Respiratory syncytial virus (rsv) anti-g protein antibodies

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an isolated monoclonal antibody or its immunoreactive fragment binding to the respiratory syncytial virus (RSV) G protein epitope of the strain A2. There are also described a nucleic acid molecule coding it, a host cell containing this nucleic acid molecule, a method for producing the antibody and a pharmaceutical composition containing this antibody.

EFFECT: presented group of inventions can be used for treating respiratory syncytial virus.

14 cl, 37 dwg, 1 tbl, 9 ex

 

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the priority of provisional applications U.S. patent No. 61/000469, filed October 25, 2007, and No. 61/089401, filed August 15, 2008, the Contents of each of these documents are incorporated herein by reference.

The technical FIELD TO WHICH the INVENTION RELATES

The invention relates to antibodies that of immunoreactive in relation to functionally important epitope contained the G protein of respiratory syncytial virus (RSV), which minimally immunogenic when administered to humans. These antibodies can be used to increase the resistance of people to RSV infection, as well as to reduce the infectious load in infected individuals, or relieve symptoms caused by RSV infection.

The LEVEL of TECHNOLOGY

Infection with RSV is a long and dangerous problem in the world, including the United States of America, Europe, Australia and Japan. The special anxiety it causes in cases of premature infants, small children and the elderly and, of course, for all people with a weakened immune system. It is estimated that about two thirds of children under the age of 1 year and almost all children aged 1 to 4 years, at least once, were infected with RSV, with the majority of cases recovery occurs is without the need of medical intervention. However, 5-10% of the observed long-term severe infection, which is considered a factor that provokes the symptoms of breathlessness and asthma in later childhood. RSV has two major surface glycoproteins, F and G. the Only one on the market monoclonal antibody against RSV only been approved for prophylactic use in premature infants for the prevention of RSV infection, and it is directed against the F protein. This antibody, palivizumab (Synagis®from MedImmune), is widely applicable due to the conservative sequence of the F protein among strains. On the contrary, G-protein is quite variable, with the exception of the Central domain CX3C, which was almost unchanged at about 100 prosecutionand strains. This area includes the motif, which has been shown to interact with receptors of fractalkine. It is believed that this interaction promotes long-term course of the disease, typical for RSV, inhibiting effective immune response to this virus: Tripp, R. A.,et al.,Nature Immunology(2001) 2:732-738. It was also shown that this area is an antagonist of Toll-like receptor 4, which, I believe, contributes to the effective suppression of the immune response: Polack,et al.,Proc. Natl. Acad. Sci. USA(2005) 102:8996-9001; Shingai,et al.,Int'l Immunology(2008) epub July 8.

Initial attempts at prevention of RSV using WACC the nation turned out to be counterproductive. Vaccination inaktivirovannye in formalin RSV or G-RSV glycoprotein was accompanied by an increase in the severity of the disease and pulmonary eosinophilia, which is explained previously noted conservative sequence region G protein, designated CX3C, which simulates chemokinesis fractalkine. (Haynes, L. M.,et al.,J. Virol.(2003) 77:9831-9844.) Passive immunization using antibodies directed against G-protein, is generally considered impractical due to the lack of conservatism sequence of this protein among strains.

Subsequently, the same group confirmed that the responses of antibodies against G-protein caused by RSV infection or vaccination, consistent with the inhibition of binding of G-protein CX3C-fractalkine receptor and modulation of chemotaxis of leukocytes mediated by G-protein of RSV (Harcourt, J. L.,et al.,J. I. D.(2004) 190:1936-1940), and that inhibition of this binding affects T-cell responses (Harcourt, J. L.,et al.,J. Immunol.(2006) 176:1600-1608). More modern research on the development of vaccines to prevent disease exacerbation associated with fixed in formalin vaccine, but it was found that the immunity gained through more new vaccines, weakening (from weeks to several months), in accordance with low immunological memory in relation to natural the RSV: Yu, et al., J. Virol.(2008) 82:2350-2357. Repeated infections are typical for this virus, unlike many others. For this effect may be responsible immunosuppressive properties of a G protein.

Monoclonal antibodies directed against G-protein has been known for over 20 years. In the work of Anderson, L. J.,et al.,J. Virol.(1988) 62:4232-4238 described the ability of mixtures of monoclonal antibodies (mAb) against protein F and G, as well as individual mAb to neutralize RSV. Related to the binding of G-protein mAb, particularly 131-2G, were later studied Sullender, W.,Virol.(1995) 209:70-79 in antigenic analysis. It was found that this antibody is associated with RSV group a and group B, representing the main strains of RSV.

In addition, the work Mekseepralard, C.,et al.,J. Gen. Virol.(2006) 87:1267-1273 summarizes earlier article, showing that passively entered antibodies against F, and against G-protein protects against experimental infection models in rodents. These articles include Routledge,et al.,J. Gen. Virol.(1988) 69:293-303; Stott, E. J.,et al.,J. Virol.(1986) 60:607-613; Taylor, G.,et al.,Immunol.(1994) 52:137-142; and Walsh, E. E.,et al.,Infect. Immun.(1984) 43:756-758. In the original article Mekseepralard and others noted that a monoclonal antibody specific against G-protein (1C2), requires glycosylation for neutralization of the virus in the presence of complementin vitroor using thein vivothe mice. The author of the note, what amino acids 173-186 G-protein conservative and that 1C2 was directed against the conserved region; however, the method of obtaining non-immunogenic antibodies was relatively coarse, namely Fab mice "chimerically" on the Fc region of a human.

In addition, in Corbeil, S.,et al.,Vaccine(1996) 14:521-525 shown that the complement system is involved in the protection of mice in the control of the RSV stimulation after passive immunization with monoclonal antibody mouse 18A2B2, even if the antibody does not show neutralizing capacityin vitro.

The PCT publication WO 00/43040 describes the use of antibodies against substance P to alleviate airway inflammation associated with RSV infection. Production of substance P, a known Pro-inflammatory mediator, is increased through the introduction of the G-protein of RSV and absent in the mutant RSV, which are not G-protein or conservative Central region is suppressive function point mutation: Haynes,et al.,J. Virol.(2003) 77:9831-9844.

U.S. patent 2006/0018925 describes and claims antibodies and small peptides that can block the interaction region CX3C G protein with its receptor. These compositions are suggested as useful for modulation of RSV infection and stimulate the immune system. Although the proposed humanization of antibodies mice, demonstrating therapeutic value the quiet antibodies actually no humanized forms were not received or described.

The PCT publication WO2007/101441, assigned company Symphogen, aimed at obtaining recombinant polyclonal antibodies for the treatment of RSV infections. Polyclonal recombinant antibodies are composed of individual monoclonal antibodies isolated from human serum. In table 5 of this publication describes 12 monoclonal antibodies that are allegedly associated with "conservative" area 164 176 amino acids of the G-protein of RSV subtype A. Five of them were tested for affinity G-protein and the affinity was in the range of 100-500 PM. Two of these antibodies were tested for the ability to neutralize the reaction suppressing belascoaran (PRNT); one showed the value EC50about 2.5 μg/ml, and more generally showed no neutralizing properties.

Description of the INVENTION

Antibodies specifically immunoreactive with respect to the G-protein of RSV compared to the F-protein, including those of immunoreactive against strains of both groups a and B and have a high affinity to G-protein and a strong neutralizing ability, have been identified in people-donors, significantly shortly infected with RSV. In addition, antibody mouse against G-protein, originally described by Anderson,et al.,J. Viol. (1988) 62:4232-4238, was modified in such a way as to minimize the possibility of immunological rejection when administered to man. Antibodies according to this invention find use as therapeutic agents, as well as to increase the resistance to RSV in humans. In particular, antibodies to the conservative motif in positions 160-176 G-protein subtype A therapeutically effective for the elimination of the virus in subjects who are already infected, and to reduce the airway inflammation characteristic of RSV infection, and for prophylactic use.

Thus, in one aspect this invention is directed to monoclonal antibodies or immunoreactive fragments that bind an epitope within about provisions 160-176 G protein of A strain RSV and that are minimally immunogenic when administered to humans. These antibodies show neutralizing ability on standardized tests of belascoaran to neutralize RSV and demonstrate in these tests EC50making <500 ng/ml, preferably <200 ng/ml, more preferably <100 ng/ml of the Antibodies according to this invention also have the affinity to the G-protein of RSV-A2, components <1 nm, preferably <500 PM, more preferably <100 PM. Antibodies according to this invention, in one embodiment, svyazyvatsyas within 30 residues chemokine CX3C motif, contained in G-protein of RSV, or directly with him, at least part of it, in an area that has a high degree of amino acid identity among the many strains of RSV. Chemokinesis the CX3C motif is approximately the provisions of amino acids 182-186 strain of RSV-A2 and in the relevant provisions of G-protein other strains. It was found that the corresponding area within which the bound antibodies according to this invention, is enclosed within residues 160-176 G-protein of RSV-A2 and related provisions G-protein other strains. This region vysokokonservativnykh in strain A and contains only a small amino acid differences between strains A and B. In particular, the highly conserved region has the sequence HFEVFNFVPCSIC in the provisions 164-176 RSV-A2. Preferably the antibodies of this invention bind an epitope comprising a sequence FEVFNF or sequence VFNFVPCSIC. In one embodiment, the antibodies according to this invention immunoreactive in relation to this area conservative amino acid identity and, thus, G-protein strains of this virus as group A and group B, and therefore G-protein of most strains.

For use in the methods according to this invention for the treatment of RSV infection or to improve resistance to RSV, monoclonal antibodies and fragments of this image is the shadow can be immunoreactive against many strains of both groups a and b, and a single monoclonal antibodies may be sufficient to produce the desired effect. Otherwise, the subject to be treated, or which needs to be done-resistant, can be entered in more than one monoclonal antibody, in particular, when one antibody in the Protocol has a higher reactivity with respect to the strains of group a and the other higher reactivity towards the strains of group B.

This invention also includes pharmaceutical compositions suitable for prevention or treatment, including a softening of inflammation, which contain as an active means of a single antibody or immunoreactive fragment according to this invention or not more than 2 antibodies or fragments according to this invention.

Other aspects of this invention include methods of using the antibodies for the treatment of RSV in humans or to induce resistance in these subjects.

Monoclonal antibodies according to this invention can be obtained recombinante, and, therefore, the invention also includes recombinant materials for getting, as well as cell lines or immortalized cells, as well as non-human multicellular organisms or cells or microbial the cells, to obtain these antibodies. In one embodiment, cells derived from the people, get in "immortalizing" form, in which their modify to ensure the secretion of antibodies for a sufficiently long period of time, so that they can be characterized and cloned the corresponding coding sequence.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 is a graph showing the frequency of occurrence in ppm of antibodies to various antigens of RSV in humans. The desired strain-independent anti-G phenotype (Gab) is quite rare, about 10 parts per million (ppm) in total, and is reduced to 1 ppm in individual subjects. "Mixed." refers to antibodies that bind both F and G; since F and G have no homology sequences, binding, probably due to the total carbohydrate determinants.

Figure 2A is a diagram of the G-protein of RSV, indicating the area CX3C and locations conservative disulfide bonds. Schematic version is universal for all strains, although the specific numbering of the provisions are slightly different from one strain to another.

In figure 2B graphically depicts the binding of sera from exposed to RSV subjects relative to the panel overlapping 12-dimensional peptide of the G protein of RSV, found weak the Yu immunogenicity conservative Central region.

In figure 2C graphically presents the frequency of polymorphism for the collection of more than 75 strains of RSV, depending on the position in the G-protein, discovered the remarkable conservatism in the conservative Central region and in the areas of alternative splicing that generates a soluble form of the G-protein.

The figure 3 shows the results of the study illustrative monoclonal antibodies (131-2G) mice relative to the matrix of peptides with overlapping sequences. This study has identified the epitope associated with this mAb. In the illustrative case, the epitope is within 30 residues of the CX3C motif.

Figures 4A-4D: panels A and B presents summary chart of blood from two donors. Panel A shows the donor with valuable frequency of occurrence Ga/Gb cross-reactive clones. Panel B shows the donor, which it is not. Each graph point represents the relative binding with 3 samples for the "molecular fingerprint" of an antibody secreted by a single clone. Panel C represents a quantitative profile of a "molecular fingerprint" secreted protein isolated B-cells, transformed with a virus Epstein-Barr (EBV). Panel D shows the profiles of the cells 4 generations from HEK293 cells, transformed genes of antibodies from cells in panel C. This profile is similar to that on the C within the accuracy of the analysis, as determined by replicate panel D.

In figures 5A-5B shows the sequence of the heavy chains (panel a) and light chain (panel B) for the representative antibodies according to this invention.

In figures 6A-6F shows the results of determining the affinity of two antibodies according to this invention, obtained using Biacore biosensor. As shown in panels E and F, the 3D3 antibody binds to G-protein and does not show directly detectable rate of dissociation. Panels A and D shows the binding of the antibody with the surface of the sensor. Panels B and E shows the increase of the sensor signal, as protein Ga runs parallel to the surface and is captured by the associated antibody, followed by reduction of the signal, as the surface is washed with a buffer, allowing associated protein Ga to decorrelates from the surface. Panels C and F similarly shows the velocity of the Association and the rate of dissociation of protein Gb.

Figure 7 is a graph showing the binding of different antibodies according to this invention compared to Synagis®F protein-binding antibody that is defined in an ELISA test using a live virus to cover the microplate.

Figure 8 is a graph on which the X axis is the affinity to the G-protein on binding Viru is ω y axis. These two indicator correlated, although 3D3 shows slightly lower affinity to the living virus than would be expected from its affinity to the G-protein.

In figures 9A and 9B show a comparison of the binding of multiple antibodies with strains A2 and A5.

The figure 10 shows the results of tests for neutralizing ability. The results are shown in terms of the number of plaques deposited depending on ug antibody.

The figure 11 shows the comparison of 3G12 antibody according to this invention with the antibody Synagis®to neutralize strain B RSV.

The figure 12 shows a comparison of the prophylactic activity of the two antibodies according to this invention with a commercial antibody Synagis®.

In figures 13A-13C shows therapeutic efficacy of mAb 131-2G model in mice post-RSV infection (treatment day +3 after infection), including dose-dependent reduction in viral load (panel A) along with other measures to reduce pneumonia: NK cells and PMN cells (panel B), and interferon-gamma (IFNγ) (panel C).

The figure 14 shows the dynamics of viral titer in the model in mice treated with antibodies 3G12, 3D3, or Synagis®in small doses, which emphasizes effective advantage of high-affinity antibodies according to this invention.

Figure 15 represents a curve of dependence "dose-effect", which evaluates to Janie antibodies on the number of copies of RSV in the lungs of RSV-infected mice during the processing day +3 after infection.

The figure 16 shows the comparative ability of Synagis®, 3D3 and 3G12 to reduce viral load in the final stages of the infection after treatment at day +3 after infection.

The figure 17 shows the effect of the control antibody, anti-F antibodies and anti-G antibodies in BAL cells in the lungs of RSV-infected mice. Processing was performed at day +3 after infection.

In figures 18A and 18B shows that the F(ab')2immunospecificity fragments of anti-G mAb as effective as the intact mAb, to reduce inflammation in RSV-infected mice, when they are given a day +3 after infection, but ineffective for reducing viral load.

In figures 19A-19C show the impact of anti-G mAb on the production of IFNγ in BAL cells at different moments of the beginning of injection of antibodies, ranging from preventive (day -1) to day +3 and day +5 after infection.

The figure 20 shows the titer of antibodies against the Central conservative area of G-protein of RSV in elderly patients infected with RSV. Patients were selected depending on the severity of clinical signs and symptoms, heavy or light. The absence of measurable titer to conservative Central region correlates with severe disease.

WAYS of carrying out the INVENTION

In this document, the term "treat" refers to the reduction of viral load in the subject, which already is adopted RSV, or alleviate the symptoms of the disease in the subject. Such symptoms include bronchiolitis, respiratory tract inflammation, congestion in the lungs and difficulty breathing.

The term gives the resistance" refers to the prophylactic effect, in which viral infection RSV in the control stimulation at least reduce its severity.

"Immortalized cells" refer to cells that can survive much more subcultures than unmodified primary isolated cells. In the context of the present invention, the term "immortalized" does not necessarily mean that the cells continue to secrete antibodies for very long periods of time, but only that they can survive longer than the culture of primary cells. The time for which there is secretion of antibodies, should only be sufficient to identify and recreate the coding nucleotide sequences.

The phrase "minimally immunogenic when administered to humans" means that the response to the introduction into the body similar to that, when such persons enter the human or humanized antibodies. It is known that the human or humanized antibodies really cause a response in 5-10% of treated people. This is true even for antibodies, which are allocated directly to the people, the AK as there is a certain level of background "noise" caused by the immune response. The immune response can be humoral or cellular, or both. In particular, this percentage of people can detect elevated levels of cytokines.

The phrase "conserved region of the G-protein of RSV" refers to the amino acid sequence containing 50 amino acids, preferably 30 amino acids, more preferably 20 amino acids, on both sides of the field CX3C that shown for a particular strain on figure 2A. Conservative region mainly extended in the 3'-5' direction from a specific area CX3C G-protein. So, using the G protein of RSV strain A2 as a model, the conserved region corresponding to the antibodies according to this invention, is from about 160 to 188 residue, preferably from 160 to 176.

Antibodies according to this invention have a number of desirable properties. First, they are immunoreactive G-protein from a variety of strains of RSV and, as a rule, immunoreactive with G-proteins and strains of type A, and type strains B. secondly, they have very high affinity to G-protein, some of them in the range of <2 PM. Thus, the antibodies according to this invention have affinity, comprising at least 10 nm, preferably 1 nm, more preferably 500 PM, more than 100 PM or 50 PM, 10 PM or 1 PM, and all values between the preferred illustrative points. It was found that commercial antibodies ynagis ®against the F-protein, have an affinity of about 5 nm. Antibodies against the F protein with a higher affinity, Numax™ (motavizumab), estimated to have an affinity of about 50 PM. Antibodies according to this invention show excellent ability to work as therapeutic agents, as well as demonstrate the ability to reduce the number of viruses in the lungs at the peak of infection. They also have this ability at the point where the infection usually exhausts itself. This is particularly useful, as subjects, recovering after RSV infection can continue to spread the virus and, therefore, can infect others in postclinical conditions. Antibodies and fragments thereof also cure the symptoms of infection, including pneumonia.

Antibodies according to this invention were obtained by two illustrative ways. In one approach, the above existing monoclonal antibodies, 131-2G, which, as you know, immunoreactive with conservative region of the G protein, were first prosecution, and then humanitarian by hybridization constant region of a person with modified variable regions of human (both heavy and light chains). Variable regions were selected based on high homology with the variable regions of antibodies 131-2G, and then modified to insert g is pervariabilis amino acids 131-2G. Well-known methods such humanization, providing the right set of amino acid substitutions that you can choose. In case 131-2G, original hybridoma line expressed more than one light chain, requiring a choice, which of them are actually responsible for binding conservative motif RSV. It was defined by the authors of the present invention, and in one embodiment the antibodies according to this invention are examples of humanized forms mAb 131-2G.

In an alternate method, antibodies of this invention were isolated from human donors infected with RSV, using proprietary CellSpot™ method, which is described in U.S. patent 7413868, international publication under PCT WO 2005/045396 and WO 2008/008858, all incorporated by reference.

In this way were analyzed 40 samples RSV-infected donors as part of the process, producing ~500000 antibody-producing cells on each blood sample. So, in total ~20000000 different B-cells were analyzed for the production of antibodies that are specific for the conserved region of the G-protein. Only ~10% of the donors had a suitable frequency Ga/Gb-specific clones (i.e. independent of the strain), and these clones were present in only ~1/50000 cells, even the representatives with the highest frequency. In General, the frequency of desirable CL the current was ~of 0.003%, which is rather low for the feasibility of a standard selection methods, but easily achievable with CellSpot™. The figure 1 shows the spectrum of the reaction of ability to antigens of RSV for 24 donors. As shown in this figure, even among those who had detected the antibodies cross-reacting with G-protein, obtained from strains of both A and B, the prevalence of these antibodies is significantly smaller than antibodies immunoreactive with the F-protein, either Ga or Gb separately. A surprisingly large number of clones recognize as protein F, and G (labeled "mixed."), which probably recognize a common carbohydrate determinants. The affinity of such anti-carbohydrate antibodies generally low and did not proceed further. The highest affinity antibodies found in this group of donors, the affinity of about 1 um, was descended from one of the donors with very low frequency Ga/Gb-specific clones, ~1 ppm namely, the detection of this highly suitable clone would be unlikely without a comprehensive screening of the full repertoire from all donors.

To perform this screening, cells were immortality virus Epstein-Barr and evaluated in accordance with the above-described methods (see example 2 for details). Identified appropriate B-cells, and were received and prosecution nucleotide sequence encoding the identified monoclonal antibodies. Then they were made manipulations in the framework of recombinant technology, so they can produce antibodies in a mammalian cell.

An important aspect of the function of G-protein characteristic Sekretareva form of the protein, s(G)generated by using the site of alternative splicing in the area of residue 50. The design of the virus, which has no s(G), resulted in lower levels of pulmonary infiltration of cells (Maher,et al.,Microbes Infect.(2004) 6:1049-1055). On the other hand, premirovanii mice by s(G) increases the production of IL-5 and eosinophilia lungs (Johnson,et al.,J. Virol.(1998) 72:2871-2880). Accordingly, suppression of the activity of s(G) is important for effective treatment of RSV. To achieve this goal, you need high-affinity antibody that is well known in this field (for example, U.S. patent 7083950). Since the Central conserved region directly involved in the function s(G) as immunomodulatory agent, an effective antibodies against s(G) should be focused on this theme.

Our study of the repertoire of human B-cells of subjects exposed to RSV, was indifferent in search of antibodies that bind to G-protein in both strains a and B (Ga/Gb cross-reactive antibodies). Because the study was extensive (40 subjects, each ~500000 tested B-cells), a remarkable finding is that in the e Ga/Gb cross-reactive antibodies binding linear epitopes suitable for mapping, recognize epitopes within a few residues from each other within the Central conservative area. This area is known to have low immunogenicity, as shown in figure 2B (Plotnicky-Gilquin,et al.,Virology(2002) 303:130-137), which is consistent with the low frequency of the high-affinity to this area of the clones, what we have reported. The authors also further characterize this area by examining the published sequences of G-proteins of >75 RSV isolates. Most residues of the protein are shown in the sample from a few to many polymorphisms. Two areas remarkably free from polymorphisms: the site of alternative splicing, which generates an s(G), and the Central conserved region, which bind all Ga/Gb cross-reactive antibodies (figure 2C). Namely, the authors found that the area that vysokokonservativnykh that is a sign of critical functionality is also netcommunity. A number of mechanisms may be the cause of such a low immunogenicity, for example, the absence of nearby sites of proteolytic cleavage suitable for effective prezentowania this area together with the histocompatibility antigen exposure for the rest of the immune system. Whatever the mechanism of this unexpected result is clear: those viruses, who survived, show low immunogenicity to this area. Therefore, the authors predicted that increased activity of the immune system against this area by passive transplantation of suitable antibodies will be effective, and animal models, it was confirmed that this is the case. The site of alternative splicing, although conservative equally, is not unusually netcommunity, indicating that its importance lies only in the creation of s(G), thus making it a poor target for passive immunotherapy.

Getting a human or humanized antibody according to this invention by conventional recombinant methods, such as getting in ovary cells Chinese hamster or other eukaryotic cell lines, such as insect cells. Alternatively, known methods of producing recombinant materials, including antibodies, in plants and in transgenic animals, for example in the milk of cattle, or in systems of single cells derived from microbes or plants, or insects.

In addition, since the nucleotide sequence encoding the antibody, the appropriate fragments that bind the same epitope, for example Fab, F(ab')2or Fvfragments can be obtained, it is recommended bindtime methods (or proteolytic processing of the protein), and antibodies can be obtained in single-stranded form. In this area there are various methods of managing production of recombinant antibodies.

For use in therapy received recombinant methods antibodies or fragments are in the pharmaceutical composition by using appropriate AIDS and administered in accordance with standard protocols. Data pharmaceutical compositions can have as its sole active ingredient a monoclonal antibody or fragment according to this invention, particularly a monoclonal antibody or fragment that cross-reactive with G-protein both A and B strains. Alternatively, two monoclonal antibodies may be a single active ingredients when one reacts more strongly with G-protein strain A, and the other more strongly with G-protein strain B. In all these cases, there may be an additional therapeutic agent, including one or more antibodies immunoreactive with the F-protein, or other drugs that are effective against RSV or inflammation. For example, such anti-inflammatory agents, both steroidal and non-steroidal anti-inflammatory compounds may be included in the song data. Also, these compounds may include pitate the performance communications substances, such as vitamins, or any other useful connection other than antibodies.

In one embodiment, when the compositions for injection are used in order to increase resistance to infection, use of antibodies, including complement-containing Fc region. Typically, these antibodies are administered at a dosage of 0.01-20 mg/kg weight of a person, or in amounts in the range from 0.01 to 5 mg/kg, or in intermediate quantities within the specified limits. In one embodiment, use the number in the range of 0.1-1.0 mg/kg May be useful for re-introduction, separated by a few days or a few weeks or a few months. Can also be offered booster injections of 1 or 2, or 5, or 10 years.

In other variants of implementation, for a therapeutic effect to reduce viral load, are also full of antibodies, including complement-containing Fc region. The number entered as part of these protocols are of the order of 0.001-50 mg/kg or intermediate values in this range, such as 0.01, 1, or 10 mg/kg can Also be applied re-introduction. Therapeutic effect appoint as soon as possible after the diagnosis of infection, although the introduction of a few days is also within the scope of this invention. Can also be applies is but a re-introduction. In order to reduce the inflammatory response in the lungs must be used only immunospecificity antibody fragments. The dosage levels are similar to those for the full antibody. Introduction mixtures immunospecificity fragments and whole antibodies are also included in the scope of this invention.

The introduction of the compositions of the antibodies according to this invention are typically provided in the form of injections, in most cases, intravenous injection. Thus, it is preferable to parenteral administration. However, included is any applicable method of administration.

These compositions are made by methods well-known in this area for the introduction of the compositions of antibodies. Suitable compounds may be found in standard pharmaceutical reference books, such asRemington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA, incorporated herein by reference. These compounds generally suitable for parenteral administration, including in the form of isotonic solutions, which include buffers, antioxidants, etc. and emulsions, which include delivery vehicles such as liposomes, micelles and nanoparticles.

The desired protocols and formulations will depend on the decision of the attending practitioner, as well as on the specific status of this subject. The dosage levels when the mu is the need will depend on age, General health and severity of infection of the subject.

The following examples are provided to illustrate, but not limit the invention.

Example 1

Cloning and humanization 131-2G

Cloning and sequencing of mAb 131-2G. Total mRNA was extracted from 131-2G hybridoma in accordance with the manufacturer's instructions (RNeasy™ kit: Qiagen, Santa Clarita, Ca). Seven specific data collection 5' VγFR1 primers designed to target a family of genes Igγ, with VH1 for VH7, and one of the consensus 3' Cγ1 primer was used for amplification and sequencing of variable regions of the heavy chain 131-2G. One of the consensus 5' Vk primer was designed to amplify each of the Vk families, and one reverse primer specific for the Kappa constant region, was used for amplification and sequencing Kappa-light chain. Transcripts of VH and VL amplified from 100 ng of total RNA using polymerase chain reaction with reverse transcription (RT-PCR).

Two PCR reaction was performed for 131-2G hybridoma: one for Kappa-light chain (κ) and one for the gamma heavy chain (γ1). For amplification used the kit QIAGEN®OneStep RT-PCR kit (Qiagen Catalog No. 210212). Extracted PCR products directly sequenced using primers specific for the constant region. The deduced sequence was compared with the WPI is unique sequences of the germline DNA of the V - and J-region of Ig using V-BASE2 and alignment of the VH and VL genes in the database germ line of mice. The sequence analysis: based on the information on the nucleotide sequence data were obtained on V - and J-gene segments of the heavy and light chain 131-2G. On the basis of sequence data has designed sets new primers that are specific to a leader sequence of the VH and VK chains Ig 131-2G. Analysis of the sequence and frequency of use of the V-genes: genes heavy chain 131-2G came from a family of genes germline VH1, germline gene D-region is a DSP2.2, and J-region was from germline JH3. The genes for the light chain came from families of genes germ lines Vκ1(K1A5) and Jκ4.

The use of V-segment IgH-VJ558 family VH1 131-2G:

The use of V-segment subgroups IgκV1 131-2G:

The humanized mAb 131-2G. The binding of an antibody (Ab) with its recognizable antigen (Ag) is a highly specific interaction. This specificity lies in the structural complementarity between the binding site of the antibody and the antigenic determinant. The binding sites of antibodies composed of residues originating mainly from the hypervariable or complementarity determining regions (CDR); sometimes the remains of negitively (or frame) areas affect the overall structure of the domain and, after vetelino, binding site.

The repertoire of VH gene segments mouse twice that of people and contains more functional genes compared to human IgH locus. The loci of the mouse and human do not have a high degree of similarity with each other. The first two CDRs of VH and VL domains have a small repertoire of structures, the conformation of the main chain, called canonical structures. The existence of a special canonical structure is mainly determined by the length of the CDR and the presence of key residues at specific locations in the sequence. The same combination of canonical structures of the family VH1 (VH1 1-2) are distributed between members of families VH1 person and VH1 mouse. Based on the analysis of sequences of the heavy and light chains 131-2G and the fact that 131-2G uses V-segments of the IgH-VJ558 family VH1 and family Igκ1, both strands were aligned and compared with members of human families VH1 and VK1. It was found that the homology of the sequences was 70% and 77% identity with the sequence of the germline of the human VH1-8 and Vk1-18, respectively. These germ lines were selected as man of the General scheme for humanized mAb 131-2G.

Mapping of the epitope mAb 131-2G. Western blot analysis using RSV lysate and purified protein Ga showed that 131-2G recognizes a linear epitope. Tie the state of the domain 131-2G mapped using a set of overlapping peptides, obtained from a sequence of G-protein of RSV-A2. Figure 2A shows a diagram of the sequence of a G protein, including localization conservative CX3C motif. In order to obtain a precise mapping of the epitope, was carried out by scanning of a family of peptides generated from dodecameric Ga, each of which is shifted by one residue. The matrix of these peptides was probed by mAb 131-2G at 1 µg/ml Binding 131-2G were detected using peroxidase labeled antibodies goat against mouse IgG in combination with supersystem detection of the chemiluminescent signal (Pierce, Rockford, IL, USA). As summarized in figure 3, the antibody 131-2G reacts with 8 consecutive peptides covering the protein of RSV-Ga residue 157 176. The epitope recognized 131-2G is on the inside of peptide sequences (157) SKPNNDFHFEVF(169) and (169)HFEVFNFVPCSI (176). Based on a single sequence of data 8 peptides binding domain 131-2G mapped residues 164-168.

To characterize the affinity of 131-2G and similar mAb man used three ways. In the first method measured the signal link from a fixed number of antibodies tested against serial dilutions of the antigen in the form of ELISA. The middle point of this titration curve is an approximation of the affinity. In case 131-2G, this median point was 4 nm. In the second method,the affinity 131-2G was measured by Biacore analysis in commercial analytical laboratory; on the basis of the relationship of the speed of the Association to the speed of dissociation calculated affinity was 7 nm. In the third method the dilution Ga protein on the beads CellSpot™ serum albumin reduces the chance of multiple copies of the protein to interact with the "molecular fingerprint" of antibodies. The final suppression effects multidentate the avidity from the raw signals allows you to rank a set of clones for affinity against a known standard. It is a measure of affinity can be used to compare human antibodies with 131-2G and effective selection of high-affinity clones. All of these methods are improved with the presence of a constant source of antigen G-protein. In early studies, the antigen was extracted from virus-infected cells. Due to the variability of the quality of the antigen thus obtained, the authors have developed a recombinant expression system for the production of G-protein, which turned out to be more reliable.

Example 2

The allocation of human B-cells secreting antibody against RSV-Ga/Gb

Mononuclear cells from peripheral blood of 40 adult subjects with confirmed RSV infection was examined in human B-cells producing anti-viral antibodies. Subjects with the desired antibodies against part of the RSV G protein used for kleinova the Oia mAb, specific against RSV-G. based On test results in ~10% of the subjects, the frequency of occurrence of desired cells was higher than 1 per 100,000. Even those with a low frequency of occurrence, however, of interest and also antibodies that have defined the highest affinity obtained from a donor with a very low frequency of the desired type B-cells, ~1 ppm

In order to complete the examination and restoration of rare matched cells, the authors used a previously described technologies CellSpot™. Test CellSpot™ effectively miniaturized equivalent test ELISA to imaginary hole the size of a single cell, capturing secreted by a single cell IgG as a "molecular fingerprint" in the immediate vicinity of the cells. The result can be easily analyzed millions of cells. Next, using the reagents for multiplex microscopy (combinatorial painted fluorescent latex microspheres, see U.S. patent 6642062), can be characterized as a "molecular fingerprint" of secreted each clone of antibody specificity and/or affinity, using multi-purpose biochemical probes. The reliability of quantitative analysis is enough to make possible the withdrawal of the extremely rare matching cells in the examined population, and expressing the cloned cell which shows the phenotype in accordance with the original identification test.

The selection criteria were: binding to G-protein from both of the two main collections of strains, denoted by Ga and Gb, and nazvanie with the F-protein (the other major viral envelope protein). Ranking of clones depending on the affinity can be achieved by dilution of the antigen on the bead serum albumin. This reduces the chances multidentate binding "molecular fingerprint" of secreted IgG (effect of "avidity"), thus selecting on the basis of higher self-affinity. G-protein was purified from Vero cells infected with one or the other of these two strains of RSV.

In relation to human B-cells, this method begins with the depletion of non-B-cells from the mononuclear cells of peripheral blood using standard methods of magnetic separation. Cells resuspendable in IMDM/20% HI-FCS to 1e6/ml; EBV (directly precipitated from the supernatant of infected B95-8 cells) was added in a dilution of 1:100 and the cells were incubated 2 h at 37°C. Excess virus was washed, and the cells either cultured at 2e6/ml in IMDM, 20% HI-FCS, 20% air-conditioned giant cell tumor environment, 2 µg/ml CpG (ODN2006) and 10 ng/ml IL-10 only for examination or further selectively IgG on the surface using magnetic positive selection. The cells were maintained at 200-300 cells/well in irradiated lung cells (MRC-5, 5000 the notches/well) in IMDM, 20% HI-FCS, 20% air-conditioned giant cell tumor environment, 2 µg/ml CpG (ODN2006) and 10 ng/ml IL-10. Wednesday met additives every 2-3 days. One half of the contents of the wells tested CellSpot™ in day 6. The rest of the cells in a small number of wells positive for the analytical results of the survey, then diluted to 10, 5, 1 and 0.5 cells/well in the same feeding cells under the same culture conditions. After 4-5 days, these tablets for a limited dilutions were again tested using ELISA method or CellSpot™.

Contents positive in limited dilution holes were then processed using PCR with reverse transcriptase to get polynucleotide encoding heavy and light chains of this antibody. Total time from thawing of mononuclear peripheral blood cells to obtain the coding sequence of mRNA by RT-PCR was 10-12 days.

The figure 4 shows illustrative data of this experiment. Examples of CellSpot™ profiling of suitable and unsuitable blood samples shown in panels A and B. the Profile of a suitable clone when the original detection is shown in panel C, along with replicative profiles panel D antibodies secreted by the offspring NECK cells transformed by cDNA cloned antibodies obtained from this cell. Profiles from the fall within the precision of this analysis, reflecting on the successful recovery of a suitable clone.

As was shown in figure 1, most of the anti-RSV antibodies directed against the F protein or antigenic determinants common to F and G (most likely, carbohydrate, because the sequences of these two proteins are not homologous). From specific to G antibodies, most links only Ga or Gb only, which is consistent with the well-known high variability of the sequence of a G protein. In the end, were tested ~20 million individual B-cells. Using RT-PCR were found on 12 of the most promising antibodies. In total, the frequency of appropriate clones was below 1 in 1 million and took more than 50 million tests, equivalent to ELISA to find these rare clones. Technology CellSpot™, thus, gave the possibility of a broader survey of the clones, than otherwise would be feasible. The quality of the clones is better than what was found with more limited screening, and comprehensive features of this high quality set has encountered an unexpected properties of the desired antibodies.

Example 3

Cloning of human antibodies against RSV-Ga/Gb

Amplification rearranging genes Ig Heavy and Ig Light of the positive holes ELISA was achieved using semiߛenclosed" polymerase chain reaction (PCR). For Ampl the pre-qualification unknown rurangirwa V-gene has designed a collection of primers, specific to families of V-genes that recognize almost all segments of the V-genes in the Igh locus of the person. Used 5'-primers together with a mixture of primers, specific for Cγ, Cκ and Cλ gene segments. Clonal properties of B-cells specific to serial dilutions of RSV-G, uniquely determined by comparing the sequences of amplification products V-genes of the individual cells of the offspring and amplificatoare full rearrangement of the V-genes cloned in expression vectors IgG. This method is also useful for solving additional problems, such as the frequency of use of the V, D and J genes, and the presence and pattern of somatic mutations.

Ways. Total mRNA isolated from B-cells was extracted using a commercially available kit RNA purification (RNeasy™; Qiagen (Germany)). PCR with reverse transcription was performed using preparations of total RNA and oligonucleotides as primers. For each sample, you must perform three reaction PCR: one for the Kappa (κ) light chain, one for the lambda (λ) light chain and one for gamma (γ) heavy chain. For amplification used the kit QIAGEN®OneStep RT-PCR kit (Qiagen Catalog No. 210212). In paired RT-PCR reactions with cDNA synthesized using a unique blend of enzymes reverse transcription (Omniscript™ and Sensiscript™) using specific anti the artisan sequence primer, the corresponding C-K, C-λ or consensus CH1 regions Cγ genes, reverse trascription was carried out at 50°C for 1 hour, followed by PCR amplification of cDNA using HotStarTaq DNA polymerase for high specificity and sensitivity. For each PCR reaction used a mixture of 5' sense primers. Sequences of the primers were based on the leader sequences of the VH, VK and VL. PCR reaction was carried out at 95°C for 15 minutes, the initial hot start, followed by 20 cycles at 95°C for 30 seconds (denaturation), 60°C for 45 seconds (annealing) and 72°C for 1 min (elongation).

"Nested" PCR for detection and cloning of the variable Ig fragments into expression vectors. The second cycle was sent aliquot of 5 μl of the first amplification reaction. Used the primers contained restriction sites 5'BgIII and 3' XbaI. Was conducted in thirty cycles of PCR. For the first and second cycles of amplification were used with the same conditions. Five μl of each reaction was applied and separated on 1% agarose gel and stained bromide by ethidium. V-C PCR product is projected amplificare rearrangeable fragments, VH and VL, 500 and 450 BP, respectively. Strip PCR, corresponding to a molecular size of about 500 BP, meant a positive result. The PCR products were purified (set Qiagen purification from the gel, catalog number 28704) and ex is legirovannye PCR products directly sequenced using specific for the constant region primers. Sequences of the cloned fragments was confirmed by sequencing the plasmid prepared for recombinant production.

Figure 5A shows the amino acid sequence of the heavy chains of the antibody according to this invention, isolated from people, and humanized 131-2G, including variable region, D - and J-region accession, the framework (FR) and complementarity determining region (CDR). All of these antibodies of immunoreactive in relation to the G-protein from both strains A and B, except antibodies 3F9, which immunoreactive only with respect to the G-protein strain A. In the figure 5B shows similar information for the sequences of the light chains of these antibodies. Dashes in printouts sequences represent amendments alignment gene sequences of different lengths.

PCR fragments described above were digested and cloned into individual expression vectors containing the constant region of the human γ1, or human κ or λ, forin vitrothe production of antibodies in mammalian cells. The expression vectors encoding the heavy and light chains together was transfusional in cell line 293 (human kidney) (Invitrogen). Expressing plasmids were introducible using cationic transfection reagent lipid based (293fectin™; Invitrogen). DL is each reaction transfection of 20 ug of purified plasmid and 40 μl of 293fectin™ was mixed with 1 ml of Opti-MEM ®(Invitrogen) and incubated for 5 minutes at room temperature before combining and allow the complexes to form for 20 min at room temperature. Complexes of DNA-293fectin was added to 3×106cells, seeded in 90 mm Petri dishes, and incubated at 37°C, 8% CO2. During the post-treatments after 72 hours after transfection the supernatant was collected by centrifugation (3000g, 15 min at 4°C) for isolation of secreted antibodies.

Example 4

Mapping the epitopes of antibodies of this invention and determination of affinity

Using the procedure described in example 1 mapping of the epitope of the antibodies of the prototype 131-2G were identified epitopes relevant antibodies according to this invention. The affinity of the antibodies according to this invention were determined using the methods described in example 1 with respect to mAb 131-2G.

As noted in table 1 below, three of the antibodies bind conformational epitopes, they do not mapped binding of overlapping peptides. Antibodies according to this invention, which are mapped in a specific sequence, shown in the table. Also given constants affinely defined using standard tests Biacore against recombinant Ga and Gb proteins expressed in PM, calculated from the measured velocities of the Association, dissocial is I. Data for two of these antibodies is shown in figure 6. Panels A, B and C shows binding data binding for 3G12, and panels D, E and F show data for the 3D3. The top row shows the loading biosensor chip antibodies, the middle row shows the signal obtained when applying Ga protein through the chip with the subsequent washing buffer, and the bottom row shows the same for protein Gb. The increase of the signal allows to calculate the velocity of the Association, while a decrease during reduction allows to calculate the dissociation rate. The ratio of the velocity of the Association to the speed of dissociation is the affinity constant, Kd.

Table 1
AntibodyChemical activityThe epitope positionThe epitope sequenceKDxGa (PM)KDxGb (PM)
1F12Ga/Gb166-172EVFNFVP
3G12Ga/Gb167-176VFNFVPCSIC173

1A5Ga/Gb161-170*HFEVF
3D3Ga/Gb164-172FEVFNF1,13
1G1Ga/GbConformational
2B11Ga/Gb162-172DFHFEVFNFVP91,6
5D8Ga/Gb160-169NNDFHFEVFN43901
2D10Ga/GbConformational
3F9GaOnly Ga
1D4Ga/GbFEVFNFV 23052
1G8Ga/Gb165 to 169NDFHFEVFNF24141
6A12Ga/GbConformational
10C6Ga/Gb164-169*HFEVF55378
* the same epitope as the 131-2G

Example 5

Comparison of binding to G-protein with the binding of virions

The figure 7 shows the results of ELISA with the use of live virus and evaluation of binding using standard analysis of horseradish peroxidase. Viral preparations from various sources used for coating the tablet with a 105PFU/well or in higher concentrations in carbonate buffer at pH 9.6 overnight at 4°C. the plates were blocked in 5% milk in PBST for one hour at room temperature. Serial dilution of antibody was added to the wells in blocking buffer for one hour at room temperature. For detection added conjugate Fcγ-HRP goat anti-man (Jackson Measurement.) in a dilution of 1:2000 in the block is the dominant buffer for one hour at room temperature. Tablets abundantly washed in PBST. The turnover of TMB substrate was measured at 450 nm. As shown in figure 7, 3D3 well-connected live virus, as do a number of other antibodies according to this invention. Antibody Synagis®, the affinity of which is considerably weaker, shows weak binding with a live virus or antibody-based test, even if a load of 104ng/ml In the figure 8 shows the interdependence of binding to recombinant protein compared to the binding of viral particles.

Figures 9A and 9B are graphs that show the comparative ability of the antibodies according to this invention to contact A2 strains compared with strains A5, using the above analysis. The figure 9 shows that the 3D3 and 3G12 good contact with the strain A2 compared to Synagis®. PAB is a commercial polyclonal antibody goat against all proteins of RSV (Chemicon, catalog No. AB1128).

Figure 9B shows that these antibodies also bind strain A5; it should be noted that the units on the X-axis here and in figure 9A differ. Similar experiments showed that the antibodies according to this invention associated with a wide variety of clinical isolates.

Example 6

Tests to neutralize

The ability of individual antibodies according to this invention to neutralize virusin vitrofound in the standard is coherent analysis belascoaran. HEp2 cells were sown in 12-hole tablets with 2×105cells/well. The next day the media was created by serial dilution of antibodies. About 200 PFU/well of RSV was added to the antibody in the presence complementarians serum rabbit for one hour at room temperature. Then or antibody-based test-virus mixture was added to cells HEp2 at 200 μl/well for 2 hours at room temperature, to ensure the infection. After period of infection medium was removed and medium containing 1% methylcellulose was added to all wells. The plates were incubated at 35°C for 6 days, after which the cells were fixed and stained to determine the number of plaques, namely: methylcellulose was removed from the cell layers and the cells were fixed in 100% methanol for 30 min at room temperature. The tablets were washed 3×with 5% milk in PBS. Primary antibodies were added at 1:500 dilution (polyclonal antibody goat against RSV (Chemicon, catalog No. AB1128)) in PBS + 5% milk protein for 1 hour. The tablets were washed again three times in 5% milk in PBS. Secondary antibodies were added at 1:500 dilution in 5% milk protein in PBS (ImmunoPure of rabbit antibodies against IgG (H+L) goat conjugated with peroxidase) (Thermo Scientific, catalog No. 31402) for 1 hour. Tablets washed three times-once by PBS. Plaques were visualized by adding one chloronaphthalene substra is (Pierce, catalogue No. 34012), 200 µl per well for 10 minutes, the Tablets were washed with water and left to dry in the open air. Plaques were counted in each well.

The figure 10 shows the results in terms of the absolute number of plaques on ug human antibodies, and antibody Synagis®included in the results. These data show that, of the tested antibody 3D3 is the most effective. The 3G12 IC50approximately 15 ng/ml or the affinity of about 100 PM in accordance with this analysis, while the commercial antibody Synagis®IC50approximately 300 ng/ml, which corresponds to the affinity of about 2 nm. In addition, it was found that Synagis®and anti-G antibodies according to this invention did not give a synergistic effect in these conditions.

The figure 11 shows the neutralizing activity of the antibodies 3G12 against strain B compared to Synagis®. Normalized data (% of control) based on the absolute number of plaques 160-180 in the experiment. Antibodies according to this invention within vitroaffiniscape from 1 PM to 5 nm (table. 1) had a value EC50between 10-100 ng/ml

Example 7

Anti-G prevention in mice

Antibodies according to this invention, Synagis®and human IgG1, were tested for their ability to prevent RSV infection in mice. Day -1 prior to infection the mice in the control group in which the Odile IPR environment and PBS. In the experimental groups injections amounted to 0.15, 1.5 and 15 mg/kg of antibody hIgG1 (immune izotopicheskii control)or 3G12, either 3D3, or Synagis®. It is about 3 μg, 30 μg, and 300 μg per mouse.

On day 0 mice were inoculable 1×106The BATTLE of long strain of RSV by the intranasal route. On days 0 and 5 lungs, bronchoalveolar allocation (BAL) and serum was collected and determined body weight, lung weight, the FIGHT in the slice of a lobe of the lung, viral load (using qPCR), histology of lung, total leukocytes, cell sorting by fluorescence activation and IFNγ in BAL.

The figure 12 shows the results based on the viral load of the lungs using analysis belascoaran from the previous list. The data in figure 12 show that 3G12 and 3D3 as effective as Synagis®in this analysis. (A typical human dose of Synagis®is 15 mg/kg for humans.)

Example 8

therapeutic efficacy of antibodies against RSV-Ga/Gb

It is shown that antibodies against the conserved motif in RSV-G have therapeutic efficacy. Mice were infected intranasally on day 0, 106The FIGHT against RSV, then day 3 was treated with 3 mg/kg of antibody, administered intraperitoneally injected, and on days 5 and 7 were analyzed for viral load in bronchoalveolar secretions. In this model, infection natural about what atom cleaned much easier than humans. However, treatment with antibodies leads to faster elimination of the virus dose-dependent manner compared with control antibody that does not bind RSV (figure 13A). Each experimental group consisted of 5 animals, and the results were statistically significant.

As described in WO 00/43040, antibodies against substance P is useful in the fight against pneumonia caused by RSV, animal model of prolonged complications that are clinically important feature of RSV infection. Increasing regulation of substance P depends on the active G-protein (Haynes, L. M.,et al.,J. Virol.(2003) 77:9831-9844). There was also a reduction in the scale of inflammation of the lungs after injection of antibodies according to this invention, including the reduction of inflammatory NK and the total PMN cells (figure 13B), as well as the reduction of cytokines, such as IFNγ (figure 13C).

In an additional test at day 0 mice were inoculable 106The BATTLE RSV long strain of type A via the intranasal route.

On the 3rd day of various groups of 4-5 mice were treated as follows:

Group 1: control group, which received no infection at day 0 and were treated with PBS.

Group 2: negative control, which received the RSV inoculation on day 0 and treatment PBS on day 3.

Group 3: RSV inoculation at day 0 and antibody Synagis®IPR in the physical and the ideological solution in quantities of 1, 10 or 100 μg per mouse either of 0.05, 0.5 or 5 mg/kg

Group 4: RSV inoculation at day 0 and introduction mAb 3D3 according to the same Protocol as group 3.

Group 5: received RSV inoculation at day 0 and introduction 3G12 in the same quantities that groups 3 and 4.

Light and liquid discharge BAL were collected on days 0, 3, 5, 7 and 10. In addition, it was determined body weight, lung weight, the FIGHT in the lobes of the lungs, viral load by qPCR, histology of lung, total leukocytes, cell sorting with activation of fluorescence, as well as IFNγ in BAL. The results of qPCR in groups, which was introduced on 10 µg mAb, shown in figure 14.

As you can see, viral titer in treated and untreated Synagis®mice behaves similarly at these relatively low doses of antibodies, while processed and 3D3, and 3G12 mice had significantly lower titers at the peak of infection in day 5. This experiment confirms that the higher the affinity ofin vitrocorrelated with higher activityin vivo.

The figure 15 shows the curve of dependence "dose-effect", showing that 3G12 and 3D3 able to reduce the number of copies of the RSV, which is determined using qPCR at day 7 at lower concentrations than Synagis®. 3D3 was particularly active, again in accordance with the fact that their affinityin vitroabove.

Similarly, when viral account when qPCR was measured at day 10, the hile viral titres at the time in nature is very low due to the natural elimination of the virus with the immune system of the mouse, 3D3 about 100 times more active than Synagis®at various concentrations, doses, as shown in figure 16. This experience underlines the suitability of high-affinity antibodies, which continue to be effective even when the concentration of antigen fall. The disease in humans is significantly longer than mice that provides explicit incentive for the use of antibodies, which continue to neutralize the virus for a longer time.

In still further experiments, mice were treated with anti-G mAb mouse or anti-F mAb mouse in groups of four individuals, each experiment was repeated three times. Mice were immunized on day 0 and were treated with antibodies at day 3, and the various manifestations of effectiveness was noted on days 3, 5 and 7.

As one indicator of the effectiveness was measured by the number of inflammatory cells in bronchoalveolar lavage fluids (BAL) in the three groups, the results of which are shown in figure 17. The number of BAL cells in terms of light plotted on the Y-axis from 0 to 140·103. The results show that anti-F mAb reduce the number of BAL cells in the lungs on day 5 compared with izotopicheskii control non-immune antibodies, whereas anti-G mAb reduce the number of BAL cells much stronger. On day 7 of infection has passed.

In figures 18A and 18B show a comparison of the effectiveness of anti-GmAb (131-2G mouse) compared with anti-G F(ab') 2derived from this antibody by personalism and removal of Fc fragments using immobilized protein A. it Was shown that the complement is essential for the antiviral effect of anti-G antibodiesin vitro. This is confirmed by figure 18A, where the antiviral effect is measured as PFU/g lung tissue. The analysis was carried out as described in example 6. F(ab')2fragment of the anti-G antibodies lacking the Fc region of IgG required for complement-mediated activity, slightly better than the control, to reduce viral load, while the anti-G mAb is very effective. However, when as a measure of results is inflammation, as shown in figure 18B, F(ab')2fragment of the anti-G mAb is as effective as the whole antibody. This experiment provides that the neutralization of the G-protein is crucial to reduce airway inflammation. Because the virus is actively secretes a soluble form of the G-protein and high-affinity binding is important for neutralization of soluble factors, high-affinity antibodies according to this invention should be particularly useful for anti-inflammatory effect.

In figures 19A, B and C shows the effect of anti-G mAb on the production of IFNγ in BAL depending on the time of administration, and the cytokines act as a marker of inflammation, digatel who's ways. Control non-immune antibodies in all cases could not reduce the growth of production of IFNγ, which is accompanied by inflammation of the Airways. However, regardless of injected whether anti-G mAb at day -1 (panel A), day +3 (panel B) or even in day 5 (panel C), there is a sharp decrease of the level of IFNγ in day 7. This experiment determines the usefulness of antibodies against conservative Central motif of G-protein of RSV for the treatment of inflammation beyond the peak viral load.

Example 9

The specificity of endogenous antibodies in infected subjects

Serum samples from 4 older adults with severe associated with RSV disease and six older adults with a moderate form associated with RSV disease were tested for immunoreactivity with synthetic peptide:

(shown disulfide bridges)

which is a conserved region of the G protein of strain A2 RSV. The analysis was performed using the ELISA Protocol described in example 5. The levels of antibodies immunoreactive against this peptide correlate with severity of disease, in which subjects with mild forms of the disease show significantly higher titers than subjects with more severe manifestations of infection (see figure 20). These results show that antibodies, immunol is active in relation to this area of the G-protein, are effective in reducing infection.

1. Isolated monoclonal antibody (mAb) or immunoreactive fragment that bind to epitope within residues 160-176 G-protein strain A2 respiratory syncytial virus (RSV), where
a heavy chain CDR1 has an area consisting of SSNYYWG (position 31-37 SEQ ID NO :29), region CDR2 comprising SIHDSGSIYYNPSLRS (position 52-67 SEQ ID NO :29), and the region CDR3 comprising HLVWFGELRNNWFDP (position 100-114 SEQ ID NO:29), and light chain CDR1 has an area consisting of RASQSVNSNLA (positions 24-34 SEQ ID NO :43), region CDR2 comprising GASTRAT (position 50-56 SEQ ID NO:43), and the region CDR3 comprising QQYNNWPL (position 89-96 SEQ ID NO:43) (3G12), or
a heavy chain CDR1 has an area consisting of ENAN (positions 31-35 SEQ ID NO :31), region CDR2 comprising GISWNSGSVGYADSVKG (position 50-66 SEQ ID NO :31), and the region CDR3 comprising MVATTKNDFHYYKDV (position 99-113 SEQ ID NO :31), and light chain CDR1 has an area consisting of KASQSVSNHLA (positions 24-34 SEQ ID NO :45), region CDR2 comprising ETSNRAT (position 50-56 SEQ ID NO :45), and region CDR3 comprising QQRNNWYT (position 89-96 SEQ ID NO :45) (3D3), or
a heavy chain CDR1 has an area consisting of TYPIS (positions 31-35 SEQ ID NO :33), region CDR2 comprising RIIPDPPMANIAQKFQG (position 50-66 SEQ ID NO :33), and the region CDR3 comprising EILQSPPFAVDV (position 99-110 SEQ ID NO :33), and light chain CDR1 has an area consisting of TGSSSDVGGYSHVS (position 23-36 SEQ ID NO :47), region CDR2 comprising EVSNRPS (position 52-58 SEQ ID NO :4), and region CDR3 comprising GSYASTNILH (position 91-100 SEQ ID NO :47) (2 B11), or
a heavy chain CDR1 has an area consisting of TYYIH (positions 31-35 SEQ ID NO :37), region CDR2 comprising VINPSGGSTTYAQKFQD (position 50-66 SEQ ID NO :37), and the region CDR3 comprising VHKGRAEQWQLLHGHFDL (position 99-116 SEQ ID NO :37), and light chain CDR1 has an area consisting of KSSQSVLYSSNNKTYLA (position 24-40 SEQ ID NO :51), region CDR2 comprising WASTRES (position 56-62 SEQ ID NO :51), and the region CDR3 comprising QQYYTTP (position 95-101 SEQ ID NO :51) (1D4), or
a heavy chain CDR1 has an area consisting of SGQYYWA (position 31-37 SEQ ID NO :38), region CDR2 comprising SIHYSGSTYQNPSLKS (position 52-67 SEQ ID NO :38), and the region CDR3 comprising QQLSLSPVENWFDP (position 100-113 SEQ ID NO :38), and light chain CDR1 has an area consisting of RASRSVGSRLA (positions 24-34 SEQ ID NO :52), region CDR2 comprising AASTRAT (position 50-56 SEQ ID NO :52), and a CDR3 region comprising QQYKEWPL (position 89-96 SEQ ID NO:52) (IG8), or
a heavy chain CDR1 has an area consisting of GYAMH (positions 31-35 SEQ ID NO :40), region CDR2 comprising VISFDGSNNYYADSVKG (position 50-66 SEQ ID NO :40), and a CDR3 region comprising PDVIAVAGTALSNPFDL (position 99-115 SEQ ID NO :40), and light chain CDR1 has an area consisting of RASQSVRSNLV (position 23-33 SEQ ID NO :54), region CDR2 comprising GASTRAT (position 49-55 SEQ ID NO :54), and a CDR3 region comprising QQNNNWPP (position 87-95 SEQ ID NO :54) (S).

2. mAb or fragment according to claim 1, where the specified mAb are 3G12, 3D3, 2 B11, 1D4, 1G8 or S and the specified immunospecificity the second fragment is from 3G12, 3D3, V, 1D4, 1G8 or S.

3. mAb according to claim 1, which is in the form of antibodies.

4. mAb or fragment according to any one of claims 1 to 3 for use in the method of treatment of RSV in subject-a person infected with RSV.

5. mAb or fragment according to any one of claims 1 to 3 for use in the method of reducing inflammation of the Airways of the subject-a person infected with RSV.

6. mAb or fragment according to any one of claims 1 to 3 for use in the method of increasing the resistance to RSV infection in the subject person.

7. Pharmaceutical composition for treatment or prevention of RSV containing isolated monoclonal antibody or fragment according to any one of claims 1 to 3 together with a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, further containing additional pharmaceutical agent, other than antibodies immunoreactive against RSV, together with a pharmaceutically acceptable excipient; or
optionally containing one or more monoclonal antibodies or their fragments, which are immunoreactive against the F-protein of RSV.

9. The pharmaceutical composition according to claim 7 or 8 for use in a method for the treatment of RSV in subject-a person infected with RSV.

10. The pharmaceutical composition according to claim 7 or 8 for use in the method of reducing inflammation of the Airways of the subject-a person infected with RSV.

11. The pharmacy is practical composition according to claim 7 or 8 for use in the method of increasing the resistance to RSV infection in the subject person.

12. Double-stranded selected nucleic acid molecule, which contains a first nucleotide sequence encoding a mAb or fragment according to claim 1 or 2, and a second nucleotide sequence complementary to the specified first nucleotide sequence along its entire length.

13. Recombinant cell host containing the system expresii and double-stranded isolated nucleic acid molecule indicated in paragraph 12, where this recombinant cell host is a mammal cells, germ cell, insect cell or plant cell.

14. The method of obtaining mAb or immunoreactive fragment comprising culturing the cell according to item 13 and the allocation of these mAb or fragment.



 

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Antibody to epha2 // 2525133

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology, medicine and biotechnology. Claimed are versions of anti-EPHA2 antibodies. Claimed antibodies are bound with polypeptide, consisting of amino acids 426-534 in SEQ ID NO:8. Also described are hybridomes, which produce such antibodies, and pharmaceutical compositions and methods of application of said antibodies and compositions.

EFFECT: invention can be used in medicine.

74 cl, 14 dwg, 14 ex, 1 tbl

FIELD: biotechnology.

SUBSTANCE: invention relates to an agent for treatment of ischemic lesions of tissues, which is a mixture with the ratio of 1.5-3 of two cultures of mesenchymal stem cells, one of which is modified by the genetic structure based on the viral vector which provides hyperproduction of vascular endothelial growth factor, and the other is modified by the genetic structure based on the viral vector which provides hyperproduction of angiopoietin, and a method of treatment of ischemic lesions of tissues by puncture of ischemic tissue, and can be used in medicine.

EFFECT: invention enables to achieve effective vascularisation and repair of ischemic tissue.

4 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: inventions relate to the field of immunology. Claimed are a single-chain antibody, specific to a carcinoembryonic antigen, a chimeric mononuclear T-cell receptor, a vector, a host cell and a method of diagnostics or treatment of diseases, characterised by the presence of antigens, capable of binding with the chimeric receptor. Described is a genetic construction, coding chimeric monomolecular T-cell receptors, in which an effector fragment of the T-cell receptor is combined with an antigen-recognising part, which represents variable fragments of two different antibodies to the carcinoembryonic antigen (CEA).

EFFECT: claimed inventions can be used in T-cell cancer therapy.

7 cl, 4 dwg, 3 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.

EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.

3 cl, 6 ex, 1 tbl, 14 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Method includes introduction of RNA molecule into a bird egg. Introduced RNA molecule contains double-stranded region and results in reduction of the level of molecule of RNA and/or protein, included into determination of sex in birds, in the egg. Invention can be used in poultry breeding.

EFFECT: claimed is method of changing sex characteristics in birds.

7 cl, 3 dwg, 6 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: invention refers to the field of biotechnology and food industry. Presented is a barley plant that yields grain and is homozygotic in at least two loci for the genetic variations having been bred, representing: a) allele wherein most of or all the genes coding B-hordein in Locus Hor2 are removed, and b) mutant allele in the barley Locus Lys3 so that the grain contains neither B-, nor C- hordeins, the said genetic variations present in Lines Riso 56 and Riso 1508 barley accordingly; absence of B-hordeins is to be revealed by absence of amplified DNA using primers: 5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3', 3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3', while absence of C-hordeins is to be revealed by absence of the 70 kDa strip during study of the grain alcohol-soluble extract by means of SDS-PAGE. Additionally presented are: barley grain cropped from the said plant; B- and C-hordein-free products produced from the said grain such as flour, malt and beer. Additionally described are methods for production of food products barley (flour, whole-grain flour, starch, malt) and beverages using grain cropped from the barley plant having the above characteristics. Proposed is a method for identification of barley grain suitable for production of a malt-based food product and/or beverage suitable for consumption by a person suffering from gluten-sensitive enteropathy which method includes: a) production of one or more materials: i) sample of a plant capable to yield the said grain, ii) grain, iii) malt produced from the grain, and/or iv) extract of the said grain; b) analysis of Stage a) material for presence of at least one hordein and/or at least one hordein-coding gene with selection of grain having the gene pattern of the above plant.

EFFECT: invention allows to manufacture B- and C-hordein-free malt-based food products or beverages.

27 cl, 14 dwg, 10 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Described is molecule of chimeric nucleic acid of porcine circovirus (PCV2Gen-1Rep), which includes molecule of nucleic acid, coding porcine circovirus of type II (PCV2), which contains sequence of nucleic acid, coding protein Rep of porcine circovirus of type 1 (PCV1). Chimeric molecule of nucleic acid is constructed by replacement of gene Rep ORF1 PCV2 with gene Rep ORF1 PCV1. Invention also includes biologically functional plasmid or viral vector, which contain unique molecules of chimeric nucleic acids, suitable host cells, transformed by plasmid or vector, infectious chimeric porcine circoviruses, which produce suitable host cells, method of obtaining immunogenic polypeptide product with application of novel chimera, viral vaccines, protecting pig against viral infection or syndrome of postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of protecting pigs against viral infection or postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of obtaining unique chimera PCV2Gen-1Rep and the like. Invention can be applied in veterinary.

EFFECT: invention additionally includes novel method of increasing level of replication and PCV2 titre in cell culture.

21 cl, 2 dwg, 6 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

Antibody to epha2 // 2525133

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology, medicine and biotechnology. Claimed are versions of anti-EPHA2 antibodies. Claimed antibodies are bound with polypeptide, consisting of amino acids 426-534 in SEQ ID NO:8. Also described are hybridomes, which produce such antibodies, and pharmaceutical compositions and methods of application of said antibodies and compositions.

EFFECT: invention can be used in medicine.

74 cl, 14 dwg, 14 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: inventions refer to biotechnology and concern a recombinant plasmid DNA pQE30/Derf2L and a bacterial strain Escherichia coli M15/pQE30/Derf2L. The characterised recombinant plasmid DNA codes protein Derf2L of a mite Dermatophagoides farinae and consists of BamHI/HindIII - a DNA fragment of a plasmid pQE30 and a DNA sequence coding BamHI/HindIII fragment including gene Derf2L Dermatophagoides farinae. The characterised bacterial strain Escherichia coli M15/pQE30/Derf2L, is produced by transformation of the cells Escherichia coli M15 by the recombinant plasmid DNA pQE30/Derf2L.

EFFECT: presented inventions can be applicable to develop a dust mite diagnosticum, as well as for conducting specific immune therapy.

2 cl, 3 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Method includes introduction of RNA molecule into a bird egg. Introduced RNA molecule contains double-stranded region and results in reduction of the level of molecule of RNA and/or protein, included into determination of sex in birds, in the egg. Invention can be used in poultry breeding.

EFFECT: claimed is method of changing sex characteristics in birds.

7 cl, 3 dwg, 6 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns an isolated polypeptide, a pharmaceutical composition containing such polypeptide, as well as a method of treating cancer. The presented polypeptide contains an amino acid sequence corresponding to SEQ. ID. NO: 2 or SEQ ID NO: 4. There are also characterised fragments or versions of the presented polypeptide.

EFFECT: group of inventions may be used to stimulate the immune system in treating malignant diseases and for diagnosing loss of immunologic activity.

7 cl, 7 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to recombinant fused protein dimers intended to inhibit or suppress immune response in a mammal, which bind human CD80 or human CD86 or the extracellular domain of any thereof, and has higher capacity for suppressing immune response than a dimer of the fused protein LEA29Y-Ig. Also disclosed are nucleic acids which code said dimers, expression vectors containing said nucleic acids, as well as recombinant host cells containing said nucleic acids and/or said vectors. Disclosed are pharmaceutical compositions for inhibiting or suppressing immune response in a mammal, which contain said fused protein dimers, as well as use of said dimers to produce drugs for inhibiting or suppressing immune response in a mammal, treating diseases or disorders of the immune system or treating organ or tissue transplant rejection in a mammal. Methods of producing said fused protein dimers are also disclosed.

EFFECT: invention provides effective inhibition or suppression of immune response in a mammal.

9 cl, 15 dwg, 11 tbl, 12 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a method for obtaining recombinant DNAse I of a human or its mutein, as well as their conjugates with polyethylene glycol, using a bacterium belonging to Escherichia class, transformed with expression plasmid, containing a promoter functioning in a bacterial cell, DNA fragment coding a hexahistidine cluster, a fragment coding enterokinase recognition sequence amalgamated in frame with human DNAse I or its functionally active mutein containing replacements of asparagine with cysteine, transcription termination section, vector pET28a(+) fragment containing initiation section of replication of bacteriophage fl, sequence coding aminoglycoside-3'-phosphotransferase, area of beginning of plasmid pBR322 replication, gene RNA-organising protein Rop, sequence coding lactose operon repressor.

EFFECT: invention allows obtaining recombinant human DNAse I or its mutein with high yield.

18 cl, 7 dwg, 1 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: isolated peptide having cytotoxic T lymphocyte (CTL) inducing capacity in the presence of an antigen-presenting cell bearing HLA-A*2402, is used to obtain antigen-presenting cells and therefore CTL. The obtained CTL are used for targeted action against CDCA1-expressing cancer cells.

EFFECT: invention provides an effective vaccine for inducing anti-tumour immunity in a subject.

16 cl, 4 dwg, 1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.

EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention represents an agent for neutralising smallpox virus representing an artificial single-chain human 1A antibody having an amino acid sequence presented in the claim material, and exposed on a surface of the filamentous phage M13.

EFFECT: invention enables neutralising smallpox virus.

7 dwg, 4 ex

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