Modified molecule tnfman, is able to induce the formation of neutralizing antibodies to human tnfdna, its coding, vector (options), a method of obtaining a vaccine tnf(options), method of testing for the presence of tnfthe way to test body fluids of a person, the method of diagnosis, method of treatment and prophylaxis medication for the treatment of
The invention relates to biotechnology, genetic engineering and medicine and can be used to obtain a modified molecule TNFman, is able to induce the formation of neutralizing antibodies. The modified molecule TNFman, can increase the amount of neutralizing antibodies to human TNFwild type after administration to a human recipient of the modified molecule TNFproduced by replacement of at least one peptide fragment of the molecule TNFperson on at least one peptide containing the immunodominant epitope for T cells, or create a truncated form of the above molecules containing the immunodominant epitope and one or both flanking region of the molecule TNFman, containing at least one TNFthe epitope for b cells, and substitution leads to a significant change in the amino acid sequence of the front-layer in any one of the connecting loops and/or in any of theperson or DNA encoding them, are used in recipes as a vaccine against TNFthe choice of pharmaceutically acceptable substances that enhance the immunogenicity of antibodies to prevent or treat chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, cancer, multiple sclerosis, diabetes, psoriasis, osteoporosis, or asthma. The liquid body is examined for the presence of TNFupon contact with the composition containing the modified TNF. The invention allows the development of tools for the treatment of chronic inflammatory diseases. 12 C. and 25 C.p. f-crystals, 16 ill., 3 table.
The technical field
This invention relates to molecules of the cytokine human Tumor Necrosis Factor(TNF), which has been modified so that they were able to raise neutralizing antibodies to human TNFwild-type after the introduction of the modified molecule TNF person on the basis of the above modified molecules TNF. Other aspects of the invention will become apparent from the following description.
The level of technology
Physiologically immune system of vertebrates serves as a protective mechanism against invasion of the body infectious objects, such as microorganisms. Foreign proteins are effectively removed by the reticuloendothelial system using highly specific circulating antibodies, and viruses and bacteria are destroyed with the help of a complex group of cellular and humoral mechanisms, including antibodies, cytotoxic T lymphocytes (CTLs), cells - natural killer cells (NK), complement, etc., a Leader in this struggle is the lymphocyte T-helper (Tx), which, together with Antigen-Presenting Cells (APC) regulates the immune protection through a complex system of cytokines.
Txcells recognize protein antigens presented on the APC surface. However, they do not recognize native antigens as such. Instead, they apparently recognize complex ligand consisting of two components, a “processed” (fragmented) protein antigen (the so-called epitope for T-kawana, ultimately, allows Txlymphocytes specifically to help B-lymphocytes to produce specific antibodies to intact protein antigen (Werdelin et al., see above). Certain T-cell recognizes only a particular combination of antigen-AGR and will not recognize the same or a different antigen presented by the product of another gene alleles AGR. This phenomenon is called a limited AGR.
AIC also represent fragments of the native protein, but the rate of such fragments are ignored or not recognized by lymphocytes, T-helpers. This is the primary reason why individuals in the serum do not usually accumulate autoantibodies, ultimately resulting in damage to the individual's own proteins (so-called private or autobake). However, in rare cases, the process goes wrong, and the immune system turns against its own components of the individual, which can lead to autoimmune disease.
The presence of some native proteins disadvantageous in situations where they are at elevated concentrations cause symptoms of the disease. So, it is known that tumor necrosis factor(TNFcan call the 2302-2306, 1991). TNFalso plays an important role in the inflammatory process (W. P. Arend et al. Arthritis Rheum. 33, 305-315, 1990), and therefore, it was demonstrated that neutralization of TNFby using monoclonal antibodies useful for patients with chronic inflammatory diseases such as rheumatoid arthritis (Elliott et al., Lancet 194, 344:1105-10) and Crohn's disease (van Dullemen et al., Gastroenterology 109 (1): 129-135 (1995). There is therefore a need for a method of inducing formation of neutralizing antibodies to such proteins TNF, this invention includes a vaccine against TNFthat possesses this property.
The TUMOR NECROSIS FACTOR
1. The main data source
The tumor necrosis factor (TNF) is a representative of the regulatory proteins of the family of cytokines (see Walsh, G. and Headon, D. E., Protein Biotechnology, 1994, John Wiley & Sons Ltd., England, p.257-267), which also includes the interferons and interleukins. Currently, there are two forms of TN, TNFand TNFrespectively. Although both proteins bind to the same receptors and cause very similar biological reactions, they are different is first referred to as TNF, more correctly be called TNF; it is also known as cachectin. On TNFalso referred to as lymphotoxin.
In addition to the cells of the brain and liver, TNFis produced by many types of cells, especially activated macrophages, monocytes and some T-lymphocytes and NK-cells. The most powerful of the known inducers of synthesis of TNFis difficult biomolecule called lipopolysaccharide (LPS). It contains both lipid and polysaccharide components and is also referred to as endotoxin. By itself, the lipopolysaccharide devoid of antitumor activity. It was found that the serum of the animals, which were injected lipopolysaccharide contains a factor toxic to cancer cells, and this factor is produced by specific cells in response to lipopolysaccharide, was called tumor necrosis factor. Many other agents, such as some viruses, fungi and parasites, also stimulate the synthesis and secretion of the cytokine. Moreover, TNFcan act in autocrine manner, stimulating its own products.
TNFcalls its biological effects by binding to specific receptors present on the surface of susceptible cells. Identified two different types of receptors for TNFthe SSA approximately 75,000 Yes. These two different types of receptors detect homology sequences not exceeding 25%. TNF-R55 is present in a wide range of cells, whereas the prevalence of TNF-R75 receptors more limited. Both receptors are transmembrane glycoproteins with an extracellular binding domain, a hydrophobic transmembrane domain and an intracellular effector domain.
The exact molecular mechanisms by which TNFinduces its biological effects remains to be determined. The binding of TNFwith its receptors, apparently, runs, in addition to activation of adenylate cyclase, phospholipases and protein kinases, a variety of phenomena that are mediated by G-proteins. The exact biological effects of TNFmay vary in different cell types. Other factors such as the presence of other cytokines, additionally modulate the observed molecular effects due to the action of TNFon sensitive cells.
Gene TNFwas cloned and introduced into a variety of recombinant expression systems, both bacterial and src="https://img.russianpatents.com/chr/945.gif" border="0">facilitated clinical study of many diseases, especially cancer. However, many of these studies using TNFseparately or in combination with interferon, gave very inconsistent results. Large amounts of TNFcan be patient because of their toxic - if not fatal - side effects.
As noted above, long-lasting products adversely elevated concentrations of TNFalso involved in the development of cachexia, wasting syndrome, often associated with chronic parasitic or other infections and cancer. TNFalso involved in the metastasis and growth of some tumors and induction of anemia. Moreover, TNFdirectly involved in the development of some chronic inflammatory diseases in humans, including rheumatoid arthritis and Crohn's disease, in which was shown the usefulness of the introduction of monoclonal antibodies to TNF. TNFalso involved in the development of osteoporosis and psoriasis. In addition, in model experiments on animals were dormancy is isagenix or transplanted tissues.
The structure of the TNF
Spatial structure factor of human tumor necrosis (TNF) was established (see the monograph "Tumor Necrosis Factors, Structure, Function and Mechanism of Action", published by Bharat B. Aggarwal and Jan Vilcek, 1992 Marcel Dekker, Ind., New York, Chapter 5 “Crystal structure of TNF"the authors Jones, E. Y., Stuart, D. I., and Walker, N. P. C.). The biological activity of TNFdepends on its interaction with its receptors. These interactions are governed by precise design correctly folded tertiary structure. Thus, to understand how the molecule TNFperforms its biological function at the level of interactions of amino acids, it is necessary not only to know the sequence of amino acids, but also the spatial structure.
II. Spatial structure
Using analytical ultracentrifugation, x-ray scattering at small angle, and gel electrophoresis, it was shown that biologically active TNFis in solution in the trimeric conformation, and research with a cross-linking showed that this active form of b is than the monomer. Experimental evidence suggests that both natural and recombinant TNFunder physiological conditions exist predominantly in the form of a trimer. Analysis of circular dichroism spectra of put TNFin the class of proteins with a layered structure (see Davis et al., Biochemistry 1987, 26, 1322-1326, which describes the results of structural analysis of purified recombinant TNFby titration sulfgidridnyh groups, gelfiltration and circular 20 dichroism). Several different crystalline forms described for recombinant TNFman. All described crystalline forms reveal crystallographic and cristallographie threefold symmetry, which is an indicator of the presence of TNFin the crystal in the form of a trimer. The TNF trimerslie in loosely Packed order, perforated by channels filled with solvent, with a diameter of 10 nm. Only a small fraction of surface molecules involved in the contacts of the crystal packing. Such contacts may slightly change the position of some of the side chains and,racemi in solution.
A. Laying the main chain of monomer TNF
The General form of the individual subunits of the trimer of TNFconsisting of 157 amino acids, resembles a wedge with a height of about 5.5 nm and a maximum width of 3.5 nm near the base. The basic topology of the circuit illustrated in Fig.1A-C; it is essentially a structure of type-sandwich formed by two antiparallelfolded layers. The laying of the main chain corresponds to the classic tune of “roll with fillings (Fig.1C) (common for viral capsid protein). The nomenclature adopted in Fig.1 for marking blocks of the secondary structure corresponds to the established order for viral structures. There are eight standardchains (from V to I), but with the insertion of a between b and C, which adds a short circuit on the edge of bothlayers and truncate the N-terminal half, so that eachfolded layer contains five antiparallel-chains, rearlayer includes-chain, S, N, E and F.
N-end is very flexible. This region up to residue 10 (see Fig.1b), is quite independent from the rest of the molecule, and the first few residues freely adopt different conformations in the solvent. In contrast, With the end submerged in the base of the backlayer and forms an integral part of this relatively flat block of the secondary structure. Striping-chains of different length and insert between the-chains b and C together form a front surface, formed almost entirely of loops, and it is “disguised” side-sandwich, in which the trimer is facing the solvent. Crystallographic data made it possible to measure the relative flexibility of different parts of the structure,-chains form a fairly rigid structure; in particular, the rearlayer is located in the core of the trimer and as a consequence especially cruel. As expected, the loop that adorn the exterior, solvent-accessible surface of the molecule, detect high levels of flexibility/mobility. In General, happens is olekuly.
B. the General topology of the TNF trimer
Three Monomeric subunit TNFconnect ecovalence with the formation of a compact, conical shape of the trimer, having a length of about 5.5 nm and a maximum width of 5 nm.-circuit three separate-sandwiches are approximately parallel (slope equal to about 30°) the common axis of the trimer. The interaction between the subunits related to the common axis, is carried out by plain packaging-sandwich-type edge-to-edge”; edge-sandwich consisting of chains F and G of one subunit, was lying across the backlayer [GDIBB] one of the three related subunits (see Fig.2). Carboxyl ends lie close to a common axis. Packing edge-to-edge” provides extremely tight bond between the subunits. Therefore, the core of the trimer completely inaccessible to solvent.
C. the Distribution of various types of amino acids
The overall distribution of residue types in the spatial structure of TNFfollows General p is colruyt surface. Therefore, the core of sandwich TNFas expected, filled with densely Packed large nonpolar residues.
The energy of the system is not conducive to the existence of TNFin the form of monomer. For large surface section, consisting of complementary residues (i.e., polar residues that form a pair with polar residues), the absence of surface accessible to solvent, provides a significant energy advantage the formation of oligomer (e.g., trimer). Exposure to solvent of a large area vysokolegirovannyh residues, normally shipped in the lower portion of the surface section of the trimer, also would destabilizers to monomer TNF.
2. Studies of the structure functions
A. interaction of TNF/antibodies
It has been observed that antibodies accumulated to TNFin one species (e.g. human), do not take cross-react with TNFother species (e.g. mouse), despite a sequence identity greater than 80%, and the ability of TNFto swanyatiputi on the spatial structure, it immediately becomes clear that the zone of the molecule with the most variable sequences correspond to surface loops that are accessible to antibodies. Region highly conserved residues within the sandwich or on the boundary surface of the trimer effectively not visible for antibodies. Therefore, the epitope for antibodies to TNFwill always contain several residues that will vary in different species, thus eliminating the binding of the antibody. This means that the characteristics of the interaction between TNFand its receptor must somehow be different from the characteristics required for binding of an antibody to TNF.
B. Site-directed mutagenesis
The role of specific residues and regions of the molecule TNFin relation to its biological (cytotoxic) activity and binding to the receptor was investigated by replacing these residues by different amino acids or removing parts of the sequence using techniques targeted mutagenesis (Jones et al, op. Cit. P. 113-119).
Remove up to eight residues from N-Terminus without any harmful effect on biological active is oncewe residues, apparently, provide indirect, secondary effects on biological efficacy of the TNF trimer.
Non-conservative substitutions in the norm of highly conserved residues that form a tightly Packed core-sandwich, disrupt the structure and, therefore, destroy the biological activity of TNF(Yamagishi et al., Protein Engineering, Vol.3, N 8, p.713-719 (1989)). Many of these mutant proteins (Malinov) cannot form a stable, properly folded molecule. Some conservative substitutions permissible within the hydrophobic area at the base of the common axis; however, apparently, considerably large variations exist in the more loosely Packed region near the top of the trimer. In particular, Cysteine 69 and Cysteine 101, which form a disulfide bridge between the two connecting loops on loosely Packed top molecules are relatively insensitive to changes (see Fig.1A). However, in General, to retain some biological activity of TNFmutations near the Central axis of the TNFmust be highly conserved, preserving the overall shape of the mutations without destructive patterns of consequences, as evidenced by the increasing number of variations of the residues in this category in different species. Therefore, a sharp decrease in the biological activity of TNFdue to substitutions in this region indicates a direct involvement of these residues in the functional interaction of the TNF trimerwith its receptor. Such remains, apparently, are the remains, including Arginine 31, Arginine 32 and Alanine 33, located in the connecting loop between In and In-chains in the rearlayer, serine 86 and Tyrosine 87, located in the connecting loop between the E and F strands FWDlayer, and Glutamic acid 146, located in the connecting loop between N-thread front-layer and I-thread back-layer (see Fig.3). They, apparently, are two different “hot zone” on the front and back sides of a monomer TNF. The distribution of all harmful mutations unrelated to the structural categories further supports this picture. The existence of such “hotspots” for the sensitivity of biological function to evolution the TNFhumans have been described in many patent publications.
Thus, in document EP 251037 described numerous site-specifically mutated molecules TNFthat were soluble and possessed cytotoxic and antitumor activity characteristic of TNFhumans, in vitro and/or in vivo.
Other mutiny with the activity of TNFdescribed in document WO 90/07579. Mutiny with higher affinity to the receptor P75 TNF man than to the receptor P55 TNF person described in the document EP-A-619 372.
None of these cited publications included in this description by reference, disclose the nature of the modification of the molecule TNFaiming to destroy the biological activity of TNFand to provide the ability to induce the formation of antibodies to human TNFwild-type.
Nevertheless, they provide useful fundamental information regarding TNFand its biological action. In addition, they revealed the nature of the receipt and expression analogues TNF. All available evidence points to the importance of the trimer as a stable natural units. It is obvious that the two “hot” area, located on different sides of the monomer TNFclosely contiguous to each other on adjacent subunits of the trimer. So functionally important region consisting of residues 31-35, 84-87 and 143-148, apparently, is located on the boundary surface between the two subunits on the bottom half of the trimer. Yamagishi et al. op. cit. reported no ability to bind to the receptor and cytotoxicity when mutations with the replacement of the Aspartic acid at position 143 to Tyrosine, a Tsujimoto et al., J. Biochem. 101, R. 919-925 (1987) described a similar effect to the replacement of Arginine 31 and Arginine 32 on Asparagine and Threonine. Thus, this site may be directly associated with binding to the receptor and cytotoxicity. Interestingly, the region of the receptor binding TNFapparently, lies on the interface between the two subunits.
In General, the detailed description of the three-dimensional structure TNFserve to explain shiroli to consider the structure together with the newly received numerous targeted mutants, the area of biological significance in relation to the receptor binding, apparently, is located on the subunits in the lower half of the trimer.
Characterization of analogs
According to the invention, opened in document WO 95/05849 which authors are some of the authors of the present invention, a method of modifying their own proteins in such a way as to induce the reaction of antibodies against unmodified native protein, where the analogue of the native protein is provided by molecular biology. In particular, one or more peptide fragments of the native protein is substituted by one or more peptide fragments containing immunodominant, alien epitopes for T-cells.
Prior to the invention described in WO 95/05849, was known for the conjugation of peptides or proteins, including native proteins, protein-carriers, comprising the epitope for T cells. Document WO 92/19746 discloses recombinant polypeptide comprising a sequence of LHRH, one or more epitopes for T-cells and the site of sterilization, which is suitable for use in vaccines for immunocastration animals.
In WO 95/one built-epitope were saved flanking region of the original protein, includes at least 4 amino acids. In other words, the epitope should not be anywhereman with the native protein as a fusion protein. Preferably the substitution must be made in a way that essentially preserve the tertiary structure of the original protein. In addition, not given specific guidance on the intramolecular position of the embedded epitope, optimal to provide the most powerful reaction of antibodies against unmodified native protein. Presumably, it will be different for different native proteins, but on the basis of the General principle of specification the most suitable position(I) can be determined without undue experimentation by selection of peptides comprising the immunodominant epitopes, replacement of the peptide sequences of essentially the same length in different parts of the molecule's own protein and evaluation of enhanced antibody responses by means of suitable methods of analysis.
With the use of modern simulation tools, is probably very difficult to predict, will replace one or more peptide fragments of the protein to changes in the tertiary structure of the original protein. Therefore, when the search key decision to hold a standard screening procedures, to assess retained whether the modified native protein tertiary structure of the original protein. This can be accomplished using several experimental techniques that have been described in numerous guidelines for characterization of proteins, such as fluorescence spectroscopy, circular dichroism in the near-UV range, infrared spectroscopy with Fourier transform and multi-dimensional NMR techniques (Physical Methods to Characterize Pharmaceutical Proteins", Biotechnology, Vol. 7 Eds. J. N. Herron, W. Jiskoot & D. J. A. Crommelin, Plenum Press, New York (1995)). Ideally, the screening process key connections should combine two or more experimental techniques mentioned above, to assess occurred or no change in the tertiary structure.
A short list of drawings
Fig.1(a) illustrates a crystalline structure of the native monomer TNF. This figure represents a schematic illustration of the stacking subunit,-chains are shown as thick arrows in the direction from the amino group to carboxypropyl, and the connecting loops are depicted as thin lines. A disulfide bridge is marked with the lightning, and high flexibility transversely �src="https://img.russianpatents.com/chr/945.gif" border="0">chain a crystal structure of the monomer TNF. This is a detailed image to be used in conjunction with Fig.1(a), to obtain an accurate comparison of amino acid sequences with more understandable, but a stylized image stacking subunit.
Fig.1(C) depicts the structure of the TNFas the motif of “roll with stuffing.” Insert between-chains b and C are shown as dashed lines; the connection between b and C goes straight across the top of the molecule.
Fig.2 depicts a packaging-sandwiches-type edge-to-face TNF trimer. View in the direction of the common axis represents a narrow slice of the trimer with-chains are depicted as ribbons, going to and from a page.
Fig.3(a) illustrates the nucleotide sequence of DNA encoding the tumor necrosis factor (TNF), having the amino acid sequence shown in Fig.3(b).
The nucleotide sequence of DNA can be obtained in the Bank of Genes under inventory No. L, SEQ ID no:339737.
The sequence was described by Wang et al., Science 228, 149-154 (1985). The sequence includes codons encoding-76-Idov gene, including introns, was described Nedwin et al., Nucleic Acids, Res. 13 (17) 6361-6373 (1985), Shirai et al., Nature 313 (6005), 803-806 (1985) and Dennica et al., Nature 312 (5996), 724-729 (1984).
Fig.3(b) shows the amino acid sequence of TNFperson, including predpolozhytelno -76--1.
Fig.4(a) schematically illustrates the replacement for immunodominant epitopes P2 and P30 in TNFwild-type (DT) with the formation of analogues TNFfrom PNO-1 to 2-7 and PNO-1 to 30-5.
Fig.4(b) shows the exact position of the substitutions in the DT sequence for the individual congeners TNF.
Figures 5(a) and 5(b) shows the structure of the analogues on the basis of Fig.1(a), where a separate replacement on P2 and P31 in chainslayers and connecting the loops, respectively, are marked in black.
Fig.6 shows the biological activity of analogues of TNFin the sample L929 (Meager, A., Leung, N., & Wooley, J. Assays for Tumor Necrosis Factor b related cytokines., J. Immunol. Meth. 116, 1-17 (1989)) compared to recombinant TNF.
Fig.7 shows the reaction of antibodies to TNFperson in rabbits by immunization of rabbits molecules modified the img src="https://img.russianpatents.com/chr/945.gif" border="0">person to induce the formation of neutralizing antibodies measured in the sample with L929 cells.
Fig.9 shows the ability of the P2/R30-modified molecules TNFperson to induce - when administered to rabbits - the formation of neutralizing antibodies measured in the sample with the receptor.
Fig.10 shows the response of mononuclear cells in peripheral blood (MCPC) from three donors on SA and P2 and P30-peptides.
Fig.11 shows the reaction of the polyclonal proliferation in two donors using different P2 and P30-modified molecules TNF.
Fig.12 shows the Index of Proliferation (PI), calculated on the 34 experiments with P2 and P30-modified molecules TNF.
Fig.13 shows the reaction MCPC on P2 and P30-modificirovannye proteins TNFin persons, specifically reacting to P2 and P30, respectively.
Fig.14 shows a similar reaction mkpk the other two donors.
Fig.15 shows the influence of the flanking amino acids on the recognition of P2 and P30 T-cells.
Fig.16 illustrates the strategy of mutations used to produce modified molecules TNF.
CE is lah total of the invention, disclosed in WO 95/05849, specific native protein, namely TNFman, so that he was biologically active and able to induce a strong reaction of neutralizing antibodies to biologically active TNFwild-type. In the context of this invention “biologically inactive” refers to various kinds of activity of TNFwild-type, mainly for its cytotoxic activity.
When discussing the tertiary structure of TNFabove, it was shown that biologically active TNFis a trimer of three of its subunits. Due to the packing edge-to-edge”, “rearlayer” is a “hidden” area of contact between the subunits, which is completely inaccessible to solvent. Significant replacement in this area almost inevitably deprive molecule TNFany biological activity. In contrast, “frontlayer, and the connecting area provide an accessible surface area, which includes area, vzaimode able to disrupt binding to the receptor and to possess the ability to neutralize TNF.
Specialists in this field of technology who want to construct detoxificating, but still immunogenic molecule TNFin accordance with the invention according to WO 95/05849 should therefore primarily embed immunodominant epitope for T cells in the rearthe layer of monomer TNF. Therefore, modification of this area most likely to destroy the biological activity of TNFand keep available for front receptors-free layer for interaction with antibodies. This is also consistent with the discussion site-directed mutagenesis in a tightly Packed inner part-sandwich, discussed above.
However, unexpectedly, this assumption is not confirmed. As will be clear from the test results below, the result was exactly the opposite, as replacement, including and With the chain in the rearlayer, unexpectedly gave analogues TNFwho were unable to induce the formation of neutralizing antibodies to TNF.
In the context of this invention “substantial change” means a change that is more than just a conservative substitution of individual amino acids wild-type. In other words, the epitope for T cells, embedded in a sequence of TNFshould preferably be embedded sequence, which very few homologous sequences TNFwild-type.
In accordance with this invention also provides a modified molecule TNFman, can increase the amount of neutralizing antibodies to human TNFwild-type after the introduction of the above-mentioned modified molecule TNFto a human recipient, in which at least one peptide fragment of the molecule TNFman was substituted by at least one peptide, which is known that it contains the immunodominant epitope for T cells, or a truncated form of the above molecules containing the immunodominant epitope and one or both flanking region of the molecule TNFperson, comprising at least one epitope of TNFfor b cells, and the above modified molecule TNFin much the spine of TNF” in the context of the present invention is intended to denote that the introduction of human being such modified molecules TNFdoes not result in significant adverse effects due to the known cytokinine the effects of native TNF. In other words, the modified molecule TNFaccording to the present invention are pharmaceutically acceptable substances.
To check out whether the modified molecule TNFman of the present invention largely devoid of activity of TNFmay be used in the bioassays L929 described next. Moreover, to confirm the immunogenic character of the modified molecule TNFantibodies to the modified molecule TNFformed from a suitable recipient must reliably inhibit the activity of TNFwild-type in the bioassays L929, as described hereinafter, and/or the above-mentioned antibodies need to reliably inhibit the binding of TNF-R55) or TNFreceptor with a molecular mass of 75 kDa (TNF-R75).
These are appropriate recipients can be, for example, primates, such as monkeys or Rhesus Cynomuleus, rodents, such as rats, mice or Guinea pigs, or Lagomorpha, such as rabbits.
Tests suitable for assessing the potential of modified molecules of the present invention, is performed by using an antibody or antisera at a concentration corresponding to sensitivity analysis. However, they should consider the ability to display physiological conditions regarding the participating reactants, or they should ensure the possibility of extrapolation to physiological conditions, based on the results obtained in the analysis. In other words, the results of the analysis must show that physiological concentrations of antibodies to TNFin vivo can reduce the activity of TNFon at least, 10, 15, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%. Specialist in the art will readily understand how to determine such conditions.
It should be understood that a suitable “reliable inhibition of TNFwild-type. Such a suitable inhibition may be the inhibition of at least 10%, at least 15%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even 100%.
Based on these premises, the characteristic of the modified molecules TNFman this invention, which has been replaced in certain areas of the molecule TNFand which include yarn forwardlayer and/or the connecting loops, so that essentially save-layered structure of any of the chains in the rearlist.
Although it has not yet been fully confirmed experimentally, it should be accepted that this feature is common to the circuits forming the rearlayer, so that preferably the replacement should be made in the areas of the molecule TNF
However, it is acceptable for the replacement was carried out in areas of the molecule TNFthat contain the segment D-thread backlayer.
In accordance with a preferred embodiment of the invention, the replacement includes at least a segment of the H chain frontlayer and connecting the loop to the 1-chain in the rearlayer, preferably amino acids 132 through 146. In accordance with another embodiment of the invention includes replacement segments H and I-circuits and all the connecting loop, preferably amino acids 132 152. In accordance with another presently preferred embodiment of the invention includes replacement segment D-chain rearlayer, at least a segment of the E-chain frontlayer and all the connecting loop, preferably amino acids 65 79 or 64 to D/chr/697.gif" border="0">and With chain front of the p-layer and the segment D-chain rearlayer, preferably amino acids from 40 to 60.
In accordance with another example embodiment of the invention, the replacement includes at least a segment of the E-chain frontlayer and one or both of the connecting loops, preferably amino acids 76 to 90.
Built-in epitope for T cells should preferably be non-uniform and known as immunogenic for most types of HLA molecules (CRC) human class II. Suitable epitopes can be obtained, for example, tetanus toxoid, preferably an epitope P2 and/or P30, Panina-Bordignon et al., Eur. J. Immunol. 19:2237-42, 1989. Can also be used epitopes derived from diphtheria toxoid.
Preferred modified molecule TNFman (analogues TNF), with the position of the substitutions indicated above, is shown in the attached sequence listing as SEQ ID NO:8 and SEQ ID NO:16.
Other suitable analogues TNFlisted as SEQ ID NO:4, 10, 14 and 16.
The invention also relates to truncated analogues of the above modified analogs of TNFcontaining random and immunodominant epitope for T cells and one or both flanking region including at least one TNFthe epitope for b-cells, preferably comprising at least five amino acids, can also be a probable active ingredient in TNFthe vaccine according to this invention. The epitope for T cells should induce the proliferation of T-cells, if it refers to molecules AGR class II, we present the APC, whereas the epitope for b-cells should potentially be recognized by the receptors of the immunoglobulin on b cells and subsequently to be presented by class I molecules AGR on these cells. This forms the basis for increasing the immune response against TNFwild-type carrying the epitope for b-cells, in accordance with WO 95/05849 and by the present invention.
The epitopes for b-cells can be identified in TNFboth theoretically and experimentally. The algorithms identify potential linear epitopes for b-cells have been published, and this forms the basis of experimental studies of the nature of these potential epitopes. Antibodies 32D/chr/945.gif" border="0">including epitopes for T-cells can be analyzed for the ability to bind native TNFhuman in vitro, preferably by neutralization. Group of monoclonal antibodies, known as the neutralization, can be screened in vitro in terms of their ability to bind potential epitopes of TNFfor b-cells. Both of these techniques are strategies identify likely and best of epitopes for b-cells.
The above analogues TNFapparently remain in the monomer form, as a modification, probably destroys characteristic of wild type TNFthe tendency to trimerization.
However, the invention also relates to dimers, oligomers, especially trimers or multimers the claimed molecules TNFthat lack TNFactivity, as well as to isolated DNA molecules that encode modified molecule TNFaccording to the invention.
Isolated DNA molecules encoding the preferred analogs of TNFhave a follower is received as SEQ ID NO:3, 9, 13 and 15.
The invention further includes vectors containing the isolated DNA molecules that encode analogs, and expression vector comprising the above DNA molecule operatively connected to a suitable sequences regulating expression.
In another aspect the invention relates to the recipient, transformed by the expression vector of the above-mentioned equivalent.
Above the recipient can be any of the recipients, usually used for expression, such as a strain of bacteria, yeast, fungi, or cell lines insects, mammals or birds.
The invention also relates to a method for production of the inventive analogs TNFaccording to which cell-recipients that have been transformed by the expression vector analogue, is cultivated under suitable conditions, providing similar products, and produced similar is retrieved.
If desired, the modified molecule TNFaccording to the present invention can be expressed as a fusion protein or to form part of a fusion protein with a suitable amplifying molecule, preferably immunologically active amplifier, such as GM-src="https://img.russianpatents.com/chr/945.gif" border="0">produced by substitutions at the corresponding segments of genes containing or representing immunodominant epitopes for T-cells within the gene encoding the native molecule TNFman. Subsequently, the modified TNFgene Express in a suitable eukaryotic or prokaryotic expression vector. Downregulation of modified molecules TNFclean and re-folded, as described below.
Although it may be preferable to embed a epitope for T cells, in some cases, it may be useful to incorporate a peptide sequence comprising the epitope and flanking region of the protein from which the epitope.
In accordance with the invention, the modified molecule TNFperson can be used in vaccines against TNF. Strategy formulations of vaccines based on purified proteins, such as modulated own proteins, primarily correspond to strategies formulations of any other drugs based on the proteins. Potential problems and guiding hydrocarbon few tutorials, for example, "Therapeutic Peptides and Protein Formulation / Processing and Delivery Systems" Ed. A. K. Banga; Technomic Publishing AG, Basel 1995. The use of reinforcing substances, such as aluminium hydroxide, aluminum phosphate (Adju-FOS), calcium phosphate, similar murmillos of the dipeptide or more recent developments in the field of amplifiers effects of the vaccines, such as biologically erodible microparticles and Claim that is the problem, well-known researchers in the field of pharmacology, working in this field.
Preparation of the vaccine of the present invention that contain peptide sequences as active ingredients is generally well known in the art that illustrate the U.S. patents№ 4608251; 4601903; 4599231; 4599230; 4596792 and 4578770 included in this description by reference. In a typical case, such vaccines are prepared in the form of injectable vaccines, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. The preparation also can be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. P is such substances and their combinations. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting and emulsifying agents, agents, buferiruemoi pH, or amplifiers, which increase the effectiveness of vaccines.
Vaccines traditionally administered parenterally, by injection, for example subcutaneously or intramuscularly. Additional formulations suitable for other routes of administration include suppositories and, in some cases, formulations for oral administration. In the case of suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in a concentration range from 0.5 to 10%, preferably 1-2%. Oral formulations include such normally used excipients, such as pharmaceutical quantity of mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, formulations with delayed allocation or powders and contain 10-95% of active ingredient, preferably 25-70%.
The polypeptides can be introduced into Retz and, formed by joining acids (formed with free amino groups of the peptide) by reaction with inorganic acids such as hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, almond, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as Isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.
Vaccines are given in a way compatible with the dosage formulation, and in such quantities that will be therapeutically effective and immunogenic. The quantity that you enter depends on the subject to be treated, including, for example, on the ability of the immune system of the individual to give the immune response, and the desired level of protection, which, in turn, depends on the level of TNFin the patient. The ranges of suitable dosages of the order of a few hundred milligrams of active ingredient per vaccination with a preferred range of 1 to 300 μg, especially in Diano represent the initial introduction and subsequent inoculations or other introductions.
The method of application can be very different. Apply any of the traditional methods of vaccine administration. It is believed that these include oral administration in solid physiologically acceptable base or in a physiologically acceptable dispersion, parenteral introduction, introduction by injection and the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person subject of vaccination, and to a lesser extent - from the body weight of the person to be vaccinated.
Some modified molecule TNFaccording to the present invention sufficiently immunogenic in the vaccine, but for some other immune response will be enhanced if the vaccine further comprises increasing the immunogenicity of a substance.
Various methods of achieving the effect of increasing the immunogenicity of the vaccine include use of agents such as hydroxide or aluminum phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffer, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment at temperatures in the range from 70 to 101°C for p is ivali treated with pepsin (Fb) antibodies to albumin, mixing with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsification in physiologically acceptable oil carrier, such as mannide monooleate (Aracel A) or emulsification with a 20% solution of perfluorocarbons (Flusol-DA) used as a blocking substituent. In accordance with the invention DDA (dimethyldioctadecylammonium bromide) is an interesting candidate substances that enhance immunogenicity, but QuilA and RIBI are also interesting possibilities. Other possibilities are monophosphorylated And (IFF) and muramyldipeptide (TIR). Other suitable adjuvants include aluminum hydroxide, aluminum phosphate (Adyu-FOS), calcium phosphate, analog muramyldipeptide. Some of the recent developments in the field of amplifiers immunogenicity of vaccines, such as biorstwami microparticles and the Claim can also be suitable for use.
Another very interesting (and therefore preferred) ability to achieve the effect of enhancing the immunogenicity is the application of the methodology described in Gosselin et al., 1992 (which is incorporated in this description by reference to it). In short, >/img>according to the present invention can be enhanced by conjugation of this antigen with antibodies (or antihistamine fragments of antibodies to FcRI receptors on monocytes/macrophages. It was demonstrated that the conjugates between antigen and anti-FcRI especially increase immunogenicity for the purposes of vaccination.
Other possibilities include the use of immunomodulatory substances, such as lymphokines (e.g., IFN-IL-2 and IL-12) or synthetic inducers of IFN-such as poly-I:C in combination with the above-mentioned adjuvants.
In many cases it will be necessary to make multiple vaccine, usually not more than six vaccinations, more often than not more than four vaccinations, and preferably one or more, usually at least three vaccinations. Vaccination usually will be made at intervals of two to twelve weeks, usually at intervals of three to five weeks. Periodic re-immunization at intervals of 1-5 years, usually after 3 years, desirable to maintain desired levels of protective immunity.
One of the reasons for mixing the polypeptides according to izaberete effect can also be obtained in other ways, for example, by expression of effective antigen vaccine in non-pathogenic microorganism. A well known example of such a microorganism is Mycobacterium bovis BCG.
Preferably, the non-pathogenic microorganism was a bacterium, for example, selected from the group consisting of the genera Mycobacterium, Salmonella, Pseudomonas and Eschericia. Particularly preferably, the non-pathogenic microorganism was Mycobacterium bovis.
Incorporation of one or more copies of the nucleotide sequence that encodes a molecule in accordance with this invention, in the presence of strain M. bovis BCG will enhance the immunogenic effect of strain BCG. It is expected that the incorporation of more than one copy of a nucleotide sequence according to the invention will further enhance the immune response, and because of this aspect of the invention is a vaccine in which at least 2 copies of the nucleotide sequence of DNA that encodes a molecule that is incorporated into the genome of the microorganism, for example at least 5 copies. Copies of the nucleotide sequences of DNA can either be identical, with identical coding molecule, or may be variants of the same nucleotide sequence of DNA encoding identical or homologous peptides, or other prinia polypeptides, among which at least one polypeptide corresponds to this invention.
Live vaccines according to the invention can be prepared by culturing transformed non-pathogenic cells in accordance with the invention, and migration of these cells in the environment for a vaccine, and optionally - added carrier, excipient and/or substances that enhance immunogenicity.
In addition to their use as starting products for the synthesis of molecules according to the invention and hybridization of samples (useful for analyses of direct hybridization or as a seed, for example, PCR or other methods of amplification molecules) fragments of the nucleic acids according to the invention can be used to effect in vivo on the expression of antigens, i.e. fragments of the nucleic acids can be used in so-called DNA vaccines. Recent studies have shown that the DNA fragment cloned in the vector, which is dereplication in eukaryotic cells can be injected into an animal (including a human being) by using, for example, intramuscular injection or percutaneous injection (so-called approach “gene guns”). DNA absorbs, for example, muscle cells, and representing interrogate gene stimulates the immune system. An overview of these newly discovered methods given in the work of Ulmer et al., 1993, which is incorporated in this description by reference.
The effectiveness of such “DNA vaccines”, apparently, can be improved by introduction of the gene encoding the expression product, together with a DNA fragment coding for the polypeptide, which is capable of modulating the immune response. For example, the gene encoding the precursor of lymphokines or lymphokines (e.g., IFN-IL-2 or IL-12), can be introduced together with the gene coding for immunogenic protein, or by introducing two separate DNA fragments, either by introducing both DNA fragments enclosed in the same vector.
Vaccines can be used for prevention/treatment of any of the diseases described above, the pathophysiology of which is characterized by the secretion of TNFespecially chronic inflammatory diseases. As examples can be mentioned rheumatoid arthritis and inflammatory bowel disease (IBD). The latter include ulcerative colitis and Crohn's disease, particularly colitis Crohn's. Other examples are cancer, cachexia, often associated with cancer, multiple sclerosis, diabetes, psoriasis, osteoporosis and sifirirati histologically malignant epithelial tumors, including carcinoma and adenocarcinoma, and malignant non-epithelial tumors, including liposarcoma, fibrosarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, gliomas, neuroblastomas, Protocol, malignant melanoma, malignant meningiomas, various leukemia, various myeloproliferative diseases, various lymphomas (hodgkinska lymphoma and nahodkinskaja lymphoma), hemangiosarcoma, sarcoma Galoshes, lymphangiosarcoma, malignant teratoma, dysgerminoma, seminoma and horiokartsinoma.
Carcinoma and adenocarcinoma are the most numerous (approximately 90% of cancer deaths) and are therefore an interesting target for the treatment of diseases/prevention according to the invention. The most important carcinomas and adenocarcinomas are tumors of the respiratory tract (especially bronchial origin), chest, colorectal and stomach. However, carcinoma and adenocarcinoma prostate cancer, ovarian cancer, lymphoid tissue and bone marrow, uterus, pancreas, esophagus, bladder and kidneys cause significant numbers of deaths and therefore also of interest.
Preferably the vaccine will assign and and recurrence they can also enter patients diagnosed with one or more of the above diseases, and they can serve to maintain the patient in remission.
Based on previously conducted studies in mice it is believed that modified analogues of TNFaccording to the invention can also be entered as part of therapeutic effects on the above-mentioned diseases in the acute stage, or at least with the aim to put the patient into remission and maintain in a stable condition.
At the present time cannot be determined a specific range of effective doses, because vaccines have not yet been tested on humans, susceptible to any diseases.
In any case, enter the dose will be prescribed by the responsible physician.
In accordance with one example embodiment of the invention, the vaccine contains a mixture of two differently modified molecules TNFcontaining various epitopes for T-cells, such as P2 and P30, which are derived from tetanus toxoid. This mixture on the selection contains the appropriate amount of pharmaceutically acceptable substances that enhance immunogenicity.
Under the man as such, but the preferred construction, including non-communicable, reintegrating DNA sequence encoding the above molecule operatively associated with a promoter sequence, which may regulate the expression of the above sequence of DNA nucleotides in humans, sufficient to allow the capture of the above construction, and was a sufficient expression to induce the reaction of neutralizing antibodies against TNF.
The usefulness of this type of vaccines, called DNA vaccines, illustrated, for example, in U.S. patent No. 5589466 and 5580859, which are both included in this description by reference, in particular, concerning methods of introduction.
DNA vaccines can include viral expression vector such as a retroviral expression vector.
In most cases, the vaccine according to the invention can be adapted for oral or parenteral, including subcutaneous, intramuscular or intradermal injection.
The invention further includes the use of antibodies, the concentration of which is increased due to the introduction of the vaccine according to the invention, preferably monoclonality human body in the presence of TNFthat includes the contact of the composition containing the modified TNFaccording to the invention with a sample fluid of the human body and the contact if the above antibodies to TNFin the above sample.
The invention also relates to a method of diagnosis involving TNFdiseases, using an immunological assay in vitro for the detection of TNFin the fluids of the human body.
The above methods can include the use of samples of sandwich, enzyme linked immunosorbent assay or equivalent tests that can be reamplification or amplificatoare, for example, using the avidin/bitenova technology.
Vaccine/recombinant proteins can be characterized using the following tests, which are well known to specialists in this field of technology:
SDS-Page gels, colored Kumasi or silver (information about the size and purity),
IEF (isoelectric focusing) (isoelectric point),
Analysis of endotoxin LAL (purity),
Protein cells of the recipient (information about chistoe profile UV detection (information on the distribution of molecular weight),
N-terminal sequence (identity),
Circular dichroism (tertiary structure),
SE-liquid chromatography high pressure detection malls (information about the tertiary structure by means of light scattering),
Amino acid composition (identity),
The immunogenicity of heterologous species (mouse, rat or rabbit),
Reactivity of antibody in Western-blot,
Full definition of amino acid sequence.
Information confirming the possibility of carrying out the invention
In a previous patent application WO 95/05849 it was shown that the epitopes for T-cells modified molecules TNFrodents can create high titers of antibodies cross-reactive with native (wild-type) TNFof rodents. These antibodies were able to prevent the interaction of TNFand its receptors in vitro and in vivo. Positive effects of immunization against TNFbeen demonstrated in several series with animal models sabitri and experimental allergic encephalomyelitis (EAE).
These results of the experiments on animals were obtained, despite the fact that the two used the modified molecule TNFmice (named MR 103 and MR 106) were not optimized in terms of immunogenicity for the laser class II haplotypes of mice DBA/1 and SJL. These mice were used for modeling of collagen arthritis and experiments with AAA respectively. In another experiment it was shown that in various ways the modified molecule TNFrodents (MR 105) caused a stronger immune response than recombinant TNFmice conjugated with proteins in E. coli using formaldehyde.
MR 103, MR 105 and MR 106 molecules were mice, and on the basis of the previous patent application WO 95/05849 it impossible to draw specific conclusions nor the relative potency for the relevant T cells modified TNFman, nor about the potential ability of these molecules to induce the formation of neutralizing TNFantibodies, as they were all active.
2. Development of designs, TNFperson
ecoli TNFthe person must meet the following requirements:
A. They must be immunogenic for a large proportion of the population.
B. They must have the best ability to induce the formation of neutralizing TNFthe antibodies.
C. They shall not retain any residual biological activity of TNF.
Moreover, the selection process can be taken into account other practical options, such as recombinant expression, ease of cleaning, solubility, etc.
2.1. Immunological heterogeneity of modified molecules TNF
During the development of vaccines against TNFman's purpose was to obtain modified molecules TNFthe person who will ultimately be immunogenic for the greatest possible part of the population of people, which certainly represents a large number of different types of HLA (CRC) class II. So instead of CRC-specific epitopes used in the previous experiments on animals were used heterogeneous epitopes for T-cells. From the earlier application WO 95/05849 was unknown, such as the epitope for T cells, P2 and P30, derived from tetanus toxoid (SA), which have been well characterized in the scientific literature. It is known that these epitopes in SA, immunodominant and capable of binding at least 80% of the molecules in the laser class II in the human population.
Moreover, when using these self-epitopes expected that it will be possible to test the immunogenicity of TNF-structures in vitro mononuclear cells of peripheral blood (MCPC) and the lines T-cells obtained from immunized SA blood donors.
Amino acid sequence of the epitope P2 looks like QYIKANSKFIGITEL, and corresponds to amino acids SA 830-844, and the sequence P30 epitope looks like FNNFTVSFWLRWPKVSASHLE, and corresponds to amino acids SA 947-967. Replacement for P2 and P30 in two different molecules TNFwill change about 10% and 15% of native sequence TNFrespectively. If both epitope embedded in one molecule TNFwill be changed about 25% of the molecule, and it can be expected that this will have too strong an adverse impact on the remaining native part of the molecule TNFwill also contribute to the immunogenicity of the resulting design will be immunogenic in almost 100% of the population.
Although it was possible to induce the formation of antibodies of all structures TNFmouse in all mouse strains tested so far (see the discussion of MR 103, 105 and 106 above), a priori one might expect that embedding alien epitope for T cells in certain provisions in TNFwill be more useful than in the other position, from the point of view of the representation of the epitope to T cells molecules AGR II class. It was therefore decided to get a set of differently modified molecules TNFman with P2 and P30-epitopes embedded in the molecule in different positions, see Fig.4. Subsequently, all the molecules were tested in vitro analyses on the basis of mononuclear cells in peripheral blood (MCPC) or P2/R30-specific the UNL been shown although there were some minor quantitative differences, the intramolecular position P2 and P30 epitopes was irrelevant to the ability to processed antigen presenting cells and subsequently be presented to the CA-specific T-cells, P2, built-in provisions 132-146, 65-79 and 76-90, and P30, built-in provisions 40-60 and 132-152 (TNF 2-5, 2-3, 2-7, 30-2, 30-5), have been processed and presented to T cells. Thus, based on above discussion, it is very likely that these molecules will eventually be universal immunogenic in the human population.
2.2. The ability of modified molecules TNFperson to induce the formation of neutralizing antibodies
As mentioned above, earlier studies in mice, described in WO 95/05849, it was impossible to predict what the situation will be most appropriate in order to ensure the ability to induce the formation of neutralizing TNFantibodies, as all three analogue 103 MR, MR 105 and MR 106 were able to induce the formation of antibodies, despite the different zones of substitution. Therefore, we created a set of different molecules TNFman with P2 or RLA, formed in rabbits after injection of these molecules subsequently tested in biochemical and biological assays in vitro for their ability to disrupt the biological activity of TNF.
In an unpublished observations of the authors of the present invention has been shown that depending on the intramolecular position of the embedded epitope was observed, overall specificity of the resulting antibodies. Contrary to what was expected based on structural information about the molecule TNFwas observed that the substitution made in the frontlayer using either P2 or P30, completely deprived of molecules of biological activity of TNFbut at the same time retained the ability of modified molecules to induce the formation of neutralizing TNFthe antibodies.
It was shown that molecules containing P2 or P30 in the provisions referred to above, is particularly effective in inducing the formation of neutralizing antibodies. Any of these molecules are therefore potential candidates for use in vaccines against TNF. Therefore, the modified molecule TNFmust be non-toxic, that is, devoid of any kind of residual activity of TNF.
All mutant TNFproteins were therefore tested in vitro TNFdependent bioassays, as well as samples for binding to receptors, to check whether they are non-toxic. It was clearly shown that all modified molecule TNFman (TNF 2-5, 2-3, 2-7, 30-2 and 30-5) were deprived of the biological activity of TNF. So we were satisfied all of the requirements of these molecules is necessary for them to be part of the universal, non-toxic vaccine capable of inducing the formation of antibodies that neutralize TNFperson.
Work on the creation of genetic constructs
It was decided to get 10 different modified molecules TNFman - five, containing P2, and five containing P30 epitope. Epitopes were races is slichnih standard restricyouth enzymes and methods of mutagenesis by PCR. Genetic structure schematically depicted in Fig.4A and 4b. Nucleotide sequence of the DNA encoding the modified molecule TNFand the corresponding amino acid sequence is included as SEQ ID NO:1 - SEQ ID NO:20. Construction of mutant gene FNO-5, the strategy of cloning and mutation and subsequent expression, isolation and purification of similar FNO-5 is explained below with an example.
Designing and obtaining FNO-5:
Genetic engineering of mutant gene FNO-5 people, the strategy of cloning and mutation
Genetic engineering gene encoding a mutant similar FNO-5 man, was based on traditional methods of mutagenesis by PCR, as with all other genetic constructions.
Native DNA sequence TNFhuman encoding a soluble part of the molecule has been obtained by traditional PCR cloning using synthetically synthesized primers I and II (table 1 and SEQ ID NO:21 and 22) from commercially available libraries of human cDNA, CLONTECH Laboratories, USA (Fig.16, 1). Native gene was integrated into a commercial expression vector E. coli pET28c, which can be purchased in the company Novagen, USA, so arowana mutant similar TNFFNO-5 was performed using the technique of PCR-mutagenesis applied to native DNA sequence. The nucleotide sequence of the nucleic acid encoding the epitope for T cells, was included in artificially synthesized 75-dimensional oligonucleotide (Primer “mut2-5”, table 1 and SEQ ID NO:27) between two 3and 5-annealed segments of homologous TNF DNA, so the “mut”-primer can anneal to the sequence of the native gene TNFperson in a specific site selected for FNO-5 (see Fig.16, 2A). In “mut”-oligonucleotide number of codons that encode the epitope for T cells, exactly corresponds to the number of TNF-codons, missed between the two 3and 5-annealed segments of homologous TNF DNA. Primer mutagenesis was used to obtain a PCR product containing the DNA encoding the epitope for T cells and the sequence of the TNFbelow (or 3the built-epitope (Fig.16, 2A).
The amount of TNFDNA above (or 5) the point of introduction of epitope was the KTA PCR eventually joined together in a final PCR reaction (Fig.16, 3) using the two most distal primers (I, II) of the two reactions. Full mutant DNA sequence FNO-5 then introduced into the commercial expression vector of E. coli by analogy with the expression of the cloned native gene so that the gene can be transcribing from IPTG inducible promoter from the transformed cells.
“ut”-primers used for constructing other analogues (TNF 2-1, 2-3, 2-4, 2-7, 30-1, 30-2, 30-3, 30-4 and 30-5), identified as SEQ ID nos:23-26, and 28-33, respectively.
Cultivation of recombinant bacteria, accumulation and dissolution inclusionin phone
The protein purification FNO-5 analog
Getting protein FNO-5 was similar to receiving other recombinant molecules TNF
1. To inoculate 20 ml of the TV medium containing 50 μg/ml of Carbenicillin, transformed line E. coli, carrier induced IPTG plasmid vector containing the gene TNF alpha recombinant protein to grow E. coli overnight at 37°C with shaking.
2. Dilute the overnight culture at a ratio of 1:25 in a 250 ml TV environment with 50 μg/ml Carbenicillin and grown culture until OD450will not be equal to 1. To induce the expression of rekombinantnyi at 37°C.
3. To assemble recombinant cells from the medium by centrifugation at 3500×g. Wash the pellet once in BSB buffer. Use 150 ml BSB 50 g wet weight of bacteria.
4. Sonicate 4 times for 30 seconds at maximum amplitude up until the bacterial suspension will not be completely homogeneous. The sonication is performed using MSE Soniprep 150 ultrasonic transducer equipped with a standard head size 9.5 mm (Soniprep 05 38 121-1154).
5. Add 8 ál PMSF (50 mm) and 80 μl of a solution of lysozyme (10 mg/ml) per gram of bacterial mass. Incubate for 30 min at room temperature.
6. Add 4 mg of deoxycholic acid per gram of bacterial mass, mix, and store at 37°C.
7. When the solution becomes viscous, add 20 ál of Gnkazy (1 mg/ml) per gram of bacterial mass and gl to a final concentration of 5 mm, mix and store at room temperature for 30 minutes
8. Sonicate in ice 5 times for 30 seconds with 30-second intervals at the maximum amplitude until the solution is liquid and non-viscous.
9. Centrifuged at 20×g for one hour to save the supernatant for later control procedures prominant in MiliQ water (1 ml H2O per gram E. coli), shake for 1 hour.
11. Centrifuged at 20×g for one hour to save the supernatant to check whether all inclusione bodies were precipitiously.
12. Re suspensionthe pellet in 1 M urea solution by adding 1 ml per gram E. coli, shake for 1 hour.
13. Centrifuged at 20×g for 1 hour, to save the supernatant to check whether all inclusione bodies were precipitiously.
14. Re suspensionthe pellet in 1 M guanidine, adding 1 ml per gram E. coli, shake for 1 hour.
15. Centrifuged at 20×g for 1 hour, to save the supernatant to check whether all inclusione bodies were precipitiously.
16. Re suspensionthe pellet in 25 ml of 6 M guanidine+20 mm Tris with pH 8.0 and stirred overnight.
17. Centrifuged at 20×g for 1 hour, to save the supernatant, containing inclusione body of recombinant protein, to keep the pellet to check whether all inclusione bodies were dissolved.
18. The protein solution is subjected to intensive dialysis against water MiiliQ, and then the solution is dried at a temperature below 0°C.
19. Dried at a temperature below 0°C, the material is dissolved astartica at night. The presence of monomers explore on a column of superdex 2000 (HC 16, Pharmacia, diameter: 1.6 cm, height: 750 cm). Mileage in the current buffer at 1 ml/min. to Compare with the standards in the same buffer.
20. The protein purification carried out by means of gel filtration on a column of superdex 200 (HC 26, Pharmacia, height: 100 cm, diameter: 2.6 cm) which is equilibrated with the equilibrium buffer. Mileage in equilibrating buffer. Use volume of sample, approximately 1% of the total volume of the column.
21. Re-folding of the recombinant protein is performed by dialysis. Protein is dissolved in a concentration of 0.1 mg/ml in equilibrating buffer, and the solution placed in boiled bag for dialysis and deleteroute against 20 mm Tris, 4 M urea (pH 8.5), with three changes of solution over night at room temperature. The dialysis bag is transferred into Tris buffer (20 mm Tris, 150 mm NaCl (pH 8.0)). To change the solution three times, of which the first change occurs at room temperature. During the night in a cold room.
Re-folding estimate column Superrose 12, equilibrated with Tris buffer (20 mm Tris, 150 mm NaCl (pH 8.0)). To compare with the standards.
The storage. Recombinant proteins stored in dried at a temperature below 0°With the condition.
Large-scale production is
500 liters of fermented culture of E. coli, diafiltration with water to a volume of about 50 liters.
1) Wash cells
A) to Thaw the frozen cells to a liquid state.
B) Centrifuge the cells for 10 minutes at 4000×g.
C) Re-resuspension the pellet in 50 mm Tris, 150 mm NaCl, pH 8.0. Repeating stages b) and C) three times.
2) Homogenization of cells
Homogenize cells by Rannie homogenizer, 5 cycles at 700 bar.
B) Centrifuged inclusione body for 30 minutes at 16.500g.
B) Wash inclusione body three times with a solution containing 3 M guanidine, 1 M NaCl, 5 mm EDTA, 20% sucrose, 50 mm Tris, pH 8. Centrifuged inclusione body for 30 minutes at 16,500g.
3) Dissolution inclusionin tel
To resuspension the pellet in 6 M guanidine, 10 mm DTT, 150 mm NaCl, 5% ethanol, 50 mm Tris, pH 8 to Use about 1 ml buffer/100 mg granules.
4) Ultrafiltration inclusionin tel
Remove coarse dirt using a 0.45 µm filter.
B) to Remove high molecular weight components by the filter 30 KD.
B) Concentrating inclusione body filter 5 KD.
5) the Replacement buffer
Prepare about the Sabbath.
6) Purification using SP-Sepharose
Download protein a column of SP-Sepharose. Wash the column with four volumes of A-buffer, eluted protein 100% B-buffer and unite all factions with TNF.
A-buffer: 6 M urea, 1 mm DTT, 50 mm Tris/Cl, pH 8.
B-buffer: 6 M urea, 1 M NaCl, 1 mm DTT, 50 mm Tris/Cl, pH 8.
7) Re-folding TNFperson
Dilute the protein pool to a concentration of about 0.1 mg TNF/ml in 6 M urea, 1 mm DTT, 50 mm NaCl, 5% ethanol in 20 mm Tris/Cl, pH 8.8, and gradually remove urea, moving to the next part of the buffer - by diafiltration.
A) 2 M urea, 1 mm DTT, 150 mm NaCl in 20 mm Tris/Cl, pH 8.8 - in for the night at 5°C.
B) 1 M urea, 1 mm DTT, 150 mm NaCl in 20 mm Tris/Cl, pH 8.8 - for 8 hours at 5°C.
B) 1 mm DTT, 150 mm NaCl in 20 mm Tris/Cl, pH 8.8 - in for the night at 5°C.
G) 150 mm NaCl in 20 mm Tris/Cl, pH 8.8 - in for the night at 5°C.
8) Storage analogues TNFperson
To concentrate the protein to 1.0 mg/ml and store the sample at -20°C.
Dissolve the broth Terrific Broth (GIBCO BRL 22711-22) in MilliQ water in accordance with manufacturer's instructions. Autoclaved at 121°C for 20 Murali in MilliQ water at a concentration of 50 mg/ml The solution was sterilized by filtration through a 0.2 μm filter (Sterifix 0409 9206).
IPTG x 100 mother solution 100 mm
Isopropyl-beta-D-thiogalactopyranoside (IPTG, USB 17884). 1.19 g IPTG was dissolved in MilliQ water to 50 ml of the Solution was sterilized by filtration through a 0.2 μm filter (Sterifix 0409 9206).
Buffer for Bacterial Suspensions
50 mm TRIS (Trisma base, SigmaT1503)
0,5 M NaCl (Ridel-de.Haen 31434)
5 mm DTT (DL-dithiothreitol, Sigma D-0632)
50 mm, phenylmethylsulfonyl, SIGMA # P-7626, dissolved in 2-propanol.
A solution of lysozyme
10 mg/ml Quality III of lysozyme protein chicken eggs, (EC 22.214.171.124) SIGMA # L-7001
(7-desoxycholic acid) Sigma # D-6750
1 mg/ml Gnkazy I, deoxyribonuclease I (EC. 126.96.36.199) Boehringer Cat # 1284932
Urea (GibcoBRL 15716-020)
Guanidine hydrochloride (Sigma G4505)
20 mm TRIS, (Trisma base, SigmaT1503)
8 M urea (GibcoBRL 15716-020)
20 mm TRIS, (Trisma base, SigmaT1503)
8 M urea (GibcoBRL 15716-020)
Expression, purification and re-folding the P2 and P30 modifitsirovannom behave during expression, cleaning and re-folding. All the proteins expressed in E. coli, and were received levels, warioware from 2 to 20%. All proteins were detected in experiments with Western blotting using commercial polyclonal rabbit antibodies to TNFperson.
Design TNFsubsequently expressed separately in 250 ml culture in batches with a volume of 3-4 HP All modified TNFproteins were expressed as inclusion bodies, and protein preparations with a purity of more than 85% and re-folded were obtained as described above.
The protein content was determined using standard ICA analysis. All protein purification were performed at least three separate runs for each molecule, and in all cases were tested by separate parties protein and found similarities. Proteins were stored at concentrations of 0.2-1.0 mg/ml in PBS at -20°C to use.
Standard tests for quality control included SDS gel electrophoresis with staining of individual protein preparations as Kumasi blue and silver. In all cases it was found that proteins have the expected size and osetrovani only with relatively low levels (approximately 2%). Moreover, these molecules were very difficult to clean, and especially difficult was re-folding. Both molecules, especially FNO-4, were not very soluble in PBS and tended to precipitate during storage.
Biological activity of different TNFstructures
Direct biological activity of purified molecules TNFwas tested bioanalysis L929, which is the standard in vitro assays to determine biological activity of TNF. As can be seen from Fig.6, none of the P2 or P30 modified molecules TNFwas not able to kill the L929 cells in the investigated concentration range (up to 60 mg/ml). Commercially available recombinant molecule TNFmaintained full activity at a concentration of about 0.6 ng/ml of the Drug TNFwild-type was also fully active in the whole studied concentration range.
The ability of modified molecules TNFto induce the formation of neutralizing antibodies
Rabbits were immunized with each of th biologically active TNFhuman in vitro. Were used in groups of three rabbits, and each rabbit received 100 mg TNFsubcutaneously in complete Freund composition of Freunds Complete Adjuvant and subsequently at 100 mg approximately every third week in incomplete Freund composition Freunds Incomplete Adjuvant.
During the whole period of immunization, is 18 weeks, at regular intervals, took blood samples and serum were investigated for anti-TNFthe activity of traditional ELISA (ELISA analysis), where the antigen was used a commercially available, pure and unmodified TNFperson.
All rabbits during the period of immunization formed antibodies to TNFand the maximum level or the antibody titers varied decimal places in each group. The average antibody titers are shown in Fig.7. FNO-5, FNO-7, PNO-3, PNO-1 and TNF wild-type induced antibody titer equal to one hundred, within 2-4 weeks, whereas FNO-4 gave significantly slower reaction. Similar kinetics were observed for proteins FNO, where a slower response was also obtained for FNO-4.
The ability of these Siva is on in L929 analysis as well as in the analysis of solid-phase receptor binding.
In the analysis of L929 divorced immunne serum and non-immune control serum was added to commercially available TNFperson before adding a mixture of L929 cells. Serum from all three rabbits in each group were tested twice and the expected average values. Since normal serum showed a tendency to increase the source levels svetootrazauschii system, they are subtracted from all values, and expected relative inhibition in percent. The results of the inhibitory capacity of 14 week program of immunization in all rabbits shown in Fig.8.
FNO-5, which did not contain substitutions in any of the segments in the rearlayer, is clearly superior to other designs, and this molecule was fully comparable with toxic molecule TNFwild type in terms of their ability to induce the formation of neutralizing antibodies (data not shown). FNO-3, which contains a small amount of the substituted segment-circuit D, and FNO-7, which does not contain any of the replaced segments in chains back-slotover has increased over the next three weeks (data not shown). FNO-4 and PNO-1, which contained G and b-chain, respectively, in the rearlayer, were unable to induce the formation of neutralizing antibodies in the analysis of L929.
The results concerning FNO proteins were more heterogeneous, although FNO-3 seemed to be the best 14 week. FNO-1 and PNA-4, which contain substitution in G and-circuits the rear of the p-layer, respectively, did not induce the formation of neutralizing antibodies in the analysis of L929.
In the analysis of solid-phase receptor binding of recombinant human TNF-1 receptor with a molecular mass of 55 kDa (TNF-R55) was immobilized on the plates for micrometrology, and commercially available biomineralogy TNFman was added at the appropriate dilution. He then determined the specific binding to receptor using streptavidin, horseradish peroxidase and a chromogenic substrate. When testing sera from immunized rabbits was added to the solution biotinylating TNFperson before adding the mixture on a plate for micrometrology covered TNF-R55. Investigated sera from all titveli as a negative control. The original values were very low, and the analysis was very sensitive. The results are shown in Fig.9.
You can see that the results obtained in L929 analysis and analysis of solid-phase receptor binding, respectively, are almost identical in relation to structures TNF2. In solid-phase analysis of the difference between PNO-2, 30-3 30-5 and was not as pronounced as in the analysis of L929. Solid-phase analysis, however, is more reproducible because of its biochemical rather than cellular character, and values for normal serum in this analysis was not subtracted.
The relative amounts of serum (in percent), which received half of the maximum level of inhibition of binding TNFvalues (IC50), were calculated for PNO-3, FNO-5, FNO-7, PNO-2, PNO-3, FNO-5 and TNFwild-type for each corresponding antisera. Assuming that a similar curve is obtained for antisera against FNO-1, FNO-4, PNO-1 and PNA-4, were performed extrapolation, and the values of the IC50were also calculated for these sera. The results are shown in table 2.
mouse in rabbits. FNO-1, FNO-4, PNO-1 and PNA-4, which all contain substitutions in the B - and G-chains in the rear-layer, very little is capable or not capable of inducing the formation of such antibodies. It suddenly shows that although the chain and G In the rear-layers are not involved in binding with the receptor, mutations in this region somehow cause damage to areas of TNFperson involved in the binding with the receptor FNO-2 and PNO-3, apparently equally well induce the formation of neutralizing antibodies.
The ability of modified molecules TNFto stimulate T cells immunized P2 and P30 healthy blood donors
As expected, the localization of P2 and P30 epitopes in the modified molecules TNFwill also affect the antigen processing and presentation built epitopes and consequently on the immunogenicity of all 10 molecules were tested in different assays with T cells. Was used polyclonal Molecular Analysis of Peripheral Blood Cells (RVMS), and the number of P2 and P30-specific line T-gif" border="0">peptides and recombinant proteins with built-P2 and P30-epitopes.
Initially, 28 healthy volunteers (donors) were tested in normal proliferation RVMS analysis on their ability to respond to SA and peptides P2 and P30. Based on these results 19 persons, able to reliably respond to SA, P2 and P30 peptides were selected for further experiments. Although the levels of reactions varied a lot, it is clearly confirmed that the P2 and P30 are different and the immunodominant epitopes for T-cells.
In addition to the 28 donors were further selected 7 donors. For the reasons explained below, some of them were selected for their ability to react only on P2 or P30. Some of them were also vaccinated CA, so that they were able to give a strong response of T cells. Some of the second group of blood donors were used for the accumulation of P2 or P30 - specific line T cells to investigate the representation of P2 and P30 modified synthetic TNFpeptides and modified molecules as antigens. In Fig.10 shows representative examples of polyclonal proliferative RVMS reaction in three donors in SA, as well as peptides P2 and P30.
is from about 100 mg/ml, and all RVMS experiments were repeated 2-3 times with each individual. Intracellular inclusion of3H-labeled thymidine was used to assess the proliferation of T-cells, and the experimental results have been received and counted in a 96-cell format. The maximum proliferation indexes (PI) of each tinational curve were calculated as the ratio of the average number of CPM in experimental cells to the average number of CPM obtained for cells without antigen (only PBS). Data on the proliferation of T-cells were repeated three times, a Con A, and P2 and P30 were used as negative and positive controls, respectively.
In Fig.11 shows three examples of experiments with two different blood donors using different P2 and P30-modified molecules TNF.
JH reacts only to P2, whereas SR reacts as P2 and P30. This is also reflected in the proliferative responses to P2 and P30-modified TNFproteins, which in varying degrees all were able to stimulate RUMS.
In Fig.12 shows the Index of Proliferation (PI), calculated on the 34 experiments. Although it is very difficult to quantitatively compare PIS from different exp is equally able to induce a proliferative response.
Among the second group of donors, some individuals were vaccinated SA 1-2 months prior to the experiments. All the PIS obtained from these individuals were among the highest values of FE observed for all modified TNFstructures, and the results were easy to evaluate. Three other individual (DL, ID and LS) were vaccinated more than 5 years ago, and all they gave levels of FE below average. This confirms that the received IP was antigen-specific. For the first group of donors, there was no data regarding the date of the last vaccination, SA, and their immunization status was clearly very different.
It is impossible to choose the preferred TNF2 or TNF30 protein based only on the average values of FE. It can be caused by various state vaccination investigated individuals and different nature RWMS-analyses, obtained from individuals with different status reactivity. In any case, it was surprising that the P2 and P30, built in TNFany provisions, is likely to be processed and presented to T cells. To exclude the possibility that, despite repeat the performance communications concentration of non-specific mitogens, were performed further experiments.
Donors from the second group, about which it was known that they do not respond to P2 and respond to P30, and donors with the opposite pattern of response were tested in RWMS-analyses. In Fig. 13 response to P2, P30 and proteins TNF2 and TNF30 shows for DL and ID.
You can see that the specific responses were received to these epitopes to T cells and, accordingly, the modified molecule TNF. However, there was a significant proliferative responses in DL protein TNF30 (upper band) and reliable proliferative reactions in ID proteins TNF2 (lower band), which confirms that purified preparations of TNFno nonspecific mitogens.
This possibility was investigated further using the P2 and P30-specific line T cells isolated from the second group of donors, which were cultured for at least six weeks with at least three rounds of stimulation corresponding synthetic P2 and P30 peptides. In Fig.14 shows the results of two tkowski P2 and P30 proteins. Moreover, it can again be seen that all TNFconstructs were able to induce proliferation of T-cells. This indicates that although the processing of the antigen can be quantitatively important to the portrayal of P2 and P30, she does not seem significant qualitative limiting factor for the presentation of antigen.
In the literature it was reported that flanking region epitopes for T-cells can affect the binding of antigenic peptides with MHC molecules of class II. Since none of the provisions in TNFthat were selected for embedding P2 or P30, apparently, is not prohibiting the introduction of antigen, it was investigated whether different flank in relation to the built epitopes sequence TNFto influence the response of T cells at P2 and P30 epitopes as a result of different binding molecules HLA class II human. Therefore, we synthesized peptides shown in table 3, which are built epitope and flanking amino acids TNFman. They are marked as PP2-5, RR-3 and so on Amino acid sequence shown in the list of sequences ka is>/img>
These peptides were used to stimulate the P2 and P30-specific line T cells. Results stimulation R30-lines shown in Fig.15. You can see that P2-specific line T cells from the MR and KG reveal parallel patterns of stimulation, being stimulated or peptide, or the corresponding TNF-protein. It is clear that FNO-5 is a good potential antigen, and these additional data confirm that the observed proliferation of T cells is antigen-specific. In General, no qualitative differences in the pattern of stimulation, if R30-specific line T cells from the MR and KG stimulated by peptides, or proteins (data not shown). R30-specific line T cells from NA preferably recognize FNO-3 and to a lesser extent reacted with FNA-2.
It was created and described ten different modified proteins TNFman. They were designed to include two well-known variety of P2 and P30 epitopes for T-cells to be potentially immunogenic at least 85% of the population.
All proteins can be Express and clear, although FNO-4 and FNO-4 with low uravneniya, resulting in proteins with low solubility. None of the modified molecule TNFit was impossible to detect the biological activity of TNF.
Rabbits were immunized all ten proteins and native TNF. 2-3 months after immunization in all sera were possible to detect high titers strongly cross-reactive antibodies to remotefilename TNFperson.
The ability of these antibodies to disrupt the biological activity of native TNFwas investigated in two different In vitro biological sample L929 and solid-phase binding with the receptor. Both analyses showed broadly the same. FNO-1, FNO-4, PNO-1 and PNA-4 were not able to induce the formation of a considerable amount of neutralizing antibodies, whereas FNO-5 was superior to all other designs. FNO-2 and PNO-3 were approximately equally effective in inducing the formation of neutralizing antibodies and about two times better FNO-5, which was good enough.
Somewhat surprisingly, it was not possible to detect reliable difference which was due to the presence of mitogens in the preparations of antigens, and on the basis of these data it was concluded that all the provisions selected for P2 and P30, allow the representation of the corresponding epitopes. The specificity of the reaction was further documented when using test modified TNFproteins using epitope-specific line T cells, and synthetic peptides containing embedded epitopes, as well as flanking sequence TNF. In these experiments, it was clearly demonstrated that FNO-5, PNO-2 and PNO-3 (among other structures) were the strongest potential immunogenum.
1. Modified molecule TNFman, is able to induce the formation of neutralizing antibodies to human TNFwild-type after the introduction of the above-mentioned modified molecule TNFto a human recipient, in which at least one peptide fragment of the molecule TNFman has been replaced by at least one peptide, which is known that it contains the immunodominant epitope for T cells, or a truncated form specified and modified molecules TNFcontaining the immunodominant epitope for T cells and one or both flanking region of the molecule f is">for b cells, thus replacing implemented in any of the circuits of the front-layer in any one of the connecting loops and/or in any of the, I or D-chains in the rearlayer, and this substitution leads to iactiveaware biological activity of TNFperson.
2. Modified molecule TNFthe person under item 1, characterized in that when tested in bioanalysis L929 it largely devoid of TNFactivity, and specified a modified molecule TNFable to induce the correct recipient antibodies to the modified molecule TNFthat significantly inhibit the activity of native TNFin the analysis of L929 and/or significantly inhibit the binding of TNFhuman wild-type TNF-receptor 1 (TNF-R55) with a molecular mass of 55 KD or TNF-receptor (TNF-R75) with molecular weight of 75 KD.
3. Modified molecule TNF
4. Modified molecule TNFman according to any one of paragraphs.1-3, characterized in that the modification done in the areas of molecules TNFthat include chain frontlayer and/or the connecting loops, so that a high degree to maintain the structure oflayer any of the circuits in the rearlayer.
5. Modified molecule TNFman according to any one of paragraphs.1-3, characterized in that the modification done in the areas of molecules TNFthat include the segment D-chain rearlayer.
6. Modified molecule TNFman according to any one of paragraphs.1-3, wherein the replacing includes at least a segment of the H chain frontlayer and connecting the loop to the I-chain, preferably amino acids 132 through 146.
7. Modified molecule TNFman according to any one of paragraphs.1-3, characterized in that the replacement on the automated molecule TNFman according to any one of paragraphs.1-3, characterized in that the replacement includes the segment D-chain, at least a segment of the E-chain and all the connecting loop, preferably amino acids 65 79 or 64 to 84.
9. Modified molecule TNFman according to any one of paragraphs.1-3, wherein the replacing includes fully With- and-chain and segment D-chain, preferably amino acids from 40 to 60.
10. Modified molecule TNFman according to any one of paragraphs.1-3, wherein the replacing includes at least a segment of the E-chain and one or both of the connecting loops frontlayer, preferably amino acids 76 to 90.
11. Modified molecule TNFman according to any one of paragraphs.1-3, characterized in that it has the amino acid sequence shown in SEQ ID NO:8.
12. Modified molecule TNFman according to any one of paragraphs.1-3, characterized in that it has the amino acid sequence shown in SEQ ID NO:10.
13. Modified molecule TNFman according to any one of paragraphs.1-3, characterized in that lekule TNFman according to any one of paragraphs.1-3, characterized in that it has the amino acid sequence shown in SEQ ID NO:20.
15. Modified molecule TNFman according to any one of paragraphs.1-3, characterized in that it has the amino acid sequence shown in SEQ ID NO:14.
16. Modified molecule TNFman according to any one of paragraphs.1-10, characterized in that the integrated epitope for T cells is heterogeneous and it is known that it is immunogenic for most types of human HLA class II.
17. Modified molecule TNFperson under item 16, wherein the epitope, preferably the epitope P2 and/or P30, derived from tetanus toxoid.
18. Modified molecule TNFman according to any one of paragraphs.1-17, characterized in that the indicated molecule is part of a fusion protein with adjuvant molecule, preferably immunologically-active adjuvant, such as GM-CSF, HSP70, or interleukin.
19. A fragment of a DNA molecule encoding a modified molecule TNFas defined in any of paragraphs.1-17, and having a nucleotide �tp://img.russianpatents.com/chr/945.gif" border="0">on PP.1-17.
20. Vector, which comprises the fragment of the DNA molecule as defined in paragraph 19.
21. The expression vector, which contains a fragment of the DNA molecule as defined in paragraph 19, operatively connected to the sequence controlling the expression.
22. The method of obtaining a modified molecule TNFperson, as defined in any of paragraphs.1-17, which comprises growing cells of the recipient, transformed by the expression vector as defined in paragraph (21, under suitable conditions, providing products of the modified TNFand the selection of the modified TNFthus obtained.
23. The method according to p. 22, characterized in that the specified recipient is selected from strains of bacteria, yeast or other fungi and cell lines insects, mammals or birds.
24. Vaccine against TNFincluding an effective immunogenic amount of one or more modified molecules TNFperson, as defined in any of paragraphs.1-18, and, optionally, a pharmaceutically acceptable adjuvant, such as aluminum phosphate, aluminum hydroxide, Fosfor promotes the secretion or activity of TNFsuch as chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, including Crohn's disease and ulcerative colitis, as well as cancer, multiple sclerosis, diabetes, psoriasis, osteoporosis and asthma.
26. Vaccine against TNFcomprising an effective amount of isolated DNA that encodes a modified molecule TNFperson, as defined in any of paragraphs.1-18 built into a suitable expression vector.
27. Vaccine for p. 26, wherein said DNA is operatively attached to a promoter sequence capable of controlling expression of the above DNA in humans, and is not integrable.
28. Vaccine for p. 26, characterized in that it contains the viral vector expression, such as a retroviral expression vector.
29. The vaccine according to any one of paragraphs.24-28 for oral or parenteral, such as subcutaneous, intramuscular or intradermal injection.
30. The way to test for the presence of TNFin a sample, comprising the following steps: bringing the antibody accumulated against the modified molecule TNFand obrazuyuscikh and determination, contact whether these antibodies to TNFin the above sample.
31. The method according to p. 30, where the antibodies are monoclonal antibodies.
32. The way to test body fluids of a person for the presence of TNFincluding the implementation of contacting the composition containing antibodies accumulated against the modified molecule TNFas defined in any of paragraphs.1-18, with a sample of body fluid of a person and the definition of contact if the above antibodies to TNFin the above sample.
33. The method according to p. 32, characterized in that it includes the use of samples of sandwich ELISA or equivalent analysis that can be reamplification or amplified, for example, with the use of avidin/bitenova technology.
34. A method for the diagnosis associated with TNFdiseases, including the implementation of immunological in vitro assays for detection of TNFin the body fluids of a person through the use of antibodies accumulated against the modified molecule TNFon any PP.1-18.
35. Ways is that it can be reamplification or amplified, for example, with the use of avidin/bitenova technology.
36. Method for the treatment or prevention of human disease, the pathophysiology of which is at least partially associated with the release or activity of TNFincludes introduction to the human an effective amount of at least one of the modified molecule TNFas defined in any of paragraphs.1-18, or vaccines, as defined in any of paragraphs.24-29, preferably in combination with a suitable adjuvant or carrier molecule.
37. Medication for the treatment or prevention of disease, the pathophysiology of which is at least partially associated with the release or activity of TNFcomprising as an active ingredient an effective amount of the modified molecule TNFas defined in any of paragraphs.1-18.