Thelen, the expression ubiquitinated then carry out protein in prokaryotic or eukaryotic organisms.

Substitution will, in particular, the amino acids on the open surface of the four beta strands of the beta reservoir or amino acids with an open surface to 3 amino acids near the thread of ubiquitin protein strands of the beta reservoir ubiquitinated protein. Every beta thread usually consists of 5-7 amino acids. With reference to SEQ ID N0:1, for example, beta-strands usually include amino acid residues 2-7, 12-16, 41-45 and 65-71. Areas that can be optionally and preferably modified to include provisions for up to 3 amino acids (i.e., 1, 2 or 3) next to the thread of the beta-layer.

The preferred areas, which can be optionally and preferably modified to include, in particular, amino acid residues 8-11, 62-64 and 72-75. Preferred fields include beta-turns, linking two beta-strands together. One preferred beta stage includes amino acid residues 62-64. The most preferred amino acid that is located near the thread of the beta reservoir, is the amino acid in position 8. Furthermore, other preferred examples of amino-acid substitutions are 36, 44, 70 and/or 71. For example, those areas which can be optionally and preferably modified to include amino acids 62, 63 � 64 (3 amino acids), 72, 73 (2 amino acids), or 8 (1 amino acid).

In preferred embodiments, amino acid residues are modified by substitution of amino acids. However, it is also possible deletions and insertions. The number of amino acids that can be added or withdrawn, limited 1, 2, 3, 4, 5, 6, 7 or 8 amino acids in Monomeric subzone of ubiquitin and respectively 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 amino acids relative to dimeric ubiquitination squirrel. In one embodiment of the insertions of amino acids do not. In yet another embodiment, the implementation does not perform withdrawals.

Provided that the modified ubiquitinate protein of the present invention includes in addition to the aforementioned substitutions defined in the claims and explained here, as well as deletions and/or additions of one or more amino acids, position source of amino acids for human ubiquitin (SEQ ID NO: 1) must be aligned with the modified ubiquitin to select proteins corresponding to each other. In the case of composite proteins (see below), the numbering (and alignment) of each of subzones monomer of ubiquitin is carried out identically, i.e., alignment of, for example, dimer begin in position 1 of the amino acid for each respective subzone.

In a Monomeric ubiquitin, preferably from mammals, nab�emer, man, at least 10% of the amino acids present in beta strands or positions up to 3 amino acids near the thread of the beta-layer, preferably at least 20%, more preferably at least 25%, can be modified, preferably substituted, according to the present invention, to create a binding property that did not previously exist. As a maximum, preferably approximately 50% of the amino acids present in beta strands or positions up to 3 amino acids near the thread of the beta-layer, more preferably at least about 40%, or about 35%, or to about 30%, or to approximately 25% are modified, preferably substituted. In one of the beta threads usually modified 1-4 amino acids. In one embodiment, the implementation of three of the six amino acids preferably in the first and fourth β strands, for example, in the field of amino acid residues 2-7 or 65-71, modified.

Modified Monomeric ubiquitin according to the invention used as a construction element for heterodimer, has in the aggregate to 20% of the amino acids. Given this, there exists a sequence identity with SEQ ID NO: 1 modified ubiquitinated protein at least 80%. In other embodiments, the sequence identity and�inoculation level is at least 83%, at least 85%, at least 87% and, in addition, at least 90%, at least 92% or at least 95% sequence identity with an amino acid sequence of SEQ ID NO: 1. The invention also encompasses the identity of the amino acid sequence of the modified ubiquitinated protein 97%" compared with the amino acid sequence of SEQ ID NO: 1.

In yet another embodiment the ubiquitin modified on 3 or 4 or 5, or 6, or 7 amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and/or 68 of SEQ ID NO: 1. In yet another embodiment of the ubiquitin that you want to modify these provisions, already pre-modified. For example, other modifications may include modifications in the amino acids 74 and 75, or amino acid 45 to create increased stability or chemical properties of the protein. Can be obtained a modified monomer of ubiquitin, which in the aggregate to 9, 10, 11, 12, 13, 14 and a maximum of 15 amino acids of ubiquitin with SEQ ID NO: 1 are modified, preferably substituted. According to one example, can be obtained a modified Monomeric ubiquitin with 14 substitutions and one withdrawal. On the basis of the total number of amino acids of ubiquitin this corresponds to approximately 20%. This was a very surprising and could not be expected �ecause usually much smaller percentage is sufficient for disorders of protein folding.

In one embodiment, these amino acids are modified to create an area that has new properties of binding ED-B, which form a continuous region on the surface of the protein. Thus can be created in a continuous area that has a property of binding to ED-B. "Continuous region" according to the invention applies to the following: because of the charge, the spatial structure and hydrophobicity/hydrophilicity of its side chains of amino acids interact with their environment accordingly. This environment can be a solvent, usually water, or other molecules, for example, spatially close amino acids. By structural information about the protein, as well as the corresponding software can be characterized surface proteins. For example, the area of the interface between the protein atoms and solvent can be visualized this way, including information about how structured this area of the interface, which parts of the surface accessible to solvent, or as distributed on the surface charges. Continuous region may be disclosed, for example, the visualization of this type using appropriate software. Such methods are known to those skilled in the art. According to izobreteny�, basically, the entire area with an open surface can be used as a continuous area on the surface that you want to modify to create new binding properties. In one embodiment, the implementation for this purpose, the modification may also include alpha-helical region. In heterodimers modified ubiquitinated protein region that defines a binding that includes two of the areas with an open surface forming together a single continuous region, which includes two lengths of one area, which determines the binding.

Modification of amino acids in at least one region of a protein with an outdoor surface, comprising at least one beta-thread region of the beta reservoir or positions up to 3 amino acids near the thread of the beta layer is very important. "Structure of beta-Plast" is defined as essentially plastopodobnye and almost fully stretched. In contrast to alpha-helices, which are formed from undisturbed segment of the polypeptide chain, beta-layers can be formed in different regions of the polypeptide chain. This image region, which are separated farther from each other in the primary structure, can enter into close proximity with each other. Beta thread usually has a length of 5-10 amino acids (usually 5-6 residues in ubiquitin) and has almost fully stretched, conformal�. Beta-strands are suitable so close to each other that are formed by hydrogen bonds between the-O one strand and the NH group of the other strand and Vice versa. Beta-layers can be formed from several threads and have plastopodobnye structure in which the position of the alpha-C atoms alternates between positions above and below the reservoir plane. Side chains of amino acids follow this model and, thus, an alternative point up or down. Depending on the orientation of the beta-strands, the layers are classified into parallel and antiparallel. According to the invention, both types can mutate and be used for preparation of the proposed proteins.

For mutagenesis of beta-strands and beta structure-formation in the ubiquitin choose the beta thread or positions up to 3 amino acids adjacent to the beta thread (which is thread beta formation), which are close to the surface. Amino acids with an open surface can be identified with regard to the available x-ray crystallographic structure. If the crystal structure is not available, you can make attempts to predict through computer analysis of the region of the beta layer on the open surface and the availability of individual provisions of amino acids with regard to the available primary structure or model the three-dimensional structure of the protein and to obtain information about the p�potential amino acids with an open surface. Further disclosure of this question can take, for example, in J. Mol. Biol, 1987 Apr 5; 194(3):531-44. Vijay-Kumar S, C. E. Bugg, W. J. Cook

However, you can also make modifications in the beta-layer or positions up to 3 amino acids adjacent to the beta thread, for which time-consuming pre-selection provisions of amino acids for mutagenesis can be omitted. Region of DNA that encode patterns of beta-layers, or up to 3 amino acids near the thread of the beta reservoir isolated from the DNA of the environment, is subjected to non-specific mutagenesis and then re-integrate into the DNA coding for the protein from which they were removed previously. This is followed by the process of selection of mutants with desired binding properties.

In yet another embodiment the beta-strands or up to 3 amino acids adjacent to the beta thread close to the surface is chosen as above and identify the position of amino acids for mutagenesis in these selected areas. The position of the amino acids selected in this way can then be subjected to mutagenesis at the DNA level or by directed to the site of mutation, i.e., a codon encoding a specific amino acid is replaced by a codon encoding another previously selected specific amino acid, or the substitution is carried out in the context of non-specific mutagenesis, which defines the substitutable position of the amino acid�located the whereabouts, but not codon, encoding a new, yet to a certain amino acid.

"Amino acids from the open surface are amino acids that are accessible to the surrounding solvent. If the availability of amino acids in the protein of more than 8% compared to the availability of amino acids in the model Tripeptide Gly-X-Gly, the amino acids referred to as" exposed surface". These areas of the protein, or position of individual amino acids, respectively, are also preferred binding sites for potential binding partners, which selection must be made according to the invention. In addition, reference is made to the publication of Custer and others (Caster et al, Science 1983, 221, 709 - 713, and Sraka and Ripley (Shrake &Rupley), 1973 J. Mol. Biol. 79(2):351-371, which for full disclosure included in this application by reference.

Variations of the frame ubiquitinated protein characterized by substitutions of amino acids in artificial binding site, newly created from the parent protein and from each other, can be created by targeted mutagenesis of the respective sequence segments. In this case, amino acids having properties such as polarity, charge, solubility, hydrophobicity or hydrophilicity can be replaced or substituted, respectively, the amino acids with the corresponding other properties. Cu�IU substitutions the terms "mutation" and "modified" and "substituted" also include insertions and deletions. On the protein level modifications can also be made by chemical alteration of the side chains of amino acids according to methods known to experts in this field.

Methods mutagenesis of ubiquitin

As a starting point for mutagenesis of the corresponding sequence segments can be used, for example, cDNA of ubiquitin, which may be prepared, modified and amplified by methods known to experts in this field. Commercially available reagents and methods for site-specific changes of ubiquitin in relatively small regions of the primary sequence (approximately 1-3 amino acids) ("Quick Change", the company Stratagene; set "Mutagene Phagemid in vitro Mutagenesis Kit", the company Biorad). For site-directed mutagenesis larger areas experts can specific variants of implementation, for example, polymerase chain reaction (PCR). For this purpose you can use a mixture of synthetic oligodeoxynucleotides having compositions degenerate base pairs in the desired positions, for example, to enter mutations. This can also be done through the use of analogues of base pairs, which in natural form are not found in genomic DNA, such as, for example,inosine.

The starting point for mutagenesis of one or more beta strands of the beta field-layer or positions up to 3 amino acids near the thread of the beta layer may be, for example, cDNA of ubiquitin or genomic DNA. In addition, the gene coding for ubiquitinated protein can also be prepared synthetically.

Known and accessible procedures for mutagenesis are methods site-specific mutagenesis, methods of non-specific mutagenesis, mutagenesis using PCR or similar methods.

In one preferred embodiment the pre-determined position of amino acids for mutagenesis. The choice of amino acids for modification carried out to fit the restrictions of paragraph 1 of the claims against those amino acids that need to modify. In each case usually create a different library of mutants, which is subjected to screening using methods known as such. Usually a pre-selection of amino acids for modification can be particularly easily performed as ubiquitinated protein, subject to the modifications there is sufficient structural information.

Methods of targeted mutagenesis, and mutagenesis of longer sequence segments, for example by PCR, chemical mutagenesis or used�isua bacterial strains gene mutator also included in the prior art and can be used according to the invention.

In one embodiment, mutagenesis is performed by assembling Oli-gonucleotide DNA carrying the NNK codon amino acids. You should understand, however, that you can use other codons (triplets). Mutations performed in such a way as to preferably maintain the structure of the beta-layer. Typically, the mutagenesis occurs on the outside of the stable region of the beta reservoir, open on the surface of the protein. This includes both site-specific and non-specific mutagenesis.

Site-specific mutagenesis, including a relatively small region in the primary structure of the (approximately 3-5 amino acids) may be created using commercially available kits Stratagene® (QuickChange®) or Bio-Rad® (Mutagene® phagemid in vitro mutagenesis kit) (see US 5,789,166; US 4,873,192).

If site-specific mutagenesis is subjected over a wider area, shall be prepared DNA cassette in which the area for the mutagenesis produced by assembling nucleotides containing mutant and unmodified provisions (Nord, etc. (Nord et al), 1997 Nat. Biotechnol. 8,772-777; McConnell and Xecc (McConell and Hoess), 1995 J. Mol. Biol. 250,460-470.). Non-specific mutagenesis can be introduced through the distribution of DNA in strains of gene-mutator or amplification PCR (error-prone PCR) (for example, Pannekoek, etc. (Pannekoek et al.), 1993 Gene 128,135 140). For this purpose, a polymerase with increased frequency of Osh�side. To enhance the degree of entered mutagenesis or to combine different mutations, respectively, mutations in PCR fragments can be combined by DNA rearrangements (Stemmer (Stemmer), 1994, Nature 370, 389-391). An overview of these mutagenesis strategies in relation to the enzymes included in this review Kuchner and Arnold (Kuchner and Arnold) (1997) TIBTECH 15, 523-530. To implement this non-specific mutagenesis in the selected region of DNA must also be designed DNA cassette, which is used for mutagenesis.

Nonspecific modification performed by methods that are well established and well known in this field. "Non-specific modified nucleotide or amino acid sequence" is a nucleotide or amino acid sequence, which in several positions was subjected to insertion, removal or substitution of nucleotides or amino acids, the nature of which cannot be predicted. In many cases, the introduced non-specific nucleotides (amino acids), or a sequence of nucleotides (amino acids) will be "completely non-specific" (e.g., as a result of a randomized synthesis or PCR-mediated mutagenesis). However, non-specific sequences may also include sequences that have a common functional feature (e.g., the ability to Rea�synchronize with ligand product expression), or nonspecific sequences can be nonspecific in the sense that the end product of gene expression is completely non-specific sequence, for example, with a uniform distribution of the different amino acids.

In order to enter the randomized fragments into the vectors correctly, according to the invention should preferably be non-specific nucleotides were introduced into the expression vector according to the principle of site-directed, PCR-mediated mutagenesis. However, the known specialist and other options, and, for example, you can enter a library of synthetic non-specific sequences in the vectors.

To create mutants or libraries by combining PCR can be performed, for example, three PCR reactions. Two PCR reactions performed to generate partially overlapping the intermediate fragments. A third PCR reaction is performed to merge the intermediate fragments.

Method of constructing a library or mutant variants may include constructing a first set of primers around the desired restriction site (primer restriction site), direct and reverse primer restriction and a second set of primers around, for example, in front and behind the codon of interest (mutagenic primers), forward and reverse mutagenic primer. In one variation�the implementation design primers immediately in front and behind, accordingly, the codon of interest. Restriction and mutagenic primers used for construction of the first intermediate and second intermediate fragments. Two PCR reactions create these intermediate linear fragments. Each of these intermediate linear fragments includes at least one mutated codon of interest flanking the nucleotide sequence and the cleavage site. A third PCR reaction using these two intermediate fragments and the forward and reverse primers restriction enzymes for the production of the joint linear product. On the contrary, still the loose ends of the linear product was digested with restriction enzymes to create sticky ends on the linear product. The sticky ends of the linear product combine by using DNA ligase to obtain a ring product, for example, the annular polynuclear-tidnyj sequence.

To construct the intermediate fragments perform the design and synthesis of two sets of forward and reverse primers, wherein the first set contains the cleavage site of restriction enzyme together with its flanking nucleotide sequence, and the second set contains at least one variant of the codon of interest (mutagenic primers). Experts in this �area will understand the number of options will depend on the number of options is desirable modifications of amino acids. The author of the present invention provides that if in this process will be used other restriction enzymes, the exact location of the cleavage site and a corresponding sequence of forward and reverse primers may be modified accordingly. The level of technology available other ways, and they can be used is outlined.

In addition to the introduction of randomized fragment of the expression product in the frame in accordance with the present invention, it is often necessary to combine non-specific sequence with a partner on the merger by combining a randomised nucleotide sequence with a nucleotide sequence that encodes at least one partner in the Association. Such partner Association may, for example, to facilitate expression and/or purification/isolation, and/or further stabilization of product expression.

Nonspecific substitution according to one example of the present invention is at least 6 amino acids in positions 2,4, 6, 8, 62,63, 64, 65, 66 and/or 68 of the monomer of ubiquitin can be performed particularly easily by means of PCR, since these provisions are localized close to the amino - or carboxy-okoncane� protein. Accordingly, the codons for manipulating located at the 5' and 3' end of the corresponding cDNA strands. Thus, the first oligodeoxynucleotide used for the mutagenic PCR reactions except codons at positions 2,4, 6 and/or 8, which will mutate, corresponds to the sequence of the coding strands for the amino-end of ubiquitin. Accordingly, the second oligodeoxynucleotide - in addition to the codons in the provisions 62,63, 64,65,66 and/or 68, which will mutate, at least in part corresponds to non-coding strands of the polypeptide sequence of the carboxy-end. Through both oligodeoxynucleotides polymer resin different chain reaction can be performed using DNA-sequence encoding the Monomeric ubiquitin, as a template.

In addition, the obtained amplification product can be added to a different polymerase chain reaction using flanking oligodeoxynucleotides, which is administered, for example, the recognition sequence for restriction endonucleases. According to the invention is preferably obtained gene cassette in a vector system suitable for use in a subsequent selection procedure for isolation of variations of ubiquitin with binding properties to a predetermined hapten or antigen.

The area subject to Modific�tion in the ubiquitin

Areas for modification can be primarily selected based on the fact that they can be available for ED-B as a communicating partner, and from the fact that the overall structure of the protein is expected to show tolerance to the modification.

In addition to modifications in surface open beta strands, can also be carried out modifications in other open surface areas of the protein, preferably in positions up to 3 amino acids adjacent to the beta thread. These modified region participate in a newly created binding with high affinity to ED-B.

In yet another embodiment of the present invention the amino acid at one or two, preferably two, of the four beta-strands in the protein or in positions up to 3 amino acids, preferably near two of the four beta-strands is modified to create a new binding property. Also, optionally, can be carried out modification in three or four of the four beta strands or positions up to 3 amino acids adjacent to three or four beta-strands to create a binding to ED-B.

Particularly preferably, the amino acids in the threads of the amino-end and carbon-sea-end or positions up to 3 amino acids near the filament amino-end and the carboxy-end have been modified, preferably substituted to create a new website svyazyvanie� with ED-B. In this regard, particularly preferred, up to 4 amino acids near the thread carboxymethyl-the end of the beta reservoir were modified, preferably substituted, and 1 amino acid near the filament amino-end of the beta reservoir was modified, preferably substituted. Particularly preferred is a modification, preferably a substitution, at least three open surface amino acids of the following provisions of ubiquitin mammal, preferably human ubiquitin: 2, 4, 6, 8, 62, 63, 64, 65, 66, 68. These at least four amino acids from the groups consisting of 5 amino acids form a continuous surface open area on the surface of ubiquitin, which has been found to be particularly suitable for the creation of modified proteins having binding affinity, which did not exist before in relation to ED-B as a communicating partner. At least 3 of these amino acid residues should be modified. By choice, 3, 4, 5, 6, 7, 8, 9 or 10 mentioned modified amino acid residues, optionally, in combination with additional amino acid residues.

After the implementation of the above modifications, the inventors have found the sequence of ubiquitin from modified amino acids described in the examples that bind ED-B with a very high crodt�ohms (values kilodaltons to 10 -9).

Integral proteins

In yet another preferred embodiment of the invention relates to a composite protein comprising a binding protein of the invention combined with a pharmaceutically and/or diagnostically active component.

In another aspect the invention relates to a composite protein comprising heterodimeric binding protein of the invention combined with a pharmaceutically and/or diagnostically active component. Integral protein of the invention may comprise a polypeptide components, for example, peptide linkers, peptide ligands, for example, a therapeutically or diagnostically relevant radionucleotides. It may also include small organic or amino acid compounds, for example, sugar, oligo - or polysaccharide, fatty acid, etc. In one preferred embodiment, the molecule on the basis of heteromera of ubiquitin binding ED-B, covalently or not covalently konjugierten to a protein or peptide having a therapeutically or diagnostically relevant properties.

Below are some examples of how to obtain integral proteins on the basis of ubiquitin with the ability to bind ED-B.

a) Protein conjugation through lysine residues present in ubiquitin.

b) Conjugation of the binding protein on the basis� heterodimers of ubiquitin via cysteine residues - can be located in the end or any other position (for example, amino acid residue 24 or 57); conjugation with maleimide selectable components.

c) a Peptide or genetic conjugation - genetic associations (preferably P - or N-end).

(d) Associations based on the "label" is a protein or a peptide located in the C - or N-end of the target protein ED-b, "Labels" Association, for example, polyhistidine (particularly relevant for radiometry).

These and other methods of covalent and covalent attachment of the protein of interest to a substrate is well known in this area and therefore more detail is not described here.

By choice, mentioned active ingredient is a cytokine, preferably a cytokine selected from the group consisting of the tumor necrosis factor (e.g., TNF-alpha, TNF-beta), interleukins (e.g., IL-2, IL-12, IL-10, IL-15, IL-24, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-8, IL-1-alpha, IL-1-beta), interferon (e.g., IFN-alpha, IFN-beta, IFN-gamma), GM-CSF, GRO (GRO-alpha, GRO-beta, GRO-gamma), MIP (MIP-1-alpha, MIP-1-beta, MIP-3-alpha, MIP-3-beta), TGF-beta LIF1 CD80, CD-40 ligand, 70, LT-beta, Fas-ligand, ENA-78, LDGF-PBP, GCP-2, PF4, Mig, IP-10, SDF-1 alpha/beta, BUNZO/STRC33,l-TAC, BLC/BCA-1, MDC, TASK, TARC, RANTES, NAC-1, NCC-4, DC-CK1, MCP-1-5, Eotaxin, Eotaxin-2,1-309, MPIF-1, 6Ckine, STACK, MONTHS, lymphotactin, fractalkine, etc.

One of the most preferred cytokine� for use in the present invention is TNF-alpha. The inflammatory cytokine TNF has numerous activities in the body of a mammal, including anti-tumor effect, which is currently clinically irrelevance because of unacceptable toxicity effective dose for humans. Currently TNF therapeutically used in combination with cytotoxic substances such as melphalan.

Next on the choice referred to the active ingredient, which can be conjugated to heteropolymer binding protein on the basis of ubiquitin, is a toxic compound, preferably a small organic compound or a polypeptide, at the option of the toxic compound, for example selected from the group consisting of saporin, truncated Pseudomonas exotoxin A, recombinant gelonin, chain of ricin-A, calicheamicin, neocarzinostatin, espiramicina, Dynami-Qing, cedarcide, naturopatia, doxorubicin, daunorubicin, auristatin, cholera toxin, modeccin, diphtheria toxin.

In yet another embodiment, heteropolymeric binding protein through the ubiquitin according to the invention may contain artificial amino acids.

In other embodiments, a compound of the protein of the present invention mentioned active component is a fluorescent dye, preferably� component, selected from the group consisting of a radionuclide, or from the group of gamma-emitting isotopes, preferably 99Tc, 123i, 111Inor from the group of positron emitters, preferably 18F, 64Cu, 68Ga, 86Y, 124Ior from the group of beta-emitter, preferably 131I90Y, 177Lu, 67Cuor from the group of alpha-emitter, preferably 213Bi, 211At; dyes Alexa Fluor or cyanic (Berlier, etc. (Berlier et al.), J Histochem Cytochem. 51 (12): 1699-1712, 2003); the photosensitizer; Proma-golanty factor, preferably the tissue factor (for example, tTF-truncated tissue factor); an enzyme for prodrug activation, preferably an enzyme selected from the group consisting of carboxypeptidases, glucuronides and glycosides; and/or a functional Fc domain, preferably a functional Fc domain of human rights.

Another variant of implementation relates to integral proteins according to the invention, additionally comprising a component, modulating the half-life of serum, preferably a component selected from the group consisting of polybag.-langille, albumin-binding peptide, and immunoglobulin.

The specificity of binding (dissociation constants)

The specificity of binding protein component according to the invention are the same as defined above for a compound Belk�, expressed in kDa. In accordance with the invention, the term "kilodaltons" defines the specific binding affinity, which in accordance with the invention is in the range from 10-7up to 10-12M. a Value of 10-5M and below can be considered Express quantitatively the binding affinity. Depending on the application, a value of 10-7M-10-11M is preferably, for example, for chromatographic applications or 10-7-10-12M for example, for diagnostic or therapeutic applications. Other preferred values of the binding affinities are in the range from 107up to 10-10M, preferably up to 10-11M.

Methods of determining the values of the binding affinity are known as such and can be selected, for example, of the following: ELISA, technology based on surface plasmon resonance (PPR) (proposed, e.g., Biacore®), fluorescence spectroscopy, isothermal titration calorimetry (ITC), analytical ultracentrifugation, the excited fluorescence of the sorted cells.

After the implementation of the above modifications, the inventors have found that the sequence of ubiquitin from modified amino acids described in examples bind their targets with very high affinity (values in kDa to 1010 M).

Dimerization of ubiquitin

In the present invention a "dimer" is a protein that comprises two Monomeric ubiquitin protein. If the dimer comprises two differently modified monomer, it is called "heteromeric dimer" or "heterodimer". Thus, "heterodimer" of the invention is considered to be the Union of two differently modified Monomeric ubiquitin proteins, combined with monovalent binding property for specific binding partner ED-B.

It must be emphasized that the modified heterodimeric binding ED-B ubiquitinate protein of the invention cannot be obtained by screening of each separate monomer ubiquitinated protein and combining two of them later, but can be obtained by screening heterodimeric proteins consisting of the first and second Monomeric level, which together exhibit a monovalent spasieuse activity with the ligand ED-B. it Should be expected that each of these subzones shows quite a limited binding affinity to ED-B, whereas only the United dimeric modified ubiquitinate protein will have an excellent binding properties described herein (see, for example, Fig.4).

According to the invention, two differently modified monomer ubiqui�ina, genetically related by combining "head to tail" bind to the same epitope of the ED-B and is effective only if both the field binding domain act together. GSO monomers form one continuous region of the binding.

Thus, ubiquitinate protein, modified in accordance with the invention, dimerizer for efficient binding ED-B of fibronectin. The monomers can be connected directly or via linkers as described above. You can use many known linkers.

Each Monomeric ubiquitin modification has at least six amino acids 2, 4, 6, 8, 62, 63, 64, 65, 66, 68. Monomeric proteins genetically combined with each other. Binding with target mediated GSO mentioned together, i.e., the OSD interact with each other and form a common binding region, is able to communicate with the said domain ED-B fibronectin monovalent manner.

Heterodimer modified ubiquitin associated with ED-B Heterodimer of ubiquitin according to the invention, binding to ED-B with a KD=10-7-10-2M and active monovalent binding in respect of the said extradoses B (ED-B) of fibronectin, choose from the two following alternatives:

(1) in the first Monomeric unit substitution, at least in amino acid positions 6, 8, 63, 64,65 and 66; and

in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; optionally 2, and

(2) in the first Monomeric unit substitution, at least in amino acid positions 2,4, 6, 62, 63, 64, 65, and 66; and

in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; optionally 2.

In one embodiment of the compound, the protein is genetically combined heterodimers mentioned monomer of ubiquitin, having a substitution in the amino acids in positions 6, 8, 63-66 first monomer of ubiquitin and substitution in amino acid residues in positions 6, 8, 62-66, and optionally in position 2 of the second monomer of ubiquitin, preferably:

- in the first monomer of ubiquitin replacement

lysine (K) to tryptophan (W) or phenylalanine (F) in regulation 6,

Leikin (L) to tryptophan or phenylalanine (W, F) in position 8, 30,

lysine (K) to arginine (R) or histidine (H) at position 63,

glutamic acid (E) lysine (K), arginine (R) or histidine (H) at position 64,

series (S) to phenylalanine (F) or tryptophan (W) at position 65 and threonine (T) to Proline (P) at position 66;

- in the second monomer of ubiquitin preferred substitution

lysine (K) to threonine (T), asparagine (N), series (S) or glutamine (Q) at position 6, Leikin (L) to glutamine (Q) or threonine (T) or �asparagin (N) or series (S) in position 8,

glutamine (Q) to tryptophan (W) or phenylalanine (F) at position 62,

lysine (K) series (S), threonine (T), asparagine (N) or glutamine (Q) at position 63,

glutamic acid (E) asparagine (N), series (S), threonine (T), or glutamine (Q) in position 64,

series (S) to phenylalanine (F) or tryptophan (W) at position 65, and

threonine (T) to glutamic acid (E) or aspartic acid (D) at position 66, and

by choice, glutamine (Q) to arginine (R), histidine (H) or lysine (K) in position 2.

These alternative substitution in each monomer may be combined with each other without any restrictions, provided that the resulting heterodimer modified ubiquitin have a specific activity of binding with said extradata B (ED-B) of fibronectin of KD=10-7-10-12M and are active monovalent binding in the context of these extradata B (ED-B) of fibronectin, and provided that the structural stability ubiquitinated protein is not destroyed or affected.

Most preferred are the following overrides:

(1) in the first Monomeric unit at least K6W, L8W, K63R, E64K, S65F and TR;

and in the second Monomeric unit at least K6T, L8Q, Q62W, K63S, E64N, S65W, and TE; additionally Q2R, or

(2) in the first Monomeric unit at least Q2T, F4W, K6H, Q62N, K63F, E64K, S65L, and T66S;

and in the second Monomeric �Vienna at least K6X, L8x, Q62X, K63X, EH, S65X, and TH; additionally Q2X, where X can be any amino acid (see Fig.2).

Particularly preferred are the following substitution in the first monomer of ubiquitin to generate proteins binding to ED-B

2: Q→T, 4: F→W, 6: K→N, 62: Q→N, 63: K→F, 64: E→K, 65: S→L, 66: T→S

To connect the two monomers "head to tail" can be use or not use the linker. Preferred are linkers SEQ ID NO: 32 or a sequence GIG or SGGGGIG, or SGGGGSGGGGIG.

In one preferred embodiment of implementation, heterodimer of ubiquitin with two fields that determine the binding (GSO) and acting together to bind ED-B, includes the amino acid sequence of SEQ ID NO: 33 or 34. Another preferred protein represented by the following sequence, where XXXX can be any amino acid (SEQ ID NO: 47). Here as the linker used SGGGGSGGGGIG. It is understood that also any other type of linkers or, alternatively, the absence of the linker.

:MTIWVHTLTGKTITLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNINFKLSLHLVLRLRGGSGGGGSGGGGIG

MQIFVXTXTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNINXXXXXLHLVLRLRGG

Consensus sequences of examples of proteins with these sequences is shown in Fig.2.

The preferred compound is a protein of the invention, including TNF-alpha as a pharmaceutically active component�the one has the sequence of SEQ ID NO: 35 or 36.

In another aspect, the present invention also encompasses polynucleotides which are responsible for the creation of a protein or protein composite as described above. Additionally, the invention encompasses vectors comprising the aforementioned polynucleotide.

In an additional aspect of the present invention are covered by the host cell, which contain a protein or compound is a protein described herein, and/or the polynucleotide to create the said recombinant protein protein or compound of the invention or the vector containing the polynucleotide.

The use of proteins of the invention, for example, binding proteins on the basis of heterodimers of ubiquitin, specifically for the unification of the ED-B with an effector, such as TNF-alpha

Proteins binding modified ubiquitin with ED-B according to the invention to be used, e.g., for the preparation of a diagnostic means for use in vitro or in vivo, and therapeutic agents. Proteins according to the invention can be used, for example, how molecules direct effector (modulator, antagonist, agonist) or antigen-recognizing domains. Examples of tumors with abundant expression of ED-B antigen are shown in the table in Fig.1.

Depending on the chosen partner for associations, pharmaceutical composition of the invention �adaptirovana to focus on cancer treatment, for example, breast and colorectal cancers, or any other neoplastic disease with abundant expression of ED-B (see examples in Fig.1).

Compositions adapted for the maintenance of a therapeutically effective dose. The value assigned to the dose depends on the patient, type of disease, age and body weight of the patient, and other factors, known as such.

The compositions contain a pharmaceutically or diagnostically acceptable carrier and, optionally, can contain other auxiliary agents and fillers known per se. They include, for example, but without limitation, stabilizing agents, surfactants, salts, buffers, tinted agents, etc.

The pharmaceutical composition may be in the form of a liquid preparation, cream, lotion topicale applications, aerosols, powders, granules, tablets, suppositories or capsules, emulsions or liposomal preparation. The compositions are preferably sterile, non-pyrogenic and isotonic and contain traditional and pharmaceutically acceptable additives, known per se. Additionally, reference is made to the rules of the United States Pharmacopoeia or the publication "Pharmaceutical Sciences Remington", May Publishing Company (1990).

In human and veterinary therapy and prevention pharmaceutically effective medications containing �about at least one heteromeric ubiquitinate protein modified in accordance with the invention, for binding to ED-B can be prepared by methods known per se. Depending on herbal drug, these compositions can be administered by parenteral introduction in case of injection or infusion, systematically, rectally, intraperitoneally, intramuscularly, subcutaneously, intradermal or other traditionally used methods. Type pharmaceutical preparation depends on the type of disease, severity of disease, the patient and other factors known to specialists in the field of medicine.

In one embodiment of the pharmaceutical composition contains a protein or compound is a protein of the invention or a combination and in addition contains one or more chemotherapeutic agents, preferably selected from the following table:

Class contentExamples
Alkylating agents (ATC L01 (A)melphalan, cyclophosphamide
Antimetabolites (ATS L01B)5-fluorouracil, gemcitabine
Taxanes(ATS L01CD)paclitaxel
Cytotoxic antibiotics (ATC L01D)doxo�ubizen, liposome docking zorubicin
Platinum compounds (ATS L01XA)cisplatin

In one preferred embodiment of implementation, the chemotherapeutic agent is selected from melphalan, doxorubicin, cyclophosphamide, dactinomycin, fergeson-ciorcila, cisplatin, paclitaxel and gemcitabine; or from the group of inhibitors of key is called.

"Pharmaceutical composition" according to the invention can be present in the form of a composition in which different active ingredients and diluents and/or carriers are mixed with each other, or may take the form of a combined preparation, in which the active ingredients are present in partially or completely separate form. An example of such a composition or combined preparation is set.

"Composition" according to the present invention includes at least two pharmacologically active compounds. These compounds can be administered simultaneously or separately with a time interval from one minute to several days. Connections can be entered in the same way or in different ways; for example, oral administration of the same active compounds and parenteral introduction in case of introduction of a different connection. Also, the active compounds can be in the same medication, for example, in one solution for infusion or �AK kit, containing both compounds separately. It is also possible that both compounds were present in two or more packages.

In particular, a preferred combination is a compound protein according to the invention and melphalan and/or (liposomal) doxorubicin. Besides neoplastic agents from the class PBX L01, TNF-integral protein of the invention may be combined with other antineoplastic substances, including cytokines and their derivatives, radiopharmaceuticals, cell therapeutic drugs and nanoparticles.

Because of its activity penetration into the tumor, TNF-integral protein of the invention (as well as other recombinant proteins/integral proteins of the present invention) may be combined with antineoplastic agents listed in category L01 classification System of anatomic therapeutic chemical (ATC) provided by the world health organization.

The inventors unexpectedly found that a compound protein from heterodimer of ubiquitin, combined with TNF-alpha, and composite protein preferably has the sequence SEQ ID NO: 35 or 36, can be profitably applied in therapy. TNF-alpha is a highly toxic substance and, thus, can be given only in small doses that are usually lower than the minimum therapeutic for�yeah (and, thus, a therapeutically inactive). Due to the toxicity of TNF-alpha to achieve a therapeutically effective concentration, currently when using TNF-alpha have chosen the approach of perfusion in the isolated limb. Perfusion of the limb is a medical technique that can be used to deliver anticancer drugs directly to an arm or a leg. The blood flow in the limb and it temporarily stopped with a tourniquet and anticancer drugs are administered directly into the blood of the limb. This allows the patient to receive a large dose of TNF-alpha in the area where the cancer occurs.

However, by application of TNF-alpha protein component of the present invention, it is possible to introduce TNF-alpha in non-toxic but therapeutically effective concentration. Because TNF-alpha is connected to the (binding) integral protein of the present invention, it can be directly active in place of the disease (e.g., location of the tumor and, thus, the number of "free" TNF-alpha can be drastically reduced.

Systemic side effects of TNF-alpha can be markedly reduced by the introduction of TNF-alpha as an integral protein of the present invention. By the use of TNF-alpha protein composite of the invention, the total dose of TNF-alpha to achieve therapeutic EF�EKTA thus can be significantly reduced and can profitably be used for the treatment of systemic tumors (without the need and limitations of perfusion in the limb), in particular in combination with chemotherapeutic agents (see above).

In yet another embodiment of the pharmaceutical composition is in the form of a set of parts providing for a separate position for recombinant ubiquitinated protein/protein composite of the invention and one or more chemotherapeutic agents.

A method of producing heterodimeric binding ED-B protein of the invention

Proteins that bind ED-B according to the invention, can be prepared by any of many conventional and well known methods such as the simple strategy of organic synthesis, solid-phase synthesis methods or commercially available automated synthesizers. On the other hand, they can also be prepared in a conventional recombinant methods as such or in combination with traditional methods of synthesis.

In another aspect of the present invention a method of creating recombi-nantongo modified ubiquitinated protein. This method includes at least the following steps:

a) providing a population of differently modified dimeric ubiquitin proteins originating from Monomeric ubiquitin proteins, and the population includes dimeric ubiquitinate proteins comprising two modified monomer of ubiquitin, tie�pick up will be together in the location of "head to tail", each monomer mentioned dimeric protein is differently modified by substitution of at least 6 amino acids in positions 2,4,6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO: 1

moreover, the above-mentioned substitution include:

In one aspect of the invention referred to the population of differently modified proteins produced by the genetic Association of two DNA libraries, each of which is responsible for receiving differently modified Monomeric ubiquitin proteins.

In another aspect mentioned method is adapted to combine the mentioned modified heterodimeric ubiquitinate protein with a pharmaceutically active component, optionally a cytokine, preferably TNF-alpha or diagnostic component, or in which the mentioned recombinant modified heterodimeric ubiquitinate protein is formed through mentioned pharmaceutically active compound, which, optionally, is TNF-alpha, or referred to by the diagnostic component.

According to the invention, a modified protein may also be prepared by chemical synthesis. In this embodiment, the implementation steps (C) - (d) of paragraph 1 of the claims then performed in one stage.

In another aspect of the present invention is directed to a library containing the DNA responsible for poluchenierazreshenija Monomeric ubiquitin proteins as mentioned above, which form the basis for obtaining heterodimeric ubiquitin proteins of the invention.

In another aspect of the invention provides a library of associations containing DNA obtained by combining the two libraries, as mentioned above, each library is responsible for obtaining links differently modified Monomeric ubiquitin proteins to get heterodimerize integral proteins of ubiquitin, and their monomer units are linked at the location of the "head to tail", and mentioned the library is responsible for heterodimerize integral proteins of ubiquitin exhibiting monovalent binding activity against the mentioned extradata B (ED-B) of fibronectin. This binding between a run or using any of the linkers known to the person skilled in the art, or a linker described herein. In one embodiment, TNF-alpha is used as the linker, while acting as a pharmaceutically active compound.

Example 1 describes the preparation of a complex library. However, caution should be exercised in relation to the quality of the library. The quality of the library in frame technology is the first, depending on its complexity (number of separate variants), and functionality (structural�RNA and protein-chemical integrity of the resulting candidates). Both features, however, can have negative effects on each other: the increasing complexity of the library by increasing the number of modified positions on the frame can lead to deterioration of protein-chemical characteristics of options. This can cause reduced solubility, aggregation and/or low product yield. The reason for this is the high deviation from the original frames, having energetically favorable packing of the protein.

Therefore, when designing such a library of frameworks requires a suitable balance between the extreme positions of the introduction of the greatest possible number of variations in the original sequence to optimize it for the target and, on the other hand, to keep the original primary sequence to the maximum extent to avoid negative protein-chemical effects.

It should be said that the present disclosure also covers every possible combination of signs described here in terms of the aspects or embodiments of the invention.

The choice of modified ubiquitin proteins with binding affinity against the target ED-B and identification of modified amino acids responsible for binding affinity After, for example, create at least two different DNA libraries responsible for receiving g�trademark modified ubiquitin proteins by various modifications of selected amino acids in each monomer unit of ubiquitin these libraries are genetically combine, for example, linker technology to obtain DNA molecules responsible for receiving heterodimeric modified ubiquitin proteins. The DNA of these libraries Express in proteins, and the thus obtained modified dimeric proteins brought into contact according to the invention with ED-B, in order, optionally, to enable binding of the partners to each other if a binding affinity exists.

A very important aspect of the invention is that the process of contact and screening perform in relation to heterodimers ubiquitination squirrel. This process allows screening for those ubiquitin proteins that have activity monovalent binding to ED-B.

According to the invention the contact is preferably performed using a suitable method of representation and choice, such as phage display, ribosomal display, mRNA display or display surfaces of cells that display surfaces of yeast or display surfaces of bacteria, preferably by phage display method. For completeness of disclosure reference is also made to the following publications: Hoess, Curr. Opin. Struct. Biol. 3 (1993), 572-579; Wells and Lowmann, Curr. Opin. Struct. Biol. 2 (1992), 597-604; Kay et al., Phage Display of Pepties and Proteins - A Laboratory Manual (1996), Academic Press. The above methods known to experts in this field and can be used according to the invention, including their modifications.

Determining whether the modified protein is calculated quantitatively the binding affinity relative to a previously established binding partner may be accomplished according to the invention, preferably one or more of the following methods: ELISA, spectroscopy method, surface plasmon resonance, fluorescence spectroscopy, the excited fluorescence of the sorted cells (FACS), isothermal titration calorimetry and analytical ultracentrifugation.

Selection method rahovym display

One type of phage display procedure, adapted to the present application is described below as an example of the selection according to the invention with respect to variations of ubiquitin, which exhibit binding properties. In the same way can be applied, for example, how to represent the bacteria (bacterial surface display; Daugherty et al., 1998, Protein Eng. 11(9):825-832) or yeast cells (yeast surface display; Kieke et al, 1997 Protein Eng. 10(11): 1303-10) or cell-free system of choice, such as ribosomal display (Hanes and Pluckthun, 1997, Proc Natl Acad Sci USA. 94(10):4937-4942; He and Taussig, 1997_Nucleic 5 Acids Res. 25(24):5132-5134) or cis display (Odegrip et al, 2004 Proc Nal Acad Sci USA. 101 (9):2806-2810) or mRNA display. In the latter case, a temporary physical linkage of genotype and phenotype is achieved by connecting the variations of the protein with a suitable mRNA through the ribosome.

In the procedure described here phage display recombinant variations of ubiquitin are presented on the filamentous phage, whereas encoding DNA presents variations are presented at the same time Packed in single-stranded form in ragovoy shell. Thus, in the framework of affine enrichment variations that have certain properties, can be selected from the library, and their genetic information can be amplified by infection of suitable bacteria or added to another cycle of enrichment, respectively. Representation of the mutated ubiquitin on the surface of phage is achieved through a genetic Association with a signal sequence from the amino end, preferably with a PelB signal sequence, and the capsid or preferred surface protein of the phage is Kar-bocciarelli Association with capsid protein FMC or its fragment.

In addition, the obtained composite protein may contain other functional elements, such as a label affinity or epitope antibodies for the detection and/or purification affinity chromatography, or the recognition sequence of the protease to specific�die splitting compound protein in the affinity enrichment. In addition, the UAG terminator can be represented, for example, between the genome for variations of ubiquitin and the coding region of the capsid protein of the phage or its fragment, which is not recognized during translation in a suitable suppressor strain, partly due to the introduction of a single amino acid.

Bacterial vector, suitable for the selection procedure in the context of the isolation of the variations of ubiquitin with the properties of binding to ED-B and in which the introduced gene cassette described for compound, called Femidom. Among others, it contains the intergenic region of the filamentous phage (e.g. M13 or f1) or its part, which in the case of a superinfection of the bacterial cells, carrier phagemid by phages-

helpers, such as e.g. M13K07, leads to the packaging of a closed DNA strands phagemid in the phage capsid. Thus created phagemid are secreted by bacteria and represent the corresponding variation of ubiquitin coded because of its Association with capsid protein pIII or its fragment on their surface. Native capsid proteinspIIIpresent in Vegemite, so his ability to reinfiltrate suitable bacterial strains and, therefore, the ability to amplificatoare corresponding DNA remains. Thus, the physical link between the phenotype variations of ubiquitin, i.e., its potential svyazyvayuscyego, and its genotype.

Received phagemid can be selected in relation to the binding of the variations of ubiquitin with ED-B by methods known to experts in this field. For this purpose presents variations of ubiquitin can be temporarily immobilized on the target substance, and associated, for example, in microtiter tablets, and can be specifically-eluted after separation is not communicating variations. The elution is preferably performed by alkaline solutions, such as, for example, 100 mm triethylamine. Alternatively, elution can be performed in acidic conditions, by proteolysis or direct upload infected with bacteria. Phagemid, thus obtained, can be reamplification and enriched by successive cycles of selection and amplification variations of ubiquitin with the properties of binding to ED-B.

Further characterization of the variations of ubiquitin, thus obtained, can be made in the form phagemid, i.e., combined with phage, or after cloning of the corresponding gene cassette expressing in a suitable vector in the form of soluble protein. Suitable methods known in the art or described in the literature. Characterization may include, for example, determining the DNA sequence and, thus, the primary sequence�eljnosti isolated variations. In addition, the affinity and specificity of isolated variations can be used to detect standard biochemical methods such as ELISA or spectroscopy surface plasmon resonance, fluorescence spectroscopy, FACS, isothermal titration calorimetry, analytical ultracentrifugation, etc. In light of the stability analysis, for example, spectroscopic methods in connection with chemical or physical deployment known to specialists in this field.

The method of selecting ribosomal display

In yet another embodiment, variations of ubiquitin procedures for ribosomal display is prepared by a cell-free system of transcription/translation, and present as a complex with the corresponding mRNA and the ribosome. For this purpose, a DNA library, which is described above, is used as the basis in which the genes of variations are present in the form of Association with appropriate regulatory sequences for expression and protein biosynthesis. Because of withdrawal, a termination codon at the end of the 3' gene libraries, as well as suitable experimental conditions (low temperature, high concentration of Mg) three-part complex consisting of emerging protein, mRNA and ribosomes, is maintained during transcription/translation in vitro.

After creating man likes�theca proteins containing heterodimers modified ubiquitinate proteins by different modifications of selected amino acids in each monomer unit of ubiquitin-modified dimeric proteins brought into contact according to the invention, with ED-B to allow partners to communicate with each other if there exists a binding affinity. These libraries of proteins can be in the form of a display library display method, or you can use any other way representing modified proteins in such a way as to ensure contact between the modified protein and protein-target ED-B, and mentioned display method is, by choice, rahovym display, ribosomal display, phage display TAT, yeast display, bacterial display or mRNA display.

The selection is modified by variations of ubiquitin relative to their activity of binding to ED-B 20, with specific binding affinity to KD in the range of 10-7-10-12M can be made by methods known to experts in this field. For this purpose, variations of ubiquitin, represented, for example, the ribosomal complexes, can be temporarily immobilized on the target substance, and associated, for example, in microtiter tablets, or may be associated with magnetic particles after binding in solution, respectively. On�Les offices nezvasimoe varying variations of genetic information variations with binding activity may be specifically-eluted in the form of mRNA through degradation of the ribosomal complex. Elution is preferably carried out with 50 mm EDTA. The thus obtained mRNA can be isolated and transcribed back into DNA by using appropriate methods (reverse transcriptase reaction), and DNA, thus obtained, can be reamplification.

Through consecutive cycles of in vitro transcription/translation, selection and amplification variations of ubiquitin with the properties of binding with a specific hapten or antigen may be enriched.

Characterization of proteins binding to EDB

Further characterization of the variations of ubiquitin, thus obtained, can be made in the form of soluble protein, as detailed above, after cloning of the corresponding gene cassette expressing in a suitable vector. Suitable methods known in the art or described in the literature.

Preferably, the step for detecting proteins having binding affinity to specific binding partner, the next step of isolation and/or enrichment of another protein.

After expression ubiquitinated protein, modified according to the invention, it may be further purified and enriched by methods known as such. Choose the methods depend on several factors, known as such specialists in the region.� area for example, used expressing vector, of the host body, intended field of use, size, protein, and other factors. To simplify purification of the protein, modified according to the invention may be combined with other peptide sequences with a high affinity with the materials division. Preferably selected such associations, which have no adverse effect on the functionality ubiquitinated protein or can be separated after cleaning due to the introduction of specific cleavage sites of the protease. Such methods are also known as such to specialists in this field.

BRIEF description of the DRAWINGS

Fig.Table 1 shows the h indicating the occurrence of ED-B in different tumors.

Fig.2 shows a consensus position and amino acid substitution in 16 sequences, which were found to have a surprisingly strong binding affinity to ED-B. the provisions of the Consensus amino acids in the first monomer region, which determines the binding are 2,4,6, 62,63, 64, 65, 66, and consensus substitutions of amino acids are Q2T, F4W, K6H, Q62N, K63F, E64K, S65L, and T66S. As can be understood from Fig.2, 4 families of sequences can be enriched (consensus sequences, the letters correspond to the frequency of occurrence of amino acids). Regulations 85 and 87 are profession� provisions in heterodimer protein; in regard to the second monomer corresponding provisions are 6 and 8; 141-145 comply with the provisions 62-64). TWH NFKLS displayed in dark blue, comes from 1071-12. Residues marked in red belong to one of the four families of sequences. Residues marked in red, were mainly enriched (178/457 sequences) and include, according to ELISA, the strongest binding molecule.

Fig.3 shows that tetramerization leads to increased affinity.

The table shows values into CD modified by monomers of ubiquitin compared to tetramers, consisting of modified monomers of ubiquitin. As examples, shown the options E and N of ubiquitin. Binding ED-B is compared to the binding of c-FN (cellular fibronectin). These Figures show a significantly higher affinity in binding tetrameric variant (for example, 56 nm for E or 1.4 nm for N) with the target ED-B in comparison with the monomer (4,51 µm for E or 9,98 µm for N).

Fig.4 shows that the recombination front (first) modified monomer of ubiquitin (having BDR1) with another, modified with a rear (second) monomer of ubiquitin (having BDR2) to create heterodimer leads to a significant increase in affinity and specificity. Molecules modified ubiqui�ina analyzed by Biacore, fluorescence anisotropy binding on cells and tissue sections. Shown concentration-dependent ELISA results (conc. - ELISA) binding of several variants with ED-B person.

Fig.4A shows the binding affinity of KD=9,45 µm for monomer V.

Fig.4B it is shown that the binding affinity KD=131 nm for 41 B10, combined with another, the second monomer, leads to N.

Fig.5 shows specific variants, combined with a cytokine (e.g., TNF-alpha). These integral proteins will timeresult modified monomer of ubiquitin and are biologically active molecules.

Fig.5 A shows a schematic drawing of a composite protein - effector binding ED-B based on modified ubiquitin; green (structure above) - effector, e.g., a cytokine, preferably TNF-alpha; brown: light brown: structure modified by monomers of ubiquitin (Affilin®).

Fig.5 it is shown that effector conjugate of the modified ubiquitin A-TNF-conjugate has Pro-ipoptions activity (measured in the analysis of apoptosis in L929).

Fig.5 shows high-affinity binding N-tar-alpha Association with ED-B (KD=15,1 nm) (filled circles, United-fitting line). Binding to BSA is plotted on a graph as a control (filled circles connected by a line).

Fig.6 shows the affinity and �aktivnosti molecules heterodimer, binding to ED-B, on the basis of the modified ubiquitin, combined with the cytokine, e.g., TNF-alpha.

- By inducing apoptosis activity of the Association of cytokine that binds to ED-B, on the basis of the modified ubiquitin: EU500,78±0,24 PM

- By inducing apoptosis activity of the free cytokine: EU503,14±3,59 PM

Fig.6A shows the affinity of heterodimer N (KD of 50.7 nm), binding ED-B, on the basis of the modified ubiquitin.

Fig.6B shows the affinity of heterodimer N binding ED-B, on the basis of the modified ubiquitin, genetically combined with the cytokine TNF-alpha to lead to multimerization of heterodimer N (KD=5,6 nm).

Fig.6C shows an analysis of examples of candidates from the selected library heteroge-dimensional modified ubiquitin, for example, clones A, 22D1, N, V of heterodimer. The value of ELISA in kDa is increased to the target ED-B compared with the cytosolic fibronectin used as a control, confirming the specific binding with the target.

Fig.6D shows the results of the analysis of molecules A modified hetero-dimeric ubiquitin analyses of interactions without labels using Biacore®. Analyzed different concentration options heterodimers of ubiquitin (see legend to the Figure: 0-15 µm E) �La binding to ED-B, immobilized on the chip (Biacore) to analyze the interaction between heterodimers option A and ED-B. the value in the kDa cannot be determined from the curve analysis of Association and dissociation.

Fig.6E shows the results of the analysis of molecules 41 B10 modified heterodimers of ubiquitin through analyses of interactions without labels using Biacore®. Analyzed different concentration options heteroge-dimensional ubiquitin (see legend to the Figure: 0-15 µm 41 B10) for binding to ED-B immobilized on the chip (Biacore) to analyze the interaction between heterodimers option B and ED-B. curve Analysis of Association and dissociation gave a KD value of 623 nm (623×10-9M to 6.2×10-7M).

Fig.7 shows the contribution of different variants on the basis of the modified ubiquitin in binding affinity and specificity. These different versions share the modules of the overall sequence, which are marked with lowercase letters. The options were analyzed for their binding to ED-B. Fig.3 shows other combinations of monomers giving heterodimer modified ubiquitin. Heterodimerize options 46-A5, 50-G11 and 46-N4 have the same first (front) modified monomer with BDR1 (marked "a" in this Figure), but the second (rear) monomer of ubiquitin modified BDR2 in �different positions. Options 52-D10 and 52-B3 have another first (front) modified monomer, compared to 46-H9 with BDR1, but the same second (rear) monomer of ubiquitin with BDR2 (marked "e").

Heterodimer modified ubiquitin have the following sequence:

46-H4: SEQ IDNO: 25, 45-H9: SEQ IDNO: 26, 46-A5: SEQ IDNO: 27, 50-G11: SEQ ID NO: 28, 52-B3: SEQ ID NO: 29, 52-D10: SEQ ID NO: 30

The above sequences have been modified during the experiments by adding a His-Tag sequence LEHHHHHH (SEQ ID NO: 31).

As can be seen from Fig.Of 7.46-N4 has excellent binding affinity to ED-B (KD=189 nm); 46-A5 and 52-D10 does not have binding activity, whereas other modified ubiquitinate small proteins have binding activity with ED-B, compared to 46-H4. Thus we can conclude that both MS room heterodimers version required for high-affinity binding to a target; both monomers exhibit monovalent binding to the target.

Heterodimer modified ubiquitin with high activity of binding to ED-B, named N, identified the following substitutions of amino acids in about(their fields binding domain of two monomers in comparison with the monomers of ubiquitin wild type:

in the first module (BDR1) (a) Q2G, F4V, K6R, Q62P, K63H, EA, S65T, T66L in the second module (BDR2) (e) CN, L8M, Q62K, K63P, E64I, S65A, THE

50G11

in the first module (N)(a) Q2G F4V, K6R, Q62P, K63H, ER, S65T, T66L in the second module (C) K6M L8R, Q62M, K63N, EA, S65R, T66L

N

in the first module (N)(a) Q2G, F4V, K6R, Q62P, K63H, ER, S65T, T66L in the second module (d) K6G, L8W, Q62T, K63Q, E64Q, S65T, T66R

V

in the first module (g) Q2R, F4P, K6Y, Q62P, K63P, E64F, S65A, T66R in the second module (N) K6H, L8M, Q62K, K63P, E64I, S65A, THE

52D10 (not associated with ED-B)

in the first module Q2V, F4C, K6R, Q62T, K63A, ER, S65G, T66D

in the second module (N) (e) K6H, L8M, Q62K, K63P, E64I, S65A, THE

A (not associated with ED-B)

in the first module (N)(a) Q2G, F4V, K6R, Q62P, K63H, ER, S65T, T66L in the second module (b) K6L, L8M, Q62L, k63 A, E64F, S65A,

Fig.8 shows the sequence alignment. Line 1: two monomer ubiquitinated wild-type protein (1st line) associated with 12-aminokislotnym the linker SGGGGSGGGGIG starting at position 77 and ending at position 88; and a second monomer with BDR2 starts at position 89 with methionine. This dimeric ubiquitinates wild-type protein is aligned with heterodimers option 46-H9 modified ubiquitin (2nd line) with different modifications in the first and second monomers, giving two of the OSD. Both GSO act together in the binding of the target due to monovalent binding with the target.

Fig.9 shows the sequence alignment heterodimeric variant 1041-D11 (1st line) modified with ubiquitin "Ub2_TsX9" (ubiquitin modified in position 45 in both monomers in tryptophan, showing the linker between GIG d�umya monomers (regulations 77 - 79; the second monomer starts with a methionine at position 80), and the substitution of glycine by alanine in the last C-terminal amino acids of the 2nd monomer. The third line shows "UBI-dt dimer, the dimer of ubiquitin wild type; not showing the alignment of the linker (thus, the second monomer starts at position 77 with methionine). 4 line shows "UBI-dt monomer", which is a human wild-type ubiquitin.

Fig.10 shows concentration-dependent ELISA of the binding of variant 1041-D11 heterodimers of ubiquitin with ED-B person. Variant 1041-D11 has a high-affinity binding to ED-B (KD=6,9 nm=6,9×10-9M). Closed circles show the binding affinity of variant 1041-D11 heterodimers of ubiquitin with ED-B containing a fragment of fibronectin (named V-tO), compared to the lack of binding of this variant with negative control (named 6789-tO) (unfilled circles).

Fig.11 shows the results of the competitive, concentration-dependent ELISA of the binding of variant 1041-D11 heterodimers of ubiquitin with immobilized ED-B containing a fragment of fibronectin (B), in the presence of increasing amounts of free target. Variant 1041-D11 heterodimers of ubiquitin shows a very high-affinity binding to ED-B (IC50=140 nm).

Fig.12 shows the result of the analysis of molecules 1041-D11 mo�aficionado heterodimers of ubiquitin in the analyses of interactions without labels using Biacore®. Analyzed different concentrations of this option heterodimers of ubiquitin (see legend in the Figure: 0-200 nm 1041-D11) for binding to ED-B containing a fragment of fibronectin (named 67 V89) immobilized on the chip SA (Biacore). The curve analysis of Association and dissociation gave a KD value of 1 nm (1×10-9M) and indicator'koff of 7.7×10-4with-1that shows a long half-life of the complex 1041-D11 and ED-B.

Fig.13 shows the binding of variant 1041-D11 heterodimers of ubiquitin with ED-B in a concentration-dependent ELISA simultaneous analysis of serum stability of the binding activity. Shows different conditions, such as pre-incubation for 1 hour at 37°C this option in the serum of mice or rats in PBST as the control. The values in CD are in the range from 10 to 20 nm. Thus, we can conclude that the binding of heterodimer 1041-D11 with ED-In the serum of affected slightly.

Fig.14 shows the analysis of the complex formation of variant 1041-D11 heterodimers of ubiquitin fragments of fibronectin due to the CO-GHWR.

Fig.14A illustrates the formation of a complex of 1041-D11 with ED-B. Three chromatograms GHWR superimposed on each other: the blue peak with a retention time of 21,651 min is pure 1041-Dl 1; black peak with a retention time of 26,289 min represents the fragment of fibronectin 67 V89; CME�ü 1041-D11 and V gives red peak with a retention time of 21,407 min after CO-GHWR. The shift of the peak of 1041-D11 to shorter retention time, as well as the disappearance of the peak V indicates the formation of a complex of 1041-D11 soluble and ED-B.

Fig.14 shows an overlay of three chromatograms CO-GHUR 1041-D11 (blue, 21,944 min), the fragment of fibronectin 6789 without ED-B (black, 26,289 min) and a mixture of 1041-D11 and 6789 (red line with peaks in 21,929 26,289 mines and min). Almost not observed shift of the peak of 1041-D11. This fact together with the lack of disappearance of the peak 6789 indicates a slight binding of the free fragment of fibronectin 6789 ED-B.

Fig.15 shows the binding of variant 1041-D11 heterodimers of ubiquitin with cells of the cell culture.

Fig.15A shows binding of variant 1041-D11 heterodimers of ubiquitin on fibroblast cells (Wi38) embryonic human lung, which were recorded. The first column in Fig.15 shows a control that uses anti-ED-B antibodies, the second column shows the incubation of this option at protein concentration of 58.7 nm, the third column shows a tenfold higher concentration of protein 1041-D11 (587 nm), the fourth column is a negative control with saline solution with phosphate buffer. In the first row fibroblast Wi38 cells of the person shown in the phase contrast; the second row shows immunofluor-indicator, and the third row shows the labeling of nuclei with DAPI. It can be concluded that option 101-D11 Wi38 associated with high specificity to ED-B, containing extracellular matrix.Was performed a control analysis using NHDF cells, which Express low levels of ED-B (data not shown). These variants do not bind to these cells.

Fig.15B shows the binding to fibroblast cells (Wi38) viable embryonic human lung. Negative control cells TnnaNHDF are the primary normal fibroblast cells, which Express low levels of EDB-fibronectin. The first and third lines show this option when different concentrations of protein and a negative control. The second and fourth lines indicate incubation control with the use of antibodies to EDB. The first 2 lines show the option and the positive control cell line Wi38. The third and fourth lines indicate incubation NHDF - cells. You can see that the variant 1041-D11 Wi38 associated with high specificity to ED-B containing extracellular matrix.

Fig.15C shows the binding fixed nipping cells of Balb 3T3. Were tested three concentrations of protein (1,10, 50 nm) of this option. The first row shows this option (SPVF-28-1041-41 1-TsX9) on cells, the second row shows the positive control (antibody to Fv28-EDB-15), the third row shows the incubation with the negative control (UB2_TsS9; unmodified ubiquitin corresponding to SEQ ID NO:1). You can see that the variant 1041-D11 binds to misname cells Balb ztd with high specificity to ED-B, containing extracellular matrix.

Fig.15D shows the binding fixed nipping cells ST-2. Were tested three different concentrations of protein (1,10, 50 nm) of this option. The first row shows the option (SPVF-28-1041-411-TsX9) on cells, the second row shows the positive control (antibody to Fv28-EDB), the third row shows the incubation with the negative control (UB2_TsS9; unmodified ubiquitin corresponding to SEQ ID NO: 1). You can see that the variant 1041-D11 heterodimers of ubiquitin binds to murine cells Balb ST-2 with high specificity to ED-B containing extracellular matrix.

Fig.16 A shows the specificity of variant 1041-D11 heterodimers of ubiquitin to the target in mammalian tissue sections. Were evaluated F9 tumor tissue from seven samples. Immunohistochemistry with different concentrations from 10 nm to 100 nm variant 1041-D11 heterodimers of ubiquitin resulted in specific vascular staining of ED-B in F9 tumors from mice. ED-B is a highly specific marker for tumor vasculature. Protein-target ED-B is located on abdominales side of the vessels. Variant 1041-D11 specifically decorates vasculature in tissue sections from tumors F9. The results obtained are comparable to the fragment L19 antibody. Additionally were checked 48 tissues; no nonspecific staining in any of the 48 tissues in relay�annoy FDA panel were observed.

Fig.16 b shows the accumulation of 1041-D11 in tumor tissue compared with wild-type ubiquitin (in this Figure, Ub2 (NCP2). Tumor tissue F9 was analyzed for the presence of 1041-D11 and ubiquitin wild type at different time points between 30 min and 16 h. the Greatest accumulation of 1041-D11 in tumor tissue was observed after 30 min and 16 h after injection, whereas accumulation of wild-type ubiquitin in tumor tissues F9 was low. This variant is enriched in tumors expressing ED-B, compared with wild-type ubiquitin. This is proof of the orientation of 1041-D11 to the tumor tissue. In addition, the ratio of the tumor-blood 1041-D11 in cancer models clearly demonstrates in vivo activity of variant 1041-D11 animals (data not shown).

Fig.17 shows high selectivity and specificity protein composite 1041-D11-TNF-Alfa for ED-B.

Fig.17A and 17B: Inducing apoptosis activity of TNF-alpha integral protein 1041-D11-TNF-alpha was tested in cell analysis (L929 cells). These Figures clearly show that the composite protein 1041-D11-TNF-alpha (Fig.17B) is the same active as the free TNF-alpha (Fig.17B) in cell culture.

Fig.17C demonstrates the high selectivity of the composite protein heterodimers of ubiquitin 1041-D11-TNF-alpha with the target ED-b Domain B fibronectin ED-B person is associated with an apparent KD value of 1.8 nm with option 041-D11 (shaded circles), showing high affinity for the target. Human without fibronectin domain ED-B (h6789) is not associated variant 1041-D11-TNF-alpha (unfilled circles).

Fig.17D+E shows the analysis of the binding of compound protein 1041-D11-TNF-alpha on the basis of the modified ubiquitin with ED-B by using the Biacore assays. The results demonstrate the high affinity compound of protein 1041-D11-TNF-alpha with a KD value=1,13 nm.

Fig.17F shows a high specificity of binding observed with the variant 1041-D11, in cell culture, which is preserved when 1041-D11 integrated with TNF-alpha. This composite protein specifically binds to expressing cells EDB. Thus, a composite protein 1041-D11-TNF-alpha binds with very high affinity and specificity to the target ED-B ("target(+)"). In serum without ED-B ("target(-)"), cross-reaction was observed.

Fig.18 shows the relative tumor growth in vivo during treatment of mice for 7 days variant 1041-D11, combined with TNF-alpha in combination with melfa-lang. The data clearly show that the variant 1041-D11-TNF-alpha in combination with cytotoxic agent melphalan reduces the relative tumor growth more effectively than mTNF-alpha in combination with melphalan or than melphalan. Kinetics of tumor growth within 7 days after treatment shows effective reduction of tumor variant 1041-D1-mTNFa. This is a clear proof of the effectiveness of treatment of tumors of the composite protein 1041-D11-TNF-alpha in combination with melphalan. ED-B is identical in several species of mammals, including mice and humans, and thus, these results predict the effect of variant 1041-D11-TNF-alpha for people.

EXAMPLES

The following Examples are provided to further illustrate the invention. The invention, in particular, demonstrated in relation to the modification of ubiquitin as an example. The invention, however, it is not limited to, the following Examples will just show the feasibility of the invention in practice on the basis of the above description. For full disclosure of the invention reference is also made to the publications mentioned in this application and in the application, are all incorporated in full into this application by reference.

Example 1. Identification heterodimeric proteins, binding to ED-B, on the basis of modified ubiquitin proteins

Construction and cloning of the library

Unless otherwise noted, use a well-known recombinant genetic methods, which are described, for example, in the publication of Sambrook and others (Sambrook et al). Nonspecific library heterodimers of human ubiquitin with high complexity was prepared by concerted mutagenesis of in total webrunner provisions of amino acids.A modified amino acid, which were replaced by NNK triplets, comprised of at least 3 amino acids selected from the provisions 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 in the proximal (first) monomer of ubiquitin and at least 3 amino acids selected from the provisions 2,4, 6, 8, 62, 63, 64, 65, 66, 68 in the distal (second) monomer of ubiquitin. Both monomers of ubiquitin were genetically related (head to tail) the linker glycine/serine at least with the sequence GIG or a linker glycine/serine at least with sequence SGGGG, for example GIG, SGGGG, SGGGGIG, SGGGGSGGGGIG (SEQ ID NO: 32) or SGGGGSGGGG, but any other possible the linker.

Selection of phage display TAT

Library heterodimers of ubiquitin was enriched on the use of the target, for example, TAT phage display as the system of choice. Can be used other ways, known in this field. The target can be immobilized on nonspecific binding surfaces of the protein or via biotinylated residues that were covalently linked to protein. The preferred immobilization via Biotin on the pellet streptavidin or strips neutravidin. Phage binding to the target, either in solution or immobilized target; for example, biotinylated and immobilized target with phage were incubated by washing phages associated with the matrix, and p�the elution of phages, associated with the matrix. In each cycle after incubation of the target pellets were separated from the solution magnetically and washed several times. In the first cycle of selection biotinylation target was immobilized on strips neutravidin, whereas in cycles 2-4 were performed elections in solution, followed by immobilization of complexes of the target-phage coated with streptavidin on the granules Dynabeads® (Invitrogen). After washing in the first two cycles of selection of phages binding to the target of the modified ubiquitin was released by elution with an acidic solution. In cycles 3 and 4 elution of phages was performed by competitive elution with excess target. - Eluted phages were reamplification. For directions to the specificity of the binder during the selection, you can include protein, similar to the target.

Alternative choice TAT phage display: vibrissaphora display

Library of ubiquitin was enriched on the use of the target, for example, ribosomal display as the system of choice (sand, etc. (Zahnd et al.), 2007), Ohashi and others (Ohashi et al.), 2007). Can be used other ways, known in this field. The target is biotinylated according to standard methods and immobilized on coated with streptavidin pellets Dynabeads® (Invitrogen). Ternary complexes including ribs�we mRNA and ARIS ing ubiquitinates polypeptide, were assembled using the kit PURExpressTMIn Vitro protein synthesis (NEB). We conducted two primary cycle of selection, which were incubated ternary complexes, after which howled executed two similar cycle of selection. In each cycle after incubation of the target pellets were separated from the solution by magnetic separation and washed with buffer ribosomal display with increasing thoroughness. After washing in the first two cycles of selection granules are again separated from the solution magnetically, and mRNA molecules of the modified ubiquitin that communicates with the target, was released from ribosomes by adding 50 mm EDTA. In cycles 3 and 4 select elution of mRNA was carried out by competitive elution with excess target (Lipovsek and Planton (Lipovsek and Pluckthun), 2004). After each cycle performed the purification of RNA and synthesis of cDNA using the kit RNeasy MinElute Cleanup Kit (Qiagen company, Germany), set "Turbo DNA-free Kit (Applied Biosystems, USA) and reverse transcriptase of transcriptor (Roche company, Germany).

Cloning of enriched pools

After the fourth cycle of selection of the synthesized cDNA was amplified by PCR using primers F1

(GGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG ATATACATATG) (SEQ ID NO: 9) and

WUBI(co)RD_xho (AAAAAAAAACTCGAGACCGCCACGCAGACGCAGAACCAG) (SEQ ID NO: 10), cut with restriction nucleases Ndel and hol (Promega, USA) and Legerova in expressing vector pet-20b(+) (Merck, Germany) using a compatible sticky ends.

The analysis of coincidences cloned colonies

After transformation the cells NovaBlue(DE3) (Merck, Germany) resistant to ampicillin cloned colonies were grown for 6 h at 37°C in 200 µl medium AG SOB (SOB medium containing 100 μg/ml ampicillin and 20 g/l glucose), expression of the modified ubiquitin that binds to ED-B was achieved by culturing for 16 h at 37°C in 96-well deep plates (the company Genetix, UK) using 500 ál autoinduction medium ZYM-5052 (Studier (Studier), 2005). The cells were collected by centrifugation for 15 min at 4°C and 3600 g and then literally by incubation for 30 min at 37°C with 300 µl of lysis buffer per well containing 0.2× BugBuster® (Merck, Germany), 0.3 mg/ml of lysozyme (VWR, Germany), 0.2 mm of phenylmethylsulfonyl (company Roth, Germany), 3 mm MgCl2and 0.2 units/ml benzonase (VWR, Germany) in 50 mm NaH2PO4, 300 mm NaCl, pH 8. After centrifugation for 30 min at 4°C and 3600 g of the obtained supernatants were subjected to screening by ELISA, using tablets Nunc MediSorp (Thermo Fisher Scientific, USA) coated with 4 µg/ml ED-B and ubiquitin-specific Fab fragment, conjugated with horseradish peroxidase. As detecti�following reagent used TMB-Plus (Biotrend, Germany), and the yellow color was obtained, using 50 µl/well of 0.2 M H2SO4, were measured in the spectrophotometer to read the tablets at 450 nm against 620 nm.

Usually performed several, e.g., four cycles of selection display against ED-B. In the last two cycles of selection of binding molecules has suirable with plenty of free ED-B. Were identified, among others, the following binding ED-B options.

The sequence N

MGIVVRTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSD YNIPHPTLLHLVLRLRGGSGGGGSGGGGIGMQIFVHTMTGKTITLEVEPSDTIENVKA KIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIKPIAELHLVLRLRGG (SEQ ID NO: 6)

The sequence E

MRIPVYTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSD YNIPPFARLHLVLRLRGGSGGGGSGGGGIGMQIFVMTRTGKTITLEVEPSDTIENVKA KIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIMNARLLHLVLRLRGG (SEQ ID NO: 7)

The sequence 22D1

MLILVRTLTDKTITLEVEPSDTIGNVKLAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSD YNISVGAMLHLVLRLRGGSGGGGSGGGGIGMQIFVLTWTGKTITLEVEPSDTIENVKA KIQDKEGIPPDQQRLIWAGKQLEDGRTLSD YNIRRLPPLHLVLRLRGG (SEQ ID NO: 8)

Sequence alignment of the monomer of wild-type ubiquitin (UBI-dt monomer) (dimer of wild-type ubiquitin (UBI-dt dimer) and ubiquitinated protein (Ub2-TsX Fig.9 with the substitution at position 45 of each monomer and with two substitutions in the C-end) with heterodimer variant 1041-D11 modified ubiquitin is shown in Fig.9. In Ub2-TsX substitution in the C-graduation (GG to AA) monomer enhance the stability in serum, as deubiquitinase disintegrate in GG of ubiquitin, but not for AA. The secondary structure of wild-type ubiquitin with�avanyu with ubiquitin with these substitutions in the C-end is almost identical.

Modified ubiquitin with an activity of binding to ED-B, as specified 1041-D11 (shown in Fig.9; SEQ ID NO: 36) or 1045-D10 are identified by the following substitutions of amino acids compared to the wild type: in the first module: K6W, L8W, K63R, E64K, S65F, TR; in the second module: K6T, L8Q, Q62W, K63S, E64N, S65W, TE; Q2R (in the variant 1041-D11, but not in variant 1045-D10).Suitable preferred linkers for this protein composite are those with SEQ ID NO: 32 or a sequence GIG. However, there are many known linkers that can be used instead.

As another preferred example of a protein represented by the following sequence, where XXXX can be any amino acid (SEQ ID NO: 47). As the linker used here SGGGGSGGGGIG (in italics). It is understood that feasible alternatives is another type of linker or no linker.

MTIWVHTLTGKTITLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNINFKLSLHLVLRLRGGSGGGGSGGGGIG

MQIFVXTXTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNINXXXXXLHLVLRLRGG

Consensus sequences of examples of proteins with these sequences is shown in Fig.2.

Example 2. Getting protein component of modified variants of ubiquitin binding ED-B, and TNF-alpha (hTNFa)

These options expressed as an integral of modified proteins of ubiquitin, for example, heterodimerisation 1041-D11, and mouse or human TNFa in E. coli. The composite analysis of protein include: the expression and purity of the protein, lack of capacity aggregation, the activity of TNFa in cell culture, affinity for the target protein ED-B, selectivity, specific binding in cell culture. A prerequisite for the experiment on animals by induction shrink tumors in mice with tumor F9 is Association with murine TNFa.

Stage 1: obtaining a vector to clone a protein component (pETSUMO-TNFa)

pETSUMOadapt is a modified vector pETSUMO (Invitrogen company), which is modified by inserting additional multiple cloning site (MCS). Since TNF-alpha, cloned in pETSUMOadapt have been introduced restriction sites for insertion of modified variants of ubiquitin binding to ED-B. the resulting construct has the structure of His6-SUMO-TNFa with the following DNA sequence (SEQ ID NO: 11):

ATGGGCAGCAGCCATCATCATCATCATCACGGCAGCGGCCTGGTGCCGCGCGGC

AGCGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTGAAG

CCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAG

AGATCTTCTTCAAGATCAAAAAGACCACTCCTttaagaaggctgatggaagcgti

CGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTAT

TAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATAT

TATTGAGGCTCACAGAGAACAGATTGGTGGTGTGCGTAGCAGCAGCCGTACCCC

GAGCGATAAACCGGTGGCGC ATGTGGTGGCGAATCCGC AGGCGGAAGGCC

AGCTGCAGTGGCTGAACCGTCGTGCGAATGCGCTGCTGGCCAACGGCGTGGAAC

TGCGTGATAATCAGCTGGTTGTGCCGAGCGAAGGCCTGTATCTGATTTATAGCCA

GGTGCTGTTTAAAGGCCAGGGCTGCCCGAGCACCCATGTGCTGCTGACCCATACC

ATTAGCCGTATTGCGGTGAGCTATCAGACCAAAGTGAACCTGCTGTCTGCGATTA

AAAGCCCGTGCCAGCGTGAAACCCCGGAGGCGCGGAAGCGAAACCGTGGTAT

GAACCGATTTATCTGGGCGGCGTGTTTCAGCTGGAAAAAGGCGATCGTCTGAGC

GCGGAAATTAACCGTCCGGATTATCTGGATTTTGCGGAAAGCGGCCAGGTGTATT

TTGGCATTATTGCGCTGTAATAA

This sequence of TNF-alpha was amplified through PCR by introducing a BamHI site and the XhoI site. The used primers are:

SUMO-EDB-TNFa-rw (SEQ ID NO: 12): TTT TTT GGA TCCGTG CGTAGCAGC AGC

SUMO-EDB-TNFa-rev (SEQ ID NO: 13): CTT GTC TCT CGA GGC GGC CGCTTA TTAC

Primer rw (direct) (SEQ ID NO: 12) recognizes the first 15 base pairs TNFa (underlined region) and has a BamHI sequence (bold). Primer rev (reverse) (SEQ ID NO: 13) contains the last couple of grounds TNFa, termination codons (underlined) and restriction site Xhol (in bold).

The reaction mixture for PCR THEN Tcl):

84,5 µl H2About; 10 μl of 10× Pwo buffer+Mg; 2 μl 10 mm deoxynucleotides (=200 μm), 0.5 μl of 100 μm primer forward/reverse (=every 0.5 μm); 2 μl of DNA (=0,25 mcg); 0.5 ál polymerase Pwo (=2,5 Ed.; Roche company)

PCR program:

3 min 94°C, 30 s 94°C, 30 s 60°C, 2 min 72°C (stages 2-4: 30 cycles), 5 min 72°C and 4°C, after purification of the PCR product using the Qiagen kit-MinElute (elution in 10 μl EB). The PCR product was introduced into MCS of the vector pETSUMOadapt by BamHI-XhoI-restriction and ligation.

A mixture of Bromphenol blue (100 ál):

Vector: 83 µl H2About; 10 μl 10× NE buffer 3; 1 µl UK BSA; 3 ál BamHI (=30 Ed.; NEB), 1,5 µl Xhol (=30 Ed.; NEB); of 1.65 μl of the vector; incubation for 3 h at 37°C.

The PCR product: 76,5 �TC H 2About; 10 μl 10× NE buffer 3; 1 μl of 100× BSA; 3 ál BamHI (=30 Ed.; NEB), 1,5 µl Xhol (=30 Ed.; NEB); 8 µl inserts; incubation for 3 h at 37°C.

Office electrophoresis in 1% agarose gel (100 V cycle 60 min); cutting the vector fragment (5659 base pairs) and insert (491 base pair); purification using Qiagen kit for the extraction of the gel (elution in 30 ál EB).

Ligation (20 µl"):

15,2 µl H2O; 2 μl 10× T4 DNA ligase buffer; 2,26 ál vector (200 ng); 0,54 µl insert (40 ng) were incubated 5 min at 65°C; cooling to 16°C; adding 1 μl T4-DNA ligase (=3 Ed.; NEB); incubation of 6 h at 16°C.

NaAc/isopropanol-precipitation: the mixture for ligation (20 µl)+2,2 ál 3M NaAc (pH 5.0)+22,2 ál isopropanol; 30 min at -20°C; 15 min at 4°C, 13000 rpm;

re-suspending the pellets in 500 ál 70% EtOH; rotation; re-suspended platforms-darovanie the pellets in 10 ál of N2O.

Transformation:

Mixing electro-competent cells Novablue(DE3) (40 μl-aliquot) with 10 µl of the ligation product; transfer to a cuvette OD cm for electroplate; vibration in the electric troposhere (1,8 kV, 50 μf, 100 Ohms); the solution for incubation with 1 ml SOC medium for 45 min at 37°C 220 rpm; 100 µl on LB-tablet with kanamycin; incubation overnight at 37°C.

Step 2: Cloning EDB-integral proteins based on modified ubiquitin

To obtain m connecting EDB options based on modified ubiquitin TNFa, interest-based sequence of the modified ubiquitin binding EDB, were amplified from pET20b vector by PCR; introduced restriction sites Bsal and BamHI. This method is suitable for Monomeric and dimeric variants that bind EDB, on the basis of the modified ubiquitin. Primer for Monomeric DT-Ubiquitin (Wubi):

Direct the linker SUMO-EDB-WUBI (SEQ ID NO: 14):: GTT CCA AGG CAT TCT GGTATG CAG ATC TTC GTG

Return linker SUMO-EDB (SEQ ID NO: 15):: GTG GTG GGA TCC GCC ACC ACC ACC AGAACC ACG GCC CAG ACG

Direct primer (SEQ ID NO: 14) recognizes the first 15 base pairs of the modified ubiquitin (underlined region) and has Bsal sequence (bold). The reverse primer (SEQ ID NO: 15) recognizes the last 15 base pairs of the modified ubiquitin and inserts an amino acid linker (sequence SGGGG) and BamHI-restricciones site (bold). For each option based on modified ubiquitin use specific exercise direct primer. The primers for Monomeric binding EDB options based on modified ubiquitin N, E and 4V10:

N (MWIKV...): primer (IMO-EBV-N-line) (SEQ ID NO: 16): GTT CCA AGG CAT TCT GGTATG TGG ATC GTG AAG

4B10 (MLILV): primer (SUMO-EDB-4B10-npflMoft) (SEQ ID NO: 17): GTT CCA AGG CAT TCT GGTATG TTG ATC CTG GTG

5E1 (MVINV...): primer (IMO-EOB-E-line) (SEQ ID NO: 18): GTT CCA AGG CAT TCT GGTATG GTT ATC AAT GTG

Reverse�first primer used for all Monomeric variants on the basis of the modified ubiquitin. The reverse primer for dimeric variants on the basis of the modified ubiquitin:

Dimer-t0a-reverse (SEQ ID NO: 19): GTG GTG GGA TCC GCC ACC ACC ACC AGAACC ACC ACG TAA ACG

Direct primer for cloning dimeric DT-ubiquitin (WubiHubi) and dimeric binding EDB options based on modified ubiquitin:

WT (MQIFV...) primer (SUMO-EDB-WUBI-line) (SEQ ID NO: 20): GTT CCA AGG CAT TCT GGTATG CAG ATC TTC GTG

(Note: direct primer for dimeric DT-of ubiquitin is identical to the direct primer for Monomeric DT-of ubiquitin.)

E (MRIPV...): primer (ESA-line) (SEQ ID NO: 21): GTT CCA AGG CAT TCT GGTATG CGTATC CCTGTG

24H12 (MVIKV...): primer (N-Sa-line) (SEQ ID NO: 22):GTT CCA AGG CAT TCT GGTATG GTT ATC AAG GTG

15G7 (MEIGV...): primer (15G7-t0a-np*Moft) (SEQ ID NO: 23): GTT CCA AGG CAT TCT GGTATG GAG ATC GGT GTG

22D1 (MLILV...): primer (2201-Sa-line) (SEQ ID NO: 24): GTT CCA AGG CAT TCT GGTATG CTT ATC TTG GTG

The PCR mixture (100 ál):

84,5 µl H2About; 10 ál 1 Oh Pwo-buffer+Mg; 2 μl 10 mm deoxynucleotides (=200 μm), 0.5 μl of 100 μm primer forward/reverse (=je 0.5 μm); 2 μl of DNA (depending on version); 0.5 μl Pwo polymerase (=2,5 Ed.; Roche company)

The PCR program;

1. Tmin°C

2. 30 C°C

3. 30 C60°C

4. 2 min 72°C (stages 2-4: 30 cycles)

5. 5 min 72°C and 4°C

Purification of PCR products in agarose gel, cutting off the required strip and purification using the Qiagen kit for gel extraction. Cloning of PCR product by BsaI-BamHI rest�iccie (pETSUMO-TNFa)

Restriction analysis (100 ál): 75 µl of N2About; 10 ál of 10x NEB buffer 3; 1 µl UK bovine serum albumin (BSA); 3 µl Bsal (=30 Ed.; NEB); 8 µl DNA (vector or PCR product) incubation 2 h 50°C, 10 min 65°C, add 3 ál BamHI (=30 Ed.; NEB), 2 h, 37°C, the separation electrophoresis in 1% agarose gel; the cutting of the tail of the vector and the insert; purification using Qiagen kit for the extraction of the gel (elution in 30 ál EB).

Subsidies (20 µl):

12,5 µl of N2About; 2 ál 10x T4 DNA legasy buffer; 5 ál vector (66 ng); 0.5 ál insert (variable), incubation of 5 min 65°C; cooling to 16°C; adding 1 μl T4-DNA ligase (=3 Ed.; NEB); incubation of 16 h 16°C

Deposition NaAc/isopropanol (see Step 1)

Transformation in electrocompetent cells Novablue(DE3) as described above. The result is the following construct merge: EDB - modified ubiquitin and TNFa in pETSUMOadapt with der His6-SUMO - modified ubiquitin-SGGGG-TNFa (359 amino acids with Monomeric modified ubiquitin, 447 amino acids with dimeric modified ubiquitin)

Example 3: Expression and purification of the compound of proteins through the ubiquitin-T No.-alpha

Analysis of DNA sequences showed the correctness of the composite sequences of proteins SUMO-TNFa. For expression of the variants clones were cultured in a flask shaker by dilution preculture 1:100 in LB/kanamycin and mixing the culture with a frequency of rotation� 200 rpm and 37°C to an optical density at 600 nm (OD600) of 0.5. Expression was induced by adding IPTG (final concentration 1 mm). The cultivation was continued for 4 hours at 30°C and 200 rpm. bacteria Cells were collected by centrifugation at 4°C, 6000× g for 20 min, the Cell pellet was suspended in 30 ml of buffer NPI-20, including benzonase and lysozyme. Cells were destructively ultrasound (3×20 seconds) on ice. The supernatant containing soluble proteins was obtained after centrifugation of the suspension: at 4°C and 40000× g for 30 min. Both proteins were purified affinity chromatography at room temperature. One column of Ni-agarose (5 ml, GE Healthcare) was equilibrated with 50 ml of NPI-20. The supernatant containing the soluble proteins, was added to the column, followed by a washing step NPI-20. Bound protein was suirable with a linear gradient to 50% NPI-500 100 ml. Fractions were analyzed by denaturing polyacrylamide gel electrophoresis with respect to their purity. The appropriate fractions were combined and applied to a gel filtration column (Superdex 75, a 1.6×60 cm, GE Healthcare), balanced SUMO-hydrolase buffer for cleavage (50 mm Tris, 300 mm NaCl, pH 8.0) with a flow rate of 1 ml/min.

The cleavage reaction was performed according to the manufacturer's instructions (Invitrogen). After cleavage of the protein were applied to Ni-agarose column (5 ml, GE Healthcare). His-tagged SUMO hydrolase and SUMO concerned�again with column, and the proper protein composite column passed (without His-tag). The purity of proteins was confirmed by analysis of liquid chromatography high-resolution gel electrophoresis. The correctness of the molecular weight of the trimer (via TNFa) was confirmed using analytical SEC analysis (10/30 Superdex G75, GE Healthcare).

Example 4: Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin with ED-B man

Example 4A. Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin by ELISA, concentration-independent.

Linking options on the basis of ubiquitin with ED-B of the person analyzed by ELISA, which depends on concentration. Increasing amounts of purified protein were applied to the plates NUNC-medisorp, coated with ED-B, BSA and cellular fibronectin (cFN). Antigenic covered with 50 μl (10 μg/ml) per well was performed at 4°With during the night. After washing tablets of phosphate-saline buffer PBS), and 0.1% Tween-20 pH 7.4 (FSBT) the wells were blocked using a blocking solution (PBS pH 7.4; 3% BSA; 0.5% of Tween-20) at 37°C for 2 h. the Wells are again three times washed FSBT. Different concentrations of protein binding to ED-B based on modified ubiquitin is then incubated in the wells at room temperature for 1 h (volume 50 μl) (Fig.10 as the starting concentration used 500 nm protein 1041D11). After washing the wells FSBT struck conjugate by peroxy duty the fuck fragment anti-Ubi fab (AbyD) in an appropriate dilution (e.g. 1:2000 or 1:6500) in FSBT. The plate was washed three times with 300 ál of buffer FSBT on the hole. To each well was added 50 µl of TMB substrate solution (KEM-EN-TEC) and incubated for 15 min the Reaction was stopped by adding 50 μl of 0.2 M H2SO4at the hole. Tablets ELISA was read using a reader ELISA TECAN Sunrise. Photometric measurements of the spectral absorption SPO strategy performed at 450 nm using 620 nm as reference wavelength. Fig.1 clearly shows the specific binding N with ED-B with the true KD value of 11 nm. Option E showed the true KD-value of 7.7 μm and 4V10 280 nm, respectively. Fig.10 shows a very high-affinity binding options 104] D11 with ED-B (KD=6,9 nm). Thus, only a few modifications (up to 8 sameshe tions in each monomer) in wild-type ubiquitin lead to very high-affinity binding to ED-B.

Example 4 Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin by competitive ELISA, concentration-independent.

Was made competitive, concentration-dependent ELISA for the analysis of binding options 1041-30 D11 of ubiquitin with immobilized ED-B containing a fragment of fibronectin (B), in the presence �veiciviomia amount of free target. The ELISA conditions were as described for Example 5A, with the exception that the protein 1041-D11 was pre-incubated with ED-B (W) (0 µm -10 µm) or with negative control 6789 (0 μm-10 μm) for 1 h and then the mixture with the target W placed on the tablet Medisorp; then this option bull detected the corresponding antibody (anti-Ubiquitin-Fab-horseradish peroxidase; dilution of 1:6500). Fig.11 shows that the variant 1041-D11 has a very high-affinity binding to ED-B (IC50=140 nm). The result, shown in Fig.10 confirmed; only a few modifications (up to 8 substitutions in each monomer) in wild-type ubiquitin lead to very high-affinity binding to ED-B.

Example 4C. Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin by a competitive ELISA, which depends on concentration, with simultaneous analysis of the stability of the binding activity in serum.

ELISA was performed using methods well known in the field as described above (Example 5A and 5B). ED-B (here named V) inflicted on the micro-titration plates, this variant was associated with ED-B and were detected by the antibody specific to ubiquitin (anti-Ubi-Fab-horseradish peroxidase). Option in this analysis were treated in different ways: variant were incubated in the serum of mice for 1 h at 37°C (see Fig.13, with�of anyway); variant were incubated in serum of rats within for 1 h at 37°C (Fig.13 red circles), or were incubated in PBS for 1 h at 37°C (Fig.13 black circles). Fig.13 shows that all the KD of the variant 1041-D11 ranged from 10.3 nm (in PBS) to 20,74 nm (in mice serum).

Example 4D. Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin by Biacore analysis.

Were analyzed variants of different concentrations (e.g., 0-200 nm variant, preferably 1041-D11) for binding to ED-B containing a fragment of fibronectin (named V) immobilized on the cm 5 chip (Biacore) using methods known to experts in this field. The obtained data were processed by the software BIAevaluation and 1:1 - Langmuir-fit. The molecular weight of the variant 1041-D11 was 1.0 nm, as shown in Fig.12. Kinetic binding constants amounted to kon=7,6×105; koff=7,7×10-4with-1. KDcomposite protein 1041-D11 - TNF-alpha amounted to 1.13 nm, as shown in Fig.17D. Kinetic binding constants amounted to kon=4,5×105M-1V-1; koff=5,0×10-4with-1.

Example 4E. Analysis of the formation of the complex variants that bind ED-B, on the basis of the modified ubiquitin by CO-GHWR.

For analysis of the complex formation was used Colo�Ki Tricorn Superdex 755/150 GL (GE-Healthcare) (volume=3 ml), the amount of protein was 50 µl. Other conditions: buffer: 1× PBS, pH 7.3, flow rate: 0.3 ml/min, cycle: 45 min (injection of the sample after 15 min). Condition: 0,72 nmole protein 1041-D11+0,72 nmole ED-B (here called W or negative control 6789) were incubated for 1 h at room temperature; then put on the column for analysis of complex formation. Fig.14 only variant is shown in black, only the target ED-B is shown in blue, binding variants, forming a complex with ED-B, shown in pink. Fig.14A shows the ED-B with this option; Fig.14 shows an embodiment without ED-B. This Figure shows that the variant 1041-D11 forms a complex with ED-B (V), but does not form complex with 6789.

Example 5: Biological analysis of TNF-alpha

The physiological activity of TNF-alpha merger, binding ED-B, on the basis of the TNF-alpha-modified ubiquitin was determined using the L929 analysis Apopis the Tosa (flick and others (Flick et al, 1984 J. Immunol. Methods. 68:167-175). In this analysis of TNF-alpha effectively stimulates cell death in cells sensitized with actinomycin D at the / EC50 values in the low picomolar range.

Cells re-suspended in medium containing the FSB and antibiotics. The cell suspension in a volume of 100 μl with a density of 3.5 x 105cells/ml seeded in the wells of 96-hole standard pad for the cultivation of cells, and then incubated for �eyes in a humidified incubator with CO 2. Thereafter, culture medium was removed and added to 50 μl of the medium containing the FSB, actinomycin D and antibiotics in each well, then incubated for another 30 min. then added 50 µl of objects, mergers, binding ED-B, on the basis of the TNF-alpha-modified ubiquitin or, as a control, recombinant TNF-alpha human in a suitable concentration range from 10-7up to 10-18M. After 48 h of incubation determined the metabolic activity as a measure of survival of cells using the reagent WST-1 (Roche company).

For each test object were performed at least three independent experiments, each of them three times. Each test compound proteins binding ED-B, on the basis of the TNF-alpha-modified ubiquitin was held in parallel with verification of the dose range of recombinant TNF-alpha man to obtain information about the variability between tests.

Quantitative assessment based on the value of EC50, i.e., the concentration value of the target, contributing to the survival of the half cell.

Table 2
The fusion of TNF-alpha mubElectrical conductivity (EC50
The merger mub® TNF-alphaThe corresponding TNF-alpha
Wubi-TNF-alpha5,18±2,84 PM7,97±12,18 PM
Wubi-Hubi-TNF-alpha32,58±11,26 PMOf 5.02±3,70 PM
SPWF-28_22-D1_NF-alpha26,15±14,41 PM2,32±2,07 PM
SPWF-28_24-H12_TNF-alpha0,78±0,24 PM3,01±4,18 PM
where: mub=binding ED-B based on modified ubiquitin.

Of the merger, binding ED-B, on the basis of the TNF-alpha-modified ubiquitin were analyzed one monomer of ubiquitin (Wubi) and construct three di-a measure of ubiquitin. Depending on the variant on the basis of the modified ubiquitin binding ED-B and connected with a part of the TNF-alpha associated with TNF-alpha activity was increased (SPWF-28_24-H12_STNF-alpha) or decreased (SPWF-28_22-D1_TNF-alpha, Wubi-Hubi-TNF-alpha) is approximately one order of magnitude. See The Fig.17 analysis of variant 1041-D11 TNF-alpha.

Example 6. Analysis of binding variants of ubiquitin in the cell culture samples

Checked the binding of variant 1041-D11 to the cells of the cell culture. Cells were analyzed once�es cell cultures, including normal fibroblast cells of the embryonic lung of a person with high levels of expression of ED-B (Wi38 cells), cell line embryonic fibroblast mice (Balb 3T3); line stromal cells derived from monocytes/macrophages (RAW264.7) bone marrow of mice (ST-2), NHDF cells and murine fibroblast cells (LM).

Variant 1041-D11 (different concentrations) or antibody specific for ED-B (500 nm FV28 CH4/F1 1× PBS were incubated (1 h, 37°C) Wi38 cells (60,000 cells/ml; from ATSS), and then performed a fixation with methanol (5 min, -20°C), blocked (5% horseradish/PBS, 1 h); incubation with a-Strep-Tag-rabbit IgG (obtained from GenScript A,1:500) for 1 h and incubation with a-rabbit-IgG*Aeha-AK rabbit (obtained from Invitrogen company Al 1008,1:1000) for 1 h. Nuclei were DAPI stained. The first column in Fig.15A shows a control that uses antibodies kEDB, the second column shows the incubation of the variant at protein concentration of 58.7 nm, the third column shows a tenfold higher concentration of protein 1041-D11 (587 nm), the fourth column shows the negative control with the FSB. In the first row fibroblast Wi38 cells of a person shown in phase contrast, the second row shows immunofluorescence and the third row shows DAPI staining. From these images it can be concluded that the variant 1041-D11 associated with fixed Wi38 cells with high spec�vechnostyu to ED-B, containing extracellular matrix.A negative control cell type are the primary NHDF normal fibroblastlike cells, which Express low levels of EDB-fibronectin (data not shown). These variants do not bind to these cells.

Fig.15 shown In the analysis of variant 1041-D11 viable Wi38 cells. A negative control cell type are the primary NHDF normal fibroblast cells, which Express low levels of EDB-fibronectin. Cells were placed in a slide chamber (NUNC, 60000 cells/ml). For analysis of potential binding cells were fixed with 100% MeOH for 5 min at -20°C. To block nonspecific binding, the cells were incubated with 5% horse serum for 1 h at 37°C. the Cells were tested with the variant 1041-D11, antibody FV28 CH4/F1 specific for ED-B as a positive control, or UB2 as a negative control at different concentrations for 1 h at room temperature. Inspection carried out by incubation with a-Strep-Tag-rabbit IgG (obtained from GenScript A,1:500) for 1 h and incubation with a-rabbit-IgG*Alexa488-AK rabbit (obtained from Invitrogen Al 1008, 1:1000) for 1 h. Nuclei were stained with DAPI. The first and third lines in Fig.15 shows In this variant at different concentrations of the protein and the negative control. The second and fourth line of the screenings�up incubation control with the use of antibodies to EDB. The first two lines show the option and the positive control cell line Wi3 8. The third and fourth lines show the incubation of cells NHDF. From the images one can see that the variant 1041-D11 binds to viable Wi38 cells with high specificity to ED-B containing extracellular matrix.Is using NHDF cells that do not contain low level EDB (data not shown). Options do not bind to these cells.

Similar experiments were performed using different cell types, for example, Balb3T3 (ATS, cat. no.30-2002), Raw (Lonza, cat. No. VE-115F/U1), ST-2 (Lonza, cat. no.BE 12-115F/U1). Fig.15C and D shows that the binding ED-B is highly specific to mouse Balb3T3 cells and ST-2. No binding was observed with monocytes/macrophages (raw) (data not shown).

As mentioned above, Fig.16A shows the specificity of 1041-D11 in tissue sections. Were evaluated tissue tumors F9 of the seven samples. Immunohistochemistry with 500 nm 1041-D11 gave specific staining of blood vessels ED-B F9 tumors from mice. ED-B is a highly specific marker for vascular network of the tumor. Protein-target EDB is located on abdominales side of the vessels. 1041-D11 specifically decorates vasculature in tissue sections from tumors F9. The results obtained are comparable with the tissue specificity of the fragment L19 antibody. Also would�and checked 48 tissues; was not observed nonspecific staining in any of the 48 tissues in the relevant panel of the FDA. Fig.16B shows the accumulation of 1041-D11 in tumor cells compared with wild-type ubiquitin. Thus, compound proteins based on modified ubiquitin specifically binding to ED-B, suitable for targeted cancer therapy based on ED-B.

Example 7: in vivo Study of the effectiveness of option 1041D11-TNF-alpha

In order to establish therapeutic efficacy of variant 1041-D11-TNF-alpha, this compound was tested for the F9 teratoma (see, Borea, etc. (Borsi et al.), 2003 Blood 102, 4384-4392) in mipping models. The expression of ED-B in mice is comparable to the human situation in vivo and is suitable for assessing therapeutic effects of 1041-D11-mTNF-alpha cancer, preferably in combination with a cytotoxic compound such as melphalan. The F9 teratoma is an aggressive tumor with a high density of blood vessels. Borea, etc. described that targeting murine TNF-alpha through EDB antibodies to enhance the effectiveness of melphalan, as demonstrated by the slowing of tumor growth. Schedule of experiments for investigating the efficiency was taken from Borea, 2003.

In phase 1 was determined pharmacologically active and tolerated dose with endpoints related to the relation of the tumour to the body weight, loss of body weight and survival. The inventors have found that 1041D11-TNF-alpha transfer at the highest dose (of 6.75 pmol/g), but not suppressing the effect on tumor growth (>10% of body weight after 3,4 and 8 days → animals were then euthanized), then kak104D11-TNF-alpha in the lowest dose (0.25 pmol/g), as it seems, slows tumor growth. Used next group of dosages reduced from 2.25 pmol/g 1041D11-TNF-alpha.

In stage 2 of the study determined the dose-dependent efficacy with melphalan at the end point of the deceleration of tumor growth (loss of body weight of the animals >10% of the tumor >10% body mass, ulceration of the tumor). In this study, were checked 1041D11/mTNFa and murine TNFa in combination with melphalan. Used 168 animals, 14 groups of dosages (8 mice in the group took when F9 tumors reached 300-400 mm3); for intravenous check the samples was followed by intraperitoneal injection of melphalan after 24 hours. Chart dosage shown in table 1.

Table 1
GroupThe object to validateDoseRoute of administrationVolume introductionCol. animals*
Melphalan** (mg/kg)
1FSB00BB10 ml/kg8
2Murine protein composite TNF-α02,25BB10 ml/kg8
3Murine protein composite TNF-α00,75BB10 ml/kg8
4Murine protein composite TNF-α00,25BB10 ml/kg8
5Murine protein composite TNF-α00,025BB10 ml/kg8
6 Murine protein composite TNF-α00,0025BB10 ml/kg8
7Melphalan4,50SP10 ml/kg8
8Melphalan/ mouse composite protein TNF-α*4,52,25SP/BB10/10 ml/kg8
9Melphalan/ mouse composite protein TNF-α*4,50,75SP/BB10/10 ml/kg
10Melphalan/ mouse composite protein TNF-α*4,50,25SP/BB10/10 ml/kg8.
11Melphalan/ mouse composite protein TNF-α*4,5 0,025SP/BB10/10 ml/kg8
12Melphalan/ mouse composite protein TNF-α*4,50,0025SP/BB10/10 ml/kg8'
13Murine TNF-α00,25BB10 ml/kg8
14Melphalan/ murine TNF-α*4,50,25SP/BB10/10 ml/kg8

Fig.18 shows the relative tumor growth during the treatment (7 days). Fig.18a shows clearly that our connection 1041-D11-TNF-alpha in combination with meliala-nom reduces the relative tumor growth more effectively than mTNF-alpha in combination with melphalan or than melphalan. Kinetics of tumor growth after 7 days after treatment shows a significant reduction of tumors when exposed 1041-D11-mTNFa. This is a clear proof of effectiveness in combination with melphalan.

1. Birchler, ., F. Viti, L. Zardi, B. Spiess, and D. Neri. 1999. Selective targeting and photocoagulation of ocular angiogenesis also been other ideas where by a phage-derived human antibody fragment. Nat Biotechnol 17:984-8.

2. Brenmoehl, J., M. Lang, M. Hausmann, S. N. Leeb, W. Falk, J. Scholmerich, M. Goke, and G. Rogler. 2007. Evidence for a differential expression of fibronectin splice forms ED-A and ED-B in Crohn's disease (CD) mucosa. Int J Colorectal Dis 22:611-23.

3. Dubin, D., J. H. Peters, L. F. Brown, B. Logan, K. C. Kent, B. Berse, S. Berven, B. Cercek, B. G. Sharifi, R. E. Pratt, et al. 1995. Balloon catheterization induced arterial expression of embryonic fibronectins. Arterioscler Thromb Vase Biol 15:1958-67.

4. Goodsell, D. S. 2001. FUNDAMENTALS OF CANCER MEDICINE: The Molecular Perspective: Antibodies. The Oncologist 6:547-548.

5. Kaczmarek, J., P. Castellani, G. Nicolo, B. Spina, G. Allemanni, and L. Zardi.

1994. Distribution of oncofetal fibronectin isoforms in normal, hyperplastic and neoplastic human breast tissues. Int J Cancer 59:11 -6.

6. Menrad, A., and H. D. Menssen. 2005. ED-B fibronectin as a target for antibody-based cancer treatments. Expert Opin Ther Targets 9:491-500.

7. Pujuguet, P., A. Hammann, M. Moutet, J. L. Samuel, F. Martin, and M. Martin.

1996. Expression of fibronectin ED-A+and ED-B+isoforms by human and experimental colorectal cancer. Contribution of cancer cells and tumor-associated myofibroblasts. Am J Pathol 148:579-92.

8. Trachsel, E., M. Kaspar, F. Bootz, M. Detmar, and D. Neri. 2007. A human mAb specific to oncofetal fibronectin selectively targets chronic skin inflammation in vivo. J Invest Dermatol 127:881 -6.

9. Van Vliet, A., H. J. Baelde, L. J. Vleming, E. de Heer, and J. A. Bruijn. 2001.

Distribution of fibronectin isoforms in human renal disease. J Pathol 193:256-62.

10. Lipovsek, D., and Pluckthun, A. (2004). In-vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290,51-67.

11. Ohashi, H., Shimizu, Y Ying, B. W., and Ueda, T. (2007). Efficient protein selection based on ribosome display system with purified components. Biochem Biophys. Res. Commun. 352,270-276.

12. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41,207-234.

13. Zahnd C, Amstutz, P. and Pluckthun, A. (2007. Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Methods 4, 269-279.

1. Protein capable of binding extradosed B (ED-B) of fibronectin comprising the modified heterodimeric ubiquitinated protein, in which two Monomeric ubiquitin link related to the location of the "head to tail" directly or through a linker, wherein each monomer mentioned dimeric protein is differently modified by substitutions of at least 6 amino acids in positions 4, 6, 8, 62, 63, 64, 65 and 66 of SEQ ID NO: 1, and the above-mentioned substitution include:
in the first Monomeric unit substitution at amino acid positions 4, 6, 62, 64, 65, and 66 in SEQ ID NO: 1,
characterized in that the substitution is as follows:
in the first Monomeric unit substitution at amino acid positions 4, 6, 62, 64, 65 and 66, and at position 4 replaced by F W, at 6 K is replaced by H, W, or F at position 62 Q is replaced by N, at position 64 E is replaced by K, R or H at position 65 S replaced by L, F, or W in position 66 T replaced by S or P;
and
in the second Monomeric unit substitution at amino acid positions 6, 8, 62, 63, 64, 65 and 66; where K at position 6 is substituted by T, N, S, or Q, L at position 8 is substituted for Q, T, N or S, Q at position 62 is substituted for W or F, K at position 63 is replaced by S, T, N or Q, E at position 64 is replaced by N, S, T or Q, S in the state� 65 being replaced with F, or W, T at position 66 is replaced by E or D; where the monomer unit optionally comprises additional amino acid substitutions that do not affect the protein activity,
and over and above protein has specific binding affinity with the domain ED-B of fibronectin of KD=10-7-10-12M and exhibits a monovalent binding activity with regard to extramenu B (ED-B) of fibronectin.

2. Protein according to claim 1, characterized in that both monomers of ubiquitin are linked through a linker having at least the sequence GIG or at least SGGGG, or preferably SGGGGIG, or SGGGGSGGGGIG (SEQ ID NO: 32).

3. Conjugate for the treatment of diseases associated with overproduction ED-b, comprising the protein according to any one of claims. 1 and 2, combined with the pharmaceutically active component, characterized in that said pharmaceutically active component is a cytokine, a chemokine, a cytotoxic compound or an enzyme.

4. The conjugate according to claim 3, which includes heterodimer of ubiquitin with SEQ ID NO: 33, or 34, or 47, or has an amino acid identity of at least 90% or 95% with the sequence SEQ ID NO: 33, or 34, or 47.

5. The conjugate according to claim 3 or 4, have a protein nature.

6. The conjugate according to claim 3, characterized in that said pharmaceutically active component is a TNF-alpha or its derivative, the implicit�plant wherein that mentioned composite protein has the sequence SEQ ID NO: 35 or 36, or wherein the SEQ ID NO: 47 is combined with TNF-alpha or its derivatives, or has an amino acid identity of at least 90% or 95% with the sequence SEQ ID NO: 35 or 36, or SEQ ID NO: 47, combined with TNF-alpha or its derivatives.

7. Conjugate for the diagnosis of diseases associated with overproduction ED-B, comprising the protein according to any one of claims. 1 and 2, combined with a diagnostically active component, characterized in that said diagnostically active component is a fluorescent compound, a photosensitizer or a radionuclide.

8. The conjugate according to claim 7, which includes heterodimer of ubiquitin with SEQ ID NO: 33, or 34, or 47, or has an amino acid identity of at least 90% or 95% with the sequence SEQ ID NO: 33, or 34, or 47.

9. Pharmaceutical composition for treating diseases associated with overproduction ED-B containing protein capable of binding domain ED-B of fibronectin, according to any one of claims. 1 and 2 in a pharmaceutically effective amount, optionally containing one or more chemotherapeutic agents and one or more pharmaceutically acceptable carriers or excipients.

10. Pharmaceutical composition for treating diseases associated with overproduction ED-B containing �anywhat according to any one of claims. 3-6 in pharmaceutically effective amounts and optionally containing one or more chemotherapeutic agents and one or more pharmaceutically acceptable carriers or excipients.

11. Pharmaceutical composition according to claim 9 or 10, characterized in that said chemotherapeutic agents are selected from melphalan, doxorubicin, cyclophosphamide, dactinomycin, ftordezoksiuridin, cisplatin, paclitaxel and gemcitabine, or from the group of kinase inhibitors, or radiopharmaceuticals.

12. Pharmaceutical composition according to claim 11, which is in the form of a combined preparation or in the form of a set of individual components.

13. The polynucleotide encoding the recombinant protein according to any one of claims. 1 and 2.

14. The polynucleotide encoding the conjugate by p 5.

15. Expression vector comprising the polynucleotide according to claim 13.

16. Expression vector comprising the polynucleotide according to claim 14.

17. A host for expression of the protein according to any one of claims. 1 and 2, comprising the polynucleotide according to claim 13.

18. A host for expression of the conjugate according to claim 5, comprising the polynucleotide according to claim 14.

19. A host for expression of the protein according to any one of claims. 1 and 2, containing the expression vector according to claim 15.

20. A host for expression of the conjugate according to claim 5, containing the expression vector according to claim 16.

21. Composition d�I diagnosis of ED-B-associirowannyh diseases comprising an effective amount of a protein according to any one of claims. 1 and 2 with a diagnostically acceptable carrier.

22. Composition for the diagnosis of ED-B-associated diseases, comprising an effective amount of a conjugate according to any one of claims. 7 and 8 with a diagnostically acceptable carrier.

23. Method of creating a protein according to any one of claims. 1 and 2, comprising the following stages:
(a) the aggregate of differently modified dimeric ubiquitin proteins originating from Monomeric ubiquitin proteins, over and above the set consists of dimeric ubiquitin proteins comprising two modified
monomer of ubiquitin linked at the location of the "head to tail", and each monomer mentioned dimeric protein is differently modified by substitution of at least 6 amino acids in positions 4, 6, 8, 62, 63, 64, 65 and 66 of SEQ ID NO: 1,
moreover, the above-mentioned substitution include:
in the first Monomeric unit substitution at amino acid positions 4, 6, 62, 64, 65 and 66, and at position 4 replaced by F W, at 6 K is replaced by H, W, or F at position 62 Q is replaced by N, at position 64 E is replaced by K, R or H at position 65 S replaced by L, F, or W in position 66 T replaced by S or P; and
in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; where KV position 6 is substituted for T, N, S, or Q, L at position 8 is substituted for Q, T, N or S, Q at position 62 is substituted for W or F, K at position 63 is replaced by S, T, N or Q, E at position 64 is replaced by N, S, T or Q, S at position 65 is replaced with F, or W, T at position 66 is replaced by E or D
where monomer unit optionally comprises additional amino acid substitutions that do not affect the protein activity;
(b) providing extradata B (ED-B) of fibronectin as a potential ligand;
c) the contact mentioned together differently modified proteins with said extradata B (ED-B) of fibronectin;
(d) identifying a modified dimeric ubiquitinated protein by the method of screening, and referred to the modified dimeric ubiquitinate protein is associated with extraluminal B (ED-B) of fibronectin with a specific binding affinity with a KD value in the range of 10-7-10-12M and active monovalent binding to the aforementioned extradata B (ED-B) of fibronectin.

24. A method according to claim 21, characterized in that said set of differently modified proteins produced by the genetic Association of two DNA libraries, each of which is responsible for receiving differently modified Monomeric ubiquitin proteins.

25. How to create a conjugate according to any one of claims. 3-6,�lichudis, what protein according to any one of claims. 1 and 2 is conjugated with pharmaceutically active component, characterized in that said pharmaceutically active component is a cytokine, a chemokine, a cytotoxic compound or an enzyme.

26. How to create a conjugate according to any one of claims. 7 and 8, characterized in that the protein according to any one of claims. 1 and 2 is conjugated to a diagnostically active component, characterized in that said diagnostically active component is a fluorescent compound, a photosensitizer or a radionuclide.

27. The application of the recombinant protein according to any one of claims. 1 and 2 or the conjugate according to any one of claims. 3-6 for the treatment of diseases associated with overproduction ED-B.

28. The recombinant conjugate according to any one of claims. 7 and 8 for the diagnosis of diseases associated with overproduction ED-B.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: strain of cells of the Chinese hamster ovaries CHO-Inflix 20/5 is produced, transfected by vectors, expressing light and heavy chains of chimeric antibody against tumour necrosis factor alpha (TNF-α) of human being is produced. The strain is deposited into the Russian collection of cellular cultures under No. RKKK(P)760D.

EFFECT: invention allows to produce chimeric antibody with the specific efficiency no less than 33,5 picograms per cell a day.

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SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

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SUBSTANCE: invention offers recombinant plasmid DNA coding a chimeric antibody against human tumour necrosis factor-alpha (TNF-alpha) based on pOptiVECTM-TOPO® plasmid. Invention refers to eukaryotic cell line as a producer of antibody to TNF-alpha, method of cell line obtainment by transfection of plasmid DNA according to the invention, and method of chimeric antibody obtainment for TNF-alpha by cultivation of cell line according to the invention.

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SUBSTANCE: invention refers to biotechnology and immunology. Described are various variants of anti-IL-1R1 antibodies. The disclosed antibodies can be applicable for treating IL-1R1-mediated disorders, including rheumatoid arthritis, asthma, and chronic obstructive pulmonary disease (COPD).

EFFECT: presented group of inventions can be used in medicine.

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FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology and can be used for the identification of heteromultimeric ubiquitins possessing an ability to bind with a ligand-antigen. The method includes the contact of a totality of heterodimeric modified ubiquitins, including two ubiquitin monomers, bound to each other by a head-to-tail scheme, with the potential ligand in a display way. Each of the said monomers is modified in a different way and contains 5-8 substitutions in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 SEQ ID NO:1. After that, a heterodimeric modified protein, which has bound with the ligand with the binding affinity Kd in the range of 10-7-10-12 M and the monovalent binding activity. Claimed are DNA-libraries, responsible for obtaining a population of the said heteromultimeric ubiquitins, as well as libraries of proteins, obtained by the expression of the said DNA-libraries.

EFFECT: invention makes it possible to obtain the novel bonding proteins based on heteromultimeric ubiquitin, capable of specific high affinity binding with the selected ligands.

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SUBSTANCE: presented invention refers to immunology. There are presented versions of antibodies neutralising a subtype group 1 and subtype group 2 influenza A virus infection. The antibody is characterised by: either a set of 3 CDR of a light and 3 CDR of a heavy chain, or the presence of variable regions of the light and heavy chains. There are disclosed: a nucleic acid molecule coding the antibody; a cell expressing the antibody; as well as a method for the attenuation of the influenza A virus infection or reducing a risk thereof with the use of the antibody in a therapeutically or preventatively effective amount.

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SUBSTANCE: inventions deal with infectious molecule of nucleic acid, coding infectious porcine Torque teNO viruses (PTTV), which contains at least one copy of genome sequence, selected from the group, consisting of sequences, corresponding to genotypes or subtypesPTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, as well as to biologically functional plasmid or viral vector, containing such infectious nucleic genome sequence, and host-cell, containing such plasmid or vector. In addition claimed inventions include live, attenuated expressible with vector application and purified recombinant capsid subunit or killed viral vaccines for protection against PTTV infection, as well as methods of immunisation of pigs against PTTV viral infection by said vaccine introduction.

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SUBSTANCE: invention relates to biotechnology and gene engineering. A method for selecting at least one transfected eukaryotic host cell expressing a target product, the eukaryotic host cells comprise at least an introduced polynucleotide encoding the target product, an introduced polynucleotide encoding a DHFR enzyme using at least one expression vector, providing a plurality of eukaryotic host cells, whose viability is dependent upon folate uptake, wherein the said host cells comprise at least a foreign polynucleotide encoding the target product, a foreign polynucleotide encoding a DHFR enzyme, culturing the said plurality of the eukaryotic host cells in a selective culture medium comprising folic acid in a concentration of 12.5-50 nM combined with a concentration of MTX of 2.3-500 nM, selecting at least one eukaryotic host cell expressing the target product.

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SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.

EFFECT: present invention can find further application in cancer therapy.

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FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology and can be used for the identification of heteromultimeric ubiquitins possessing an ability to bind with a ligand-antigen. The method includes the contact of a totality of heterodimeric modified ubiquitins, including two ubiquitin monomers, bound to each other by a head-to-tail scheme, with the potential ligand in a display way. Each of the said monomers is modified in a different way and contains 5-8 substitutions in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 SEQ ID NO:1. After that, a heterodimeric modified protein, which has bound with the ligand with the binding affinity Kd in the range of 10-7-10-12 M and the monovalent binding activity. Claimed are DNA-libraries, responsible for obtaining a population of the said heteromultimeric ubiquitins, as well as libraries of proteins, obtained by the expression of the said DNA-libraries.

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FIELD: medicine.

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SUBSTANCE: group of inventions refers to biotechnology. What is presented is a precursor protein for the recombinant preparation of peptides having a length of a sequence of 5 to 70 amino acid residues. The above protein contains a splittable recurrent sequence of target peptide elements (Pep) and auxiliary peptide elements (Aux) of general formula: (Pep-Aux)x or (Aux-Pep)x, wherein x>1. The elements Aux contain a self-assembled peptide element (SA). The elements Pep contain an amino acid sequence having a length of the sequence of 5 to 70 amino acid residues; ends of the elements comprise splittable sequences, which enable releasing the elements Pep from the precursor protein by specific splitting. There are also presented a nucleic acid molecule with a nucleic acid sequence coding the above precursor protein, an expression cartridge and a recombinant expression vector containing the sequence of the above nucleic acid. There are also presented a variant of the precursor protein, a method for producing the target peptide Pep, as well as using an amphiphilic peptide for the recombinant production of the antimicrobial target peptide.

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SUBSTANCE: invention refers to biotechnology and concerns recombinant plasmid pPA-OPRFI DNA coding hybrid recombinant F-I protein of Pseudomonas aeruginosa outer membrane, of the bacterial strain E.coli PA-OPRFI producing this hybrid protein, and a method for producing this recombinant protein. The presented plasmid DNA contains a DNA fragment containing a sequence of modified promoter of bacteriophage T5 and two lactose operons; a DNA fragment containing a ribosome entry site, an initiation ATG-codon and a sequence coding six histidines; a DNA fragment containing the full-size sequences of oprF and opri genes of P.aeruginosa; a DNA fragment containing a ribosome entry site, and a DNA fragment containing a lambda t0 transcription stop region.

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EFFECT: group of inventions can be used for preparing a therapeutic agent for reduction of cancer cell migration and the metastases inhibition.

11 cl, 36 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular biology and genetic engineering. What is presented is a method for selecting an eukaryotic host cell expressing a desired level of polypeptide of interest involving cell transfection by heterologous nucleic acid containing at least one cartridge containing at least one first polynucleotide coding the polypeptide of interest, a stop codon arranged towards the expression in relation to the first polynucleotide, a second polynucleotide arranged towards the expression in relation to the stop codon coding an immunoglobulin transmembrane anchor domain, culturing host cells for the expression of the polypeptide of interest so that a portion of the disclosed polypeptide is expressed in the form of a fused polypeptide containing the immunoglobulin transmembrane anchor domain; this fused polypeptide is exposed on the surface of the above host cell, selecting the cell by the presence or the amount of the fused polypeptide exposed on the cell surface.

EFFECT: invention can be used in biotechnology for selecting the high-production cell lines.

15 cl, 8 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to modified von Willebrand factor (VWF), and can be used in medicine. A recombinant method is used to preparing modified VWF fused in C-terminal of its primary translation product with N-terminal of albumin by the linker SSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS. The prepared modified VWF is used as a part of the pharmaceutical composition for treating or preventing coagulation failure.

EFFECT: invention enables preparing the modified VWF which maintains its ability to N-terminal dimerisation and C-terminal multimerisation with a prolonged half-period of functional blood plasma occurrence as compared to the half-period of functional VWF occurrence.

17 cl, 5 dwg, 4 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: inventions refer to biotechnology and concern a fused protein for the specific inhibition of blood coagulation, an expression plasmid DNA coding this fused protein, a bacterium of the genus Escherichia transformed by this DNA, and to a method for preparing the fused protein. The presented fused protein contains thioredoxin I E.coli and infestine-4 and is characterised by the sequence SEQ ID NO:2. The plasmid DNA contains the sequence SEQ ID NO:1 coding the presented fused protein and controlled by a promoter functioning in a bacterial cell. The method for preparing the above fused protein involves culturing the above bacteria in a nutrient medium, breaking the bacterial cells and purifying the above fused protein with using a metal chelate chromatography and an anion-exchange chromatography.

EFFECT: characterised solutions enables preparing the protein providing the above specificity to be used in blocking the contact activation of blood coagulation by inhibition of the XIIa factor and the absence of inhibition of the Xa factor.

4 cl, 10 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to artificial immunogenic proteins having the properties of melanoma antigens. What is declared is an artificial gene coding MEL-TCI-A0201 polyepitope immunogenic protein containing multiple cytotoxic restricted HLA-A*0201 and T-helper epitopes of NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-A11, MAGE-C1 melanoma antigens, having a sequence of 1,535 base pairs presented in Fig. 3. There are also declared a recombinant plasmid DNA containing the above artificial gene, and MEL-TCI-A0201 immunogenic protein with the properties of the melanoma antigens.

EFFECT: invention enables providing higher immunogenicity of the artificial polyepitope T-cell immunogen inducing a higher level of cytotoxic T-lymphocyte response.

3 cl, 11 dwg, 3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of biotechnology. Method of extracting DNA from blood cells is claimed. Magnetic particles and ferromagnetic nanospheres CoNiFe2O4 50 nm are added into sample. Biologic material is lysed. DNA is washed and DNA is taken off from carrier.

EFFECT: preference of claimed method consists in increase of DNA quantity in obtained sample.

3 dwg, 1 ex

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