FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology. There are presented the antibodies that bind to the P-selectin glycoprotein ligand-1 (PSGL-1), as well as methods for stimulating the death of activated T-cells and simulating T-cell immune response in a patient by the use of the antibodies under the invention, and pharmaceutical compositions containing the antibodies under the invention. There are also disclosed nucleic acids, expression vectors and host cells for producing the antibodies under the invention.
EFFECT: invention may can find further application in therapy of the PSGL-1 associated diseases.
35 cl, 4 ex, 3 tbl
The SCOPE of the INVENTION
Highly aggressive T-cells are often the cause of unwanted immunological reactions, which in turn cause various disorders, such as autoimmune diseases, transplant rejection, allergic diseases and T-cell cancers. As a consequence, the reduction in the number of aggressive T cells is an important factor in the treatment of such disorders. The activity of these cells may be limited due to immunosuppression or induce immunological tolerance. An alternative solution is the induction of apoptosis, which, apparently, is involved in the removal of unwanted cells, including highly aggressive T cells. See, for example, Kabelitz and others (1993) hnmunol Today 14, 338-340; and Raff (1992) Nature 356, 397-399.
BRIEF description of the INVENTION
The invention relates to antibodies and their derivatives, inducing apoptosis by joining glycoprotein ligand-1 P-selectin (PSGL-1) on activated T cells.
On the one hand, the invention is an immunoglobulin chain having three sequences: (1) contain, respectively, RSSQSIVHNDGNTYFE, KVSNRFS and FQGSYVPLT (SEQ ID nos 1-3); (2) contain, respectively, SFGMH, YINGGSSTIFYANAVKG and YASYGGGAMDY (SEQ ID No. 4-6); (3) contain, respectively, RASSTVNSTYLH, GSSNLAS and QQYSGYPLT (SEQ ID nos 7-9); (4) contain, respectively, AYYIH, VNPNTGGTSYNPKFKG and SGPYYRYDD (SEQ ID No. 10-12); (5) contain, respectively, RSSQSIVNSNGNTYLE, KVSNRFS and FQGSHVPWT (SEQ ID No. 13-15) or (6) contain, respectively, TNAMNWVRQAPGKGLE, TYYADSVKD and GGSYWYFDV (SEQ ID No. 16-18).
Each of these six groups of sequences corresponds to the three hypervariable sites (hv-sites) light or heavy chain antibody, which binds to PSGL-1, for example the areas of the three antibodies mouse A, B and 9F9 described below in the examples. Below shows the variable regions (V) light and heavy chains of these three antibodies (SEQ ID No. 19-26, hv-sites are underlined and highlighted):
SEQ ID No. 19 (V region light chain antibody mouse A):
SEQ ID No. 21 (V region light chain antibody mouse W):
SEQ ID No. 23 (V region light chain antibody mouse 9F9):
As antigennegative the antibody specificity is determined by the hv-areas of light and heavy chains described above hv-sites allow you to generate derivatives of antibodies that retain antigennegative specificity. As examples of derivatives of antibodies can lead to chimeric antibodies, humanized antibodies, as well as their functional equivalents. Below shows the V region of the light chain (SEQ ID No. 25) and heavy chain (SEQ ID No. 26) gumanitarnogo antibodies A, which respectively include SEQ ID nos 1-3 and SEQ ID nos 4-6:
The field of this invention relates also selected nucleic acid that includes a sequence encoding one of the above immunoglobulin chains. The term "antibody" or "immunoglobulin chain" refers to an isolated polypeptide, i.e. a polypeptide that is substantially separate from other proteins, lipids, and nucleic acids with which it is associated in nature. The polypeptide may comprise at least 50, 70 or 95% of the dry weight of the pure drug. "Isolated nucleic acid" refers to nucleic acid whose structure differs from the structure of any natural nucleic acids or any part of the natural genomic nucleic acid. Thus, the term includes, for example, (a) DNA comprising the sequence of the natural genomic DNA molecule but is not flanked coding sequences that flank the specified part of the molecule in the genome of the organism, where it is found in nature; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in such a way that the resulting molecule is not identical to any natural vector or genomic DNA; (C) a separate molecule such as a cDNA, a genomic fragment, a fragment formed through polymerase chain reaction (PCR), or fragment is restriktsii; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e. a gene encoding a protein. Nucleic acid in accordance with this invention suitable for ekspressirovali polypeptide in accordance with this invention. With this purpose, the nucleic acid may be linked with appropriate regulatory sequences to generate expressing vector.
The vector is a nucleic acid molecule capable of transporting another nucleic acid to which it is linked, and is also capable of independent replication or integration in the host DNA. As examples can be called a plasmid, cosignee and viral vectors. Vector in accordance with this invention includes nucleic acid in a form suitable for expression of the nucleic acid in the cell host. Preferably, the vector includes one or more regulatory sequence operatively linked to the nucleic acid sequence to be expression. As examples of regulatory sequences can lead to promoters, enhancers and other regulators of gene expression (e.g., polyadenylation signals). Regulatory sequences include sequences that regulate the constitutional expressiontreemodel sequence, as well as tissue-specific regulatory and/or induced sequence. The development of such expressing vector based on research, including the selection of the host cell and the desired level of expression. Expressing the vector can be introduced into the cell owner to obtain a polypeptide, a subject of the present invention. The invention also includes cell host containing the above nucleic acid. A host cell is a cell that contains an exogenous coding sequence or non-coding sequence. Exogenous sequence can be introduced into the cell by the method of transfection with calcium phosphate, DEAE-dextran or by electroporation. Such cell-hosts include bacterial cells (e.g., E. coli. Bacillus subtilis and Salmonella typhimurium), yeast cells (yeast - Saccharomyces cerevisiae and Schizosaccharomyces pombe), plant cells (tobacco - Nicotiana tabacum and cotton - Gossypium hirsutum), as well as mammalian cells (hybridoma mice, Chinese hamster ovary and fibroblasts T).
To obtain immunoglobulin chain in accordance with this invention the cell is the master of cultured under conditions that allow expression of the polypeptide encoded by the above nucleic acid, and then the polypeptide isolated from the environment of the cultivation. Instead, it is possible to transcription and translation of nuclein the howling acid the subject matter of this invention in vitro, for example, using the regulatory sequence of the T7 promoter and T7 polymerase.
The invention also encompasses antibody. It forms the first immunoglobulin chain and the second immunoglobulin chain, which contain, respectively, the hv-sections of the light chain and hv-areas of heavy chain antibodies A, B and 9F9 mice mentioned above. Preferably, this antibody is formed by light and heavy chains A. In addition, the subject invention relates another antibody that (1) specifically binds to glycoprotein ligand-1 P-selectin, without breaking the binding glycoprotein ligand-1 P-selectin and P-selectin, (2) when the binding glycoprotein ligand-1 P-selectin (PSGL-1) on activated T-cells induces death of T cells. In one embodiment, the antibody specifically binds to glycoprotein ligand-1 P-selectin person.
In addition, the invention includes another antibody that specifically binds to amino acid residues 115-126 Mature glycoprotein ligand-1 P-selectin person. Preferably, the antibody specifically binds to amino acid residues 117-123. More preferably, it specifically binds to amino acid residues 119-121 - generalizing the typical sequence p is all investigated determinants. Indeed, mutation of one or more of these amino acid residues stops the binding of the antibody. In one example, these antibodies with binding glycoprotein ligand-1 P-selectin on activated T-cell causes the death of such cells.
In one embodiment of the present invention, one of these two antibodies is formed of a light chain and heavy chain, which contain, respectively, SEQ ID nos 1-3 and SEQ ID nos 4-6 (for example, SEQ ID nos 19 and 20 or SEQ ID No. 25 and 26). Further, the invention includes a method of stimulating the death of activated T-cells. The method involves contacting one of the three above described antibodies to activated T-cell, and the binding of an antibody to activated T-cells leads to cell death.
The invention also includes a method of modulating T-cell immune response in the patient's body. The method includes (1) selecting a patient in state or having a risk of developing a condition associated with excessive T-cell immune response, and (2) introducing an effective amount of one of the three above-described antibodies. "Excessive T-cell immune response" means an immune response caused by excessive levels of activated T-cells. "Excess level" means (1) level higher than normal, and (2) a higher level than required in the body given in the om case, even if it does not exceed the normal level. As examples of such conditions can be called inflammatory diseases, autoimmune diseases, allergic diseases and T-cell cancers, as well as the presence or planning allogenic or xenogenic transplantation.
The following is a more detailed description of one or several embodiments of the invention. In the process described other characteristics, objects, effects and advantages of the present invention.
DETAILED description of the INVENTION
This invention is based, at least in part, on the surprising discovery of the ability to cause apoptosis of activated T-cells, which are removed by binding of antibodies or derivatives thereof with a ligand PSGL-1 on activated T-cells. Antibodies and their derivatives can be used to treat conditions caused by excessive or unwanted T-cell immune response or proliferation of T cells.
Accordingly, the invention relates to polypeptides containing hv-lots of light and heavy immunoglobulin chains of the antibodies anti-PSGL-1, as well as nucleic acids, their encoding. As the chain of the immunoglobulin, and nucleic acids provide antibodies and derivatives.
Immunoglobulin chain in accordance with this invention can be is obtained as a synthetic polypeptide or a recombinant polypeptide. To obtain recombinant polypeptide encoding its nucleic acid attached to another nucleic acid that encodes a partner mergers, such as glutathione-8-transferase (GST), the epitope 6x-His protein M13 Gene 3 or C-region of the heavy chain of immunoglobulin. Thus obtained fused nucleic acid introduced into a cell for expression of the protein. Protein is separated from the host cell known in the field methods. The selected protein can be processed further, for example, under the action of enzymes to remove a partner to merge with obtaining the target recombinant polypeptide. In addition, immunoglobulin chain can be obtained from the appropriate host cell by activating endogenous expression of a nucleic acid that encodes a given chain.
The amino acid composition of immunoglobulin chains in accordance with the invention may be varied without disturbing the ability of antibodies to bind to the ligand PSGL-1. For example, a product may contain one or more conservative substitutions of amino acids."Conservative substitution of amino acids" is replaced, in which amino acid residue is substituted by another amino acid residue with a similar side group. Well-known family of amino acid residue with a similar side groups. Family, not only who have an amino acid side groups, have properties of bases (e.g., lysine, arginine, histidine), acidic side groups (e.g., aspartic acid, glutamic acid), uncharged polar side groups (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side groups (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), side groups with beta-branching (e.g., threonine, valine, isoleucine) and aromatic side groups (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus given the balance of non-essential amino acids in the polypeptide is preferably replaced with another amino acid residue belonging to the same family by his side group. In addition, mutations can be introduced randomly along all or part of the polypeptide in accordance with the invention, for example, by the method of saturating mutagenesis, and the resulting mutants can be tested for the formation of antibodies capable of binding to the ligand PSGL-1 in order to detect possible variations of the present invention, as described below in the examples. Thus, the terms "immunoglobulin chain containing SEQ ID No. 19 includes chains of immunoglobulins containing different variations of SEQ ID No. 19.
The above immunoglobulin chain and their variations to pozvolyayuschty antibody in accordance with this invention and its derivatives. The term "antibody" includes whole molecules and their fragments, such as Fab, F(ab')2, Fv, scFv (single-chain antibody) and dAb (domain antibody; Ward and others (1989) Nature, 341, 544). Derived antibody means a protein or protein complex, where the polypeptide is a variation of the present invention. Antibodies and derivatives in accordance with this invention can be obtained by the method of co-expression of the corresponding polypeptide containing hv-lots of light and heavy chains in a suitable cell host, as shown in the examples below. In addition, they can be obtained well-known in the field methods of obtaining monoclonal and polyclonal antibodies and fragments. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
For preparation of antibodies in accordance with this invention the ligand PSGL-1 or antigenic fragment attached to a protein-carrier, such as KLH, mixed with adjuvant, and injected into the body of the animal. Antibodies that are produced in the body of this animal, purified using affinity chromatography of peptides. As these animals tend to use rabbits, mice, Guinea pigs and rats. The choice of adjuvant to enhance the immune response depends on the type of animal, it can be adjuvant's adjuvant (complete and incomplete), inorganic gel, such to the to aluminum hydroxide, surfactants, such as lysolecithin, plutonomy polyols, polyanion, peptides, oil emulsions, hemocyanin lymph snails and dinitrophenol. Useful adjuvants suitable for people who are BCG vaccine (Bacillus Calmette-guérin (BCG) and microorganisms of the genus Corynebacterium parvum.
Polyclonal antibodies are heterogeneous populations of antibody present in the sera of immunized objects. Monoclonal antibodies are homogeneous populations of antibodies to a particular antigen, can be prepared by standard techniques using hybrids. See, for example, Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) Eur. J. Immunol. 6, 511; Kohler et al. (1976) Eur. J. Immunol. 6, 292; and Hammerling et al. (1981) Monoclonal antibodies and T Cell Hybridomas, Elsevier, N.Y. In particular, monoclonal antibodies can be obtained by any method which allows the production of antibody molecules stable cell strain in culture, such as described in U.S. Patent No. 4376110; The human B-cell hybridoma technique (Kosbor et al. (1983) Immunol Today 4, 72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026), and The EBV-hybridoma technique (Cole et al. (1983) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may belong to any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, including any subclass of the class data. Hybridoma producing monoclonal antibodies specific to this invention, can be cultivated in vitro or in vivo. Due to the ability of the product is activated monoclonal antibodies with high titer in vivo, this method of production is effective.
In addition, you can use methods developed to obtain chimeric antibodies. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604 and Takeda et al. (1984) Nature 314, 452. A chimeric antibody is a molecule, different parts of which obtained with different species of animals, such as these, where V region is a monoclonal antibody mouse, and With the region - human immunoglobulin. In addition, techniques described for the production of single-chain antibodies (U.S. Patent No. 4946778 and 4704692), can be modified to obtain a library of phage single-chain Fv antibodies. Single-chain antibodies are formed by linking the fragments of the heavy and light chain Fv plot through amino acid bridge. Moreover, fragments of antibodies can be formed by known methods. For example, such fragments can be represented as (but not exclusively) fragments, F(ab')2that can be obtained pepsinogen hydrolysis of molecules, antibodies, and Fab fragments, which are created when restoring disulfide bridges fragments F(ab')2. Antibodies can also humanize the methods described below in the examples, or by other methods known in this field. For example, it is possible humanization of monoclonal antibodies with the desired SP is cifically binding on an industrial scale (Scotgene, Scotland and Oxford Molecular, Palo Alto, Calif). In the scope of the present invention is applicable fully humanized antibodies, for example, expressed in the organism genetically modified animals (see, for example, Green et al. (1994) Nature Genetics 7, 13 and U.S. Patent No. 5545806 and 5569825).
In addition, the subject invention includes a method of stimulating the death of activated T-cells, for example, by making activated T-cells in vitro in contact with the antibody specific to the invention, and effective amount of the antibodies in the patient's body, in this need. This effect can be used if the organism has arisen or may arise a condition associated with excessive or unwanted T-cell immune response, for example, if the patient suffers from an autoimmune disease, in the case of transplants, allergic diseases and T-cell cancers. This method can be applied separately or in combination with other drugs and therapies.
The term "impact" refers to the introduction of the composition to a patient with the purpose to cure, relieve or ease, recovery, prevent or reduce the intensity of disturbances or symptoms of disorders, secondary painful condition caused by the violation of, or predisposition to the occurrence of the violation. "Effective amount" considers the I amount of the composition, able to cause the desired medical effect in the body of the patient.
Examples of diseases for the indicated effects: diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren syndrome, Crohn's disease, aphthous ulcer, inflammation of the iris of the eye, conjunctivitis, keratoconjunctivitis, diabetes, inflammatory bowel disease, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug-induced dermatitis, erythematous leprosy nodules, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, true red cell anemia, idiopathic thrombocytopenia, polyandria syndrome's granulomatosis, chronic active hepatitis, malignant exudative erythema, idiopathic trophic atty, planus, toxic goiter, benign Wegener, primary biliary cirrhosis, ass the s uveitis, interstitial pulmonary fibrosis, graft versus host disease, cases of transplantation (including transplantation of allogenic or xenogenic tissue, such as bone marrow, liver, or transplantation of any organ or tissue, allergies, such as atopic Allergy, AIDS, and T cell neoplasm, such as leukemia or lymphoma.
According to one of the methods in vivo, therapeutic composition (e.g., a composition comprising antibodies in accordance with this invention) is injected into the patient. In General, antibodies are suspended in a pharmaceutical carrier (e.g., physiological saline) and administered orally or in the form of intravenous, subcutaneous, intramuscular, intrathecal or intraperitoneal injection; intrarectal, intrawaginalno, intranasally, intragastrically, vnutritrahealno or intra-lungs.
The dosage depends on the selected route of administration; the nature of the dosage form; specific diseases; size, weight, surface area, age and sex of the patient; other drugs taken simultaneously; and from the doctor's appointment. Doses are in the range of 0.01-100.0 mg/kg Significant changes in dosage may be associated with a variety of existing compositions and different efficiencies of various routes of drug administration. For example, doses of the oral should be higher than by intravenous infusion. Changes in dose level can be established by standard empirical optimization procedures, well known in this field. Encapsulation of the drug in the appropriate media (e.g., polymer microparticles or implantable devices) may increase the efficiency of administration, especially in the case of oral administration.
In addition, this invention includes pharmaceutical preparation containing pharmaceutically acceptable carrier and an effective amount of the antibodies in accordance with the invention. Drug suitable for the treatment of the abovementioned diseases. Pharmaceutically acceptable carrier can be a solvent, dispersion medium, coating, antibacterial and antifungal agent, an isotonic vehicle or the vehicle slow down the absorption.
Medicinal product in accordance with this invention can be made in the form of various dosage forms for specific routes of administration using standard methods. For example, for oral administration, the drug can be encapsulated in a capsule, gel, or tablet. Capsules may contain any of the usual pharmaceutical materials, such as gelatin or cellulose. Tablets can be formed in accordance with generally accepted p is acticoa by pressing a mixture of the drug with a solid filler and a lubricant. Solid filler can be a starch and bentonite with sugar. It is also possible the introduction of the drug in tablet form with a hard shell or capsules with a binder, such as lactose or mannitol, a conventional filler and teletrauma additive. The introduction of drugs parenteral way. Examples of forms for parenteral administration are aqueous solutions, isotonic saline, 5% glucose or any other excipient known in the pharmaceutical industry. For example, for injection of a medicinal product suitable cyclodextrins or other solubilizing components, well known in this field.
The efficacy of the medicinal product in accordance with this invention can be evaluated both in vitro and in vivo (see examples below). You can test the drug's ability to cause the death of activated T cells in vitro. For studies of the drug in vivo it is introduced in the body of animals (e.g. mice) and then measure therapeutic effect. On the basis of the received results to determine the appropriate interval, dosage and method of administration.
The specific examples given below are given for illustrative purposes, they do not limit in any way the description of the present invention. It is assumed that h is on the specialist, qualified in this area will have the opportunity to fully apply the invention on the basis of this description without further elaboration. All publications are fully incorporated by reference.
EXAMPLE 1: Mouse monoclonal antibody A, B and 9F9
Mouse monoclonal antibodies that specifically bind to PSGL-1 person (hCD162), obtained by the standard method. More specifically, mice were immunized membrane fraction of human T cells activated by phytohemagglutinin, and cut to obtain a hybridoma cell lines. Supernatant obtained from hybridoma cell lines were subjected to screening for binding to cells Cho (Chinese hamster ovary), which stably expressed hCD162. Those lines which have produced antibodies to bind to the cells SNO expressing hCD162, but not with the parent cells SNO identified, was subclinically and further analyzed as described below.
Among the identified lines were: m152-A, m166-V and m128-9F9. They were produced antibody to IgG1, respectively A, B and 9F9. Analysis by the method of Western blot turns showed that these three antibodies bind contained in the lysate activated T-cells protein that can be detected using antibodies anti-hCD162 (kpl-1, PharMingen, San Diego, California).
These three antibodies were tested for ability to cause apoptosis of activated T cells. Supernatant culture containing the monoclonal antibodies secreted by three hybridoma cell lines were respectively incubated with either inactivated human T cells (Day 0), or human T cells activated in vitro (Day 7) for 6 hours. Then cells were stained with annexin V and analyzed by FACS method (cell sorting with excitation fluorescence). CD3-positive cells were selectively for the purpose of counting or human T cells activated in vitro or dormant human T cells. Apoptotic cells showed positive staining with annexin V. Table 1 shows the proportion of apoptotic T cells among all studied T cells.
The percentage of apoptotic T cells
|No processing||Anti-myc||m128-9F9||No processing||Anti-myc||m152-A||M-W|
|Day 0||4,17||of 5.82||18,18||15,52||5,23||to 6.57|
|Day 7||12,63||made 13.36||28,71||24,18||results were 23.08||51,66||49,44|
These results show that antibodies mouse A, B and 9F9 (1) are specific to hCD162 and (2) may contact with activated T cells and to induce apoptosis of activated human T cells, but not dormant T cells.
Apoptotic analysis is also conducted with respect to the mononuclear cells of peripheral blood (RVMS)activated by phytohemagglutinin. Discovered that antibodies induced apoptosis only in activated T-cells, but not in the dormant T-cells, B-cells and neutrophils.
It is known that antibodies that Deplete T cells, such as anti-CD3, can cause the production of soluble factors. Therapy with the use of such antibodies usually leads to harmful syndrome cytokinesis. To check, cause antibodies anti-PSGL-1-like is associated with cytokinesis side effects, svezhesobrannye mononuclear cells peripheral blood is the ne (RVMS) were grown together with A within 24, 48 or 72 hours. Then determined the levels of the cytokines in the supernatant. In cells RVMS activated by phytohemagglutinin, produced significant amounts of IL-2, TNF-a and IFN-y (positive control), whereas the content of these cytokines in cells treated A, did not respond to discovery. These results confirmed that the anti-PSGL-1 has no effect or little effect on dormant peripheral blood cells in both the initiation of apoptosis and cell activation.
Since the above-described antibodies selectively induce apoptosis in activated T-cells without harmful effects on dormant T cells or other immune cells, it is unlikely that their introduction into the organism resulted in lymphopenia or wide immunodeficiency, as in the case of anti-CD3 or immunosuppressants.
Mapping the epitopes of antibodies anti-CD162
With the aim of mapping binding epitopes mouse A, B and 9F9 to the human gene CD162 expressed and purified a number of fused proteins corresponding to different parts of the human gene CD162. The interaction between the fused proteins and data monoclonal antibodies studied by the method of the sandwich enzyme-linked immunosorbent assay (ELISA).
Briefly, the fragments corresponding to various parts of the human gene CD162, were expressed as a fusion protein with a region of the heavy chain gamma 1 immunoglo Olina person in E. coli. cDNA encoding the constant region of the heavy chain gamma 1 immunoglobulin was amplified by PCR with primers having the BglII site and the BamHI site. The PCR product was cut restrictase BglII and BamHI and subcloned into the vector pet-32A (producer - Novagen), which was cleaved by the same enzymes. Then cDNA encoding different parts hCD162 were amplified by PCR with primers containing NdeI site at the 5' end and a BglII site - in the end 3'. The PCR products were cleaved with appropriate enzymes and merged without shifting the reading frame sequence that encodes a constant region of the heavy chain gamma 1 immunoglobulin in the vector pet-32A. The primers used in each of the structures listed in Table 2 and the sequence of the primers are shown in Table 3.
The above-described expression constructs were introduced into a strain of Escherichia coli BL21 (DE3). Transformed cells were collected 6 hours after injection IPTG (2 mm) and resuspendable in a solution of PBS (saline phosphate buffer). After cell disruption by ultrasound and centrifugation at 14000 g for 10 minutes the resulting supernatant were collected for purification of fused proteins. More precisely, supernatant was the first incubated with beads protein G or protein a for 3 hours at 4°C. Then the granules were besieged by centrifugation at 3000 g and washed 5 times with wash buffer I (0.05% Triton X-100, 50 mm Tris-HCl, pH 8.5, 400 mm NaCl, 1 mm CaCl2and 1 mg/ml of egg albumin) and wash buffer II (0.05% Triton X-100, 50 mm Tris-HCl, pH 8.5 and 150 mm NaCl). Then associated proteins were suirable buffer for elution containing 0.1 M salt glycine-HCl pH 2.7, neutralized in 1 M Tris-HCl, pH 8.6. All purified fused proteins was quantitatively determined using the test kit proteins of the company Bio-Rad (Bio-Rad Laboratories, catalog No. 500-0006) and tested method SDS page electrophoresis in the presence of sodium dodecyl sulfate (method SDS-PAGE).
To study the interaction between fragments hCD162 and each of the antibodies A, 9F9 and V conducted sandwich enzyme-linked immunosorbent assay (ELISA). In wells of 96-well microtiter tablets were adsorbed goat anti-human IgG antibody (Southern Biotechnology, catalog No. 2040-01) (2 μg/ml, 50 μl/cell) over night at 4°C. the plates were blocked by incubation with 0.25% BSA in PBS solution (150 μl/cell) for 1 hour at 37°C. Then the blocked plates were incubated for 2 hours at room temperature with fused proteins containing different fragments of the human gene CD162 (2 μg/ml). After 4 times washing with a solution of PBS containing 0.05% Tween 20 (PBST solution), the tablets were incubated with the test antibody (2 μg/ml) for the tion 1.5 hours at room temperature. After incubation tablets 4 times washed with PBST solution. Then for each cell was added 50 μl diluted in 1-3000 time goat antimisting IgG conjugated to alkaline phosphatase (Southern Biotechnology, catalog No. 1031-04), and the plates were incubated for 1 hour at 37°C. the Enzymatic reaction was performed by adding 50 μl of substrate solution of alkaline phosphatase (1 tablet substrate of alkaline phosphatase, dissolved in 5 ml of substrate buffer containing 0.012 M Na2CO3, 0.16 M NaHCO3and 1 mm MgCl2, pH 8.6) measuring the absorption at 405 nm.
It was found that antibodies V and 9F9 able to interact with all fused proteins containing residues 50 to 60 Mature human gene CD162, indicating that the epitopes of antibodies V and 9F9 are in the range of residues 50 and 60. Unlike 9F9 and V, antibodies A been associated only with the merged protein spanning residues 42 through 319, but not with the merged protein spanning residues 42-119, indicating that the epitope A is located between residues with 119 319. Then the location of the epitope A was adjusted to between residues 115 to 126. Replacement of one amino acid at position 120 (Glu→Arg) weakened the interaction between antibodies A and fused protein, which shows that the initial contact area A with the human genome CD162 is in position 120 or near his summe is of zi and that the Glu residue is essential for this interaction.
Slit proteins, covering different parts of the human gene CD162, were also expressed in mammalian cells and tested for interaction with A. Fragments encompassing these areas have been expressed in the form of a fused protein with a constant region of the heavy chain gamma 1 immunoglobulin in mammalian cells. First of all, cDNA encoding the constant region of the heavy chain gamma 1 immunoglobulin, was introduced into the vector pcDNA3 (producer - Invitrogen). Then cDNA encoding different parts hCD162 were amplified by PCR with primers containing a BamHI site at the 5' end and a XhoI site at the end 3'. The PCR products were cleaved with appropriate enzymes and subcloned into the vector pet-32A, containing the gene for the constant region of the heavy chain gamma 1 immunoglobulin. Names and sequences of all primers are shown above in Tables 2 and 3.
These vectors for expression in mammalian cells were transliterowany cells COS-7 using reagent Lipofectamine 2000 (producer - hivitrogen, catalog No. 11668-027) according to the method described in the manufacturer's manual. Transfetsirovannyh cells were grown in medium with low content of immunoglobulin (producer - Invitrogen, catalog No. 16250-078). Expressed proteins were purified and subjected to sandwich immunofermentnom is in the assay (ELISA), as explained above.
The results of the analysis using ELISA method shows that only fused proteins containing residues with 94 148 can communicate with A. These results are consistent with the idea that the epitope in A is located between residues 115 and 126.
All the above results indicate that the epitopes of antibodies 9F9, V and A are dependent protein, but not dependent modification of carbohydrate, as all three of these antibodies bind expressed in bacteria fused proteins. They also show that although antibodies A, 9F9 and V have similar properties in terms of binding specificity and function of inducing apoptosis in activated T-cells, they act through different areas of human gene CD162 and behave in a different way.
EXAMPLE 2: Chimeric antibodies A, B and 9F9
Cloning of variable (V) regions of light and heavy chains of the antibodies anti-SR
cDNA encoding V region light and heavy chains (VLand VH) antibodies A, B and 9F9, amplified by PCR. the 3'primers were hybridized to C-sections, and 5'-primers hybridized to the G-chain attached to the cDNA using terminal deoxytransferase. The PCR fragments were cloned in the vector PCR-II (producer - Invitrogen). For each circuit was sequencially and compared several clones. Was selected sequence to depict Alanna largely independent clones. Then the translated amino acid sequence was analyzed to ensure that the selected sequence exhibits properties typical V region light and heavy chains of murine antibodies and belongs to the selected subtype. After this were hypervariable sites (hv-plots) by comparison of the translated amino acid sequence with generalizing a typical sequence for each subtype. The name and sequence for each of the used primers are shown above in Tables 2 and 3. The identified amino acid sequence for the V regions of light and heavy chains A, B and 9F9 (SEQ ID No. 19-24) are given in the Abstract.
To generate vectors for expression of a chimeric antibody cDNA encoding the VLand VHlots A, B and 9F9, were amplified by PCR using primers incorporating the sequence 5'-signal peptide and 3' donor splanirovano fragment. The primers introduced an XbaI sites at both ends of the PCR products, which were then cut by the enzyme XbaI and legirovanyh with vector pVK, pVg1, pVg2, or pVg4, is decomposed by the enzyme XbaI. More specifically, VLsections cDNA A, B and 9F9 were subcloned in plasmid pVk. The indicated plasmid contains the CMV promoter and a sequence encoding a human is Constantinou region light chain. Region VHcDNA A, B and 9F9 were subcloned in the plasmid pVg1, pVg2, or pVg4. Each of the three plasmids contained the CMV promoter. They also contained, respectively, From-region heavy chain IgG1, IgG2 and IgG4 human.
Each of the three above described plasmids encoding the light chain was cotransfection with a plasmid that encodes a heavy chain in cells COS-7. Collected supernatant transfected cells. Chimeric antibodies in supernatant analyzed for the ability to bind human gene CD162 and to induction of apoptosis of activated T cells.
It was found that all chimeric antibodies derived from antibodies A, B and 9F9 related transfitsirovannykh cells Sp2/0, but not parental cells Sp2/0, stably expressed human gene CD162, thus showing that the binding specificity of the human gene CD162. Moreover, it was found that chimeric antibodies caused apoptosis in T-cells, activated within 7 days, indicating the preservation of this function in case of their murine counterparts.
For obtaining humanized antibodies were used mouse antibodies A, hv-areas which were grafted to the human frame. To save the binding capacity and specificity, it is important not to disturb the conformation of the V region for the transplant hv-past the Cove on the human frame. For the selection of donor human framework amino acid sequences of the V regions of light and heavy chain A compared with those of the 50 mouse antibodies, which were humanitarian.
Found that murine antibody mDREG-55 has a high homology sequence of the V region of the mouse antibody A as in the light and heavy chains. Below is a comparison of sequences of mouse antibodies A with the indicated antibody mDREG-55 (hv-plots highlighted):
comparison of light chain:
comparison of heavy chain:
Murine antibody DREG-55 - monoclonal IgG1 antibody against L-selectin. Sequence VLand VHregions of mouse antibodies A were homologous, respectively, to 64.3% (only for frame: 73.8%) and 70% (only for frame: 81.6%) sequences of mouse antibody DREG 55. Humanitariannet antibody DREG-55 (HuDREG-55) was designed using frame sequence VLand VHareas of human antibodies Gal. For humanization of mouse antibodies A frame sequence light and heavy chains of human antibodies Gal used to replace similar plots mouse antibodies A.
Humanized light and heavy variable regions A linked each 4 pairs of synthetic is such oligonucleotides (length ~80 bases). The oligonucleotides in each pair were blocked for approximately 20 nucleotides. Nucleotide sequences were selected and synthesized to encode the protein sequences of humanized variable regions, including signal peptides. The layout and gene amplification was carried out in four stages: (1) four pairs of complementary oligonucleotides were hybridized and enlarged fragment of maple in the 4 separate reactions; (2) received 4 fragment dzanc pairs were mixed, was denaturiruet, rehybridization and increased in two separate reactions; (3) received 2 fragment dccnk mixed, denaturiruet, rehybridization and increased with the formation of the final full dccnk and (4) the DNA amplified by PCR with primers to introduce XbaI site at both ends. The PCR fragment was then removed using XbaI and introduced into the vector pVk and pVg4 out XbaI. Then in those locations where interactions between hv-land and the frame was important residues Gal antibodies was again replaced on the remains of mouse antibodies A (i.e. I62V and D74H). Below shows a comparison of mouse antibodies A and gumanitarnogo A (Nia) mDREG-55, which plots V62 and N underlined.
Resulting plasmids encode the heavy and light chain gumanitarnogo A. These plasma is IDA were then cotransfection cells COS-7. Collected supernatant with cultured cells. Humanized A in supernatant was tested for the ability to bind to transfitsirovannykh cells SNO expressing hCD162, and to induce apoptosis in T-cells, activated within 7 days. It is shown that the data capacity stored.
Obtaining chimeric and humanized antibodies
Generated cells producing humanized and chimeric antibodies. More specifically, cells Sp2/0 (Sp2/0-Agl4; ATCC CRL 1581) were transliterowany with the appropriate plasmids by electroporation with apparatus Gene Pulser (Bio-Rad Laboratories) at 360 V and 25 μf in accordance with the methods of the manufacturer. Before transfection, the plasmids were linearized with the enzyme BamHI. All transfections were carried out with 107cells in a solution of PBS and 20 μg of plasmid DNA. Cells after each transfection were placed in two 96-well plate to cultivation. After 48 hours was applied elective medium (DMEM - modified according to the method of Dulbecco Wednesday Needle - 10% FBS/ gipoksantin/thymidine) and 1 µg/ml mycophenolic acid. Antibody productive cells were separated, the presence of antibodies in the culture supernatant was determined using sandwich enzyme-linked immunosorbent assay (ELISA).
The separated cells were cultured in serum-free medium (low Ig), collecting the supernatant of the culture. Antibodies were purified is, passing through a column of protein A-Sepharose CL-4B, and washed 5 times with buffer I (0.05% Triton X-100, 50 mm Tris-HCl, pH 8.5, 400 mm NaCl, 1 mm CaCl2and 1 mg/ml of egg albumin) and wash buffer II (0.05% Triton X-100, 50 mm Tris-HCl, pH 8.5 and 150 mm NaCl). After that, the bound antibodies were suirable buffer for elution containing 0.1 M salt glycine-HCl pH 2.7, neutralized in 1 M Tris-HCl, pH 8.6.
Changes in the affinity
Binding capacity of the above-described murine, chimeric and gumanitarnogo antibodies A was determined by the method of competitive binding.
Mouse antibodies A was biotinilated action system the EZ-Link sulfo-NHS-Biotin (manufacturer Pierce Biotechnology, catalog No. 21217). Briefly, 0.5 mg (3.3×10-6nmol) mouse antibodies A was dissolved in 187 μl PBS and mixed with 6.8×10-5nmol system sulfo-NS-Biotin. The mixture was incubated on ice for 2 hours, after which the free Biotin was removed by dialysis at 4°C against PBS, leaving for the night. Biotinylated mouse antibodies A kept at 4°C until use.
Transfetsirovannyh cells Sp2/0, stably expressing the human gene CD162, were used as the source of human antigen CD162. Biotolerance mouse antibodies A was used as a labeled drug. The increasing number of competitive antibody (murine, chimeric or gumanitarnogo A) was mixed with 35 ng etilirovannym mouse antibodies A and incubated with 1×10 -5cells Sp2/0, expressing CD162, for 1.5 h at 4°C with constant shaking. After washing, to the mixture was added to the secondary antibody streptavidin-peroxidase (manufacturer : Becton Dickinson Immunocytometry System Inc., catalog No. 349023). After incubation for 45 minutes at 4°C. cells were again washed and resuspendable in 300 μl of PBS-1% FBS, and then analyzed by FACS method (cell sorting with excitation fluorescence).
It was found that premaxilla competitive concentration of mouse antibodies A is 3.72 µg/mg, and chimeric and humanized antibodies, respectively approximately 5.71 mg/ml and 4.51 μg/ml. the results show that the affinity of murine, chimeric and humanized antibodies A comparable among themselves, in other words, the binding capacity (Ka) for mouse A is 4.03×107M-1and for chimeric and humanized antibodies A respectively 2.62×107M-1and 3.33×107M-1.
By the method of competitive analysis studied the interaction of the above three types of mouse antibodies, PSGL-1 and P-selectin.
P-selectin is a primary high-affinity ligand for PSGL-1 on most leukocytes. To determine whether these three types of antibodies to prevent binding of P-selectin with PSGL-1, measured step the HB binding of P-selectin person with activated T-cells in the presence of three kinds of antibodies. As a positive control was used antibodies KPL-1, which block the interaction of P-selectin with PSGL-1.
Mononuclear cells of peripheral blood (RVMS) activated by 1% of phytohemagglutinin within 2 days and maintained in medium containing IL-2 for 3 days. Cells were incubated with titrated antibodies 9F9, A, V, KPL-1 (antagonist PSGL-1) or with a control antibody (A) for 30 min, and then added recombinant P-selectin person (1.25 µg/ml). Binding of P-selectin with activated T-cells was measured using FACS analysis with anti-P-selectin-fluoresceinisothiocyanate.
In accordance with previous reports, antibodies KPL-1 at low concentration (0.31 µg/ml) almost completely blocked the binding of P-selectin with activated T-cells. Antibodies V prevented the binding of P-selectin with activated T-cells as effectively as KPL-1, while in the case 9F9 to achieve equal effect requires a higher concentration of antibodies. Indeed, 0.08 μg/ml of KPL or V reduces binding by 50%, and 9F9 this value is 5 µg/ml Antibody I had no inhibitory effect on the binding of P-selectin even at a concentration of 20 μg/ml. moreover, it was found that these antibodies contribute to the binding of P-selectin with PSGL-1. Thus, it is possible Zack is ucity, the mechanisms of antibody binding A and P-selectin with PSGL-1 on activated T-cells of different.
The lack of competitive binding of antibodies A and P-selectin with PSGL-1 allows us to conclude that the introduction of A in vivo, apparently, will not affect natural immunity by inhibiting P-selecteddevicename update leukocytes.
Reported the expression of PSGL-1 low platelets. We studied the inuence of antibodies A on platelets. Discovered that antibodies do not increase and not reduce platelet aggregation in humans.
EXAMPLE 3: Monoclonal antibodies hamster TAU against murine PSGL-1
Monoclonal antibodies TV to murine PSGL-1 were obtained similarly to the method described in Example 1. They caused apoptosis of T cells in vitro and eliminated T cells in vivo. To clarify, inhibit whether these antibodies binding between mouse PSGL-1 and murine P-selectin, conducted competitive analysis according to the method similar to that described in Example 2. It was found that TV did not inhibit the binding of murine P-selectin with murine PSGL-1, even at concentrations up to 20 μg/ml
EXAMPLE 4: mouse Monoclonal antibodies V, S, E, W, E and 18D12
Characterized by additional monoclonal antibodies to PSGL-1 person: V, S, E, W, E and 18D12. When binding to activated T-cells they all produced GI is spruce activated T-cells. To clarify, block whether they interaction between PSGL-1 and P-selectin, conducted competitive analysis, as described in Example 2. Found that these antibodies have a weak inhibitory effect on binding to human P-selectin from human PSGL-1 or do not have such effects even at the highest investigated concentration (5 µg/ml).
VARIANTS of the INCARNATION
The characteristics stated in this description can be applied in any combination. Each of these signs can be replaced with alternative sign, engaged in the same, equivalent or similar function. Thus, unless otherwise stated, each of these signs is only an example of a series of equivalent or similar features.
The description allows the technician to easily identify the essential characteristics of this invention and to carry out various changes and modifications of the invention without leaving the scope of its meaning and scope, to adapt the invention to various conditions of use. Thus, the embodiments also relate to the subject matter of this invention
1. The antibody that specifically binds to glycoprotein ligand-1 P-selectin (PSGL-1), where the antibody upon binding with PGSL-1 on activated T-cell causes the death enabled the T-cells, and where the antibody comprises a light chain and heavy chain, respectively, containing SEQ ID nos: 7-9 and SEQ ID NO: 10-12.
2. The antibody according to claim 1, characterized in that the light and heavy chain contain, respectively, SEQ ID nos: 21 and 22.
3. The antibody that specifically binds to glycoprotein ligand-1 P-selectin (PSGL-1), where the antibody upon binding with PGSL-1 on activated T-cell causes the death of the activated T-cells, and where the antibody comprises a light chain and heavy chain, respectively, containing SEQ ID nos: 7-9 and SEQ ID NO: 10-12.
4. The antibody according to claim 3, characterized in that the light and heavy chain contain, respectively, SEQ ID nos: 23 and 24.
5. The antibody that specifically binds to glycoprotein ligand-1 P-selectin (PSGL-1), without preventing binding of PSGL-1 with P-selectin, and where the antibody upon binding with PGSL-1 on activated T-cell causes the death of the activated T-cells, and the antibody includes a light chain and heavy chain, respectively, containing SEQ ID nos: 1-3 and SEQ ID nos: 4-6.
6. The antibody that specifically binds to glycoprotein ligand-1 P-selectin (PSGL-1), where the antibody comprises the light and heavy chain, respectively, containing SEQ ID nos: 1-3 and SEQ ID nos: 4-6, and where the antibody upon binding to PSGL-1 on activated T-cell causes the death of the activated T cells.
7. The antibody according to claim 6, characterized in that the light chain and the heavy price the b contain, respectively, SEQ ID nos: 25 and 26.
8. The antibody that specifically binds to amino acid residues 115-126 glycoprotein ligand-1 P-selectin person (PSGL-1), where the antibody upon binding to PSGL-1 on activated T-cell causes the death of the activated T-cells, and the antibody includes a light chain and heavy chain, respectively, containing SEQ ID nos: 1-3 and SEQ ID nos: 4-6.
9. The antibody of claim 8, characterized in that it specifically binds to amino acid residues 117-123.
10. The antibody according to claim 9, characterized in that it specifically binds to amino acid residues 119-121.
11. Way to stimulate the death of activated T-cells, comprising contacting the antibody according to any one of pp.5-10 with an activated T-cell, and the binding of an antibody to an activated T-cell causes the death of the activated T cells.
12. Method of modulating T cell immune response in a patient, including:
the choice of the patient, which is in the state or having a risk of developing a condition associated with excessive T-cell immune response and introducing into the patient an effective amount of the antibody according to any one of pp.5-10.
13. The method according to item 12, wherein the specified condition is an inflammatory disease, autoimmune disease and allergic disease.
14. The method according to item 12, otlichuy is the, the specified condition is a T-cell cancer.
15. The method according to item 12, wherein the specified state is a rejection of allogeneic or xenogeneic transplant.
16. A host cell expressing the antibody according to any one of pp.5-10, containing nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 19, 20, 25, or 26.
17. A host cell according to item 16, characterized in that it is a bacterial cell, yeast cell, plant cell, insect cell or a cell of a mammal.
18. A host cell according to 17, wherein the mammal cells is a cell hybridoma.
19. A host cell expressing the antibody according to any one of claims 1 and 2, containing nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 21 or 22.
20. A host cell according to claim 19, characterized in that it is a bacterial cell, yeast cell, plant cell, insect cell or a cell of a mammal.
21. A host cell according to claim 20, characterized in that the mammal cells is a cell hybridoma.
22. A host cell expressing the antibody according to any one of p and 4, containing nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 23 or 24.
23. Glue the ka-host on p.22, characterized in that it is a bacterial cell, yeast cell, plant cell, insect cell or a cell of a mammal.
24. A host cell according to item 23, wherein the mammal cells is a cell hybridoma.
25. The antibody according to claim 6, characterized in that the light chain and heavy chain contain, respectively, SEQ ID nos: 19 and 20.
26. The selected nucleic acid encoding the antibody according to any one of claims 1 to 10, containing the nucleotide sequence corresponding to the amino acid sequence represented in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25 or 26.
27. The expression vector containing the nucleic acid according p.
28. The selected nucleic acid encoding a variable domain of the antibody according to any one of claims 1 to 10, containing SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.
29. The expression vector containing the nucleic acid according p.
30. A host cell expressing the antibody according to any one of claims 1 to 10, containing nucleic acid encoding the amino acid sequence shown in SEQ ID nos: 1-3, SEQ ID nos: 4-6, SEQ ID NO: 7-9, SEQ ID nos: 10-12, SEQ ID NO: 13-15 or SEQ ID nos: 16-18.
31. A host cell according to item 30, characterized in that it is a bacterial cell, yeast cell, plant cell, insect cell or a cell of a mammal.
32. A host cell according p notable is the cell of a mammal is a cell hybridoma.
33. The selected antibody that specifically binds to glycoprotein ligand-1 P-selectin, and the antibody contains (i) a light chain comprising the variable region with the amino acid sequence SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 21 or SEQ ID NO: 23, associated with the constant region of the light chain of the Kappa immunoglobulin, and (ii) a heavy chain comprising the variable region with the amino acid sequence SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 22 or SEQ ID NO: 24.
34. The antibody according p, characterized in that it contains a constant region of the heavy chain IgG1, IgG2 or IgG4 human.
35. Pharmaceutical composition for treatment of a condition associated with excessive or unwanted T-cell immune response containing an effective amount of the antibody according p or 34.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, more specifically to expression constructs, and may be used for immunoglobulin expression. An expression vector contains one open reading frame (sORF) insert which contains a first sequence of nucleic acid coding a first polypeptide; a first intermediate sequence of nucleic acid coding a first protein cleavage site containing an autoprocessing element with an intein segment providing proteolytic sORF polypeptide cleavage between the first polypeptide and the intein segment and the second polypeptide, but not ligation of said first polypeptide with said second polypeptide; and a second sequence of nucleic acid coding the second polypeptide. The expression vector is able to express a mammalian polypeptide coding sORF and cleaved in said first protein cleavage site in a host cell; consisting of the first polypeptide - an immunoglobulin heavy chain, and the second polypeptide - an immunoglobulin light chain able to be assembled into a multimer.
EFFECT: invention provides functional antibody production with 'correct' setup and assembly.
40 cl, 9 dwg, 57 tbl, 4 ex
SUBSTANCE: cell population is recovered from peripheral blood or umbilical blood by a method involving positive cell secretion from peripheral blood or umbilical blood able for immune reaction with an antibody specified in a group consisting of aHTH-CD44, anti-CD11b and their combination. The recovered myeloid-like cell population contains the cells expressing CD44 antigen, CD11b antigen and hypoxia-induced factor lα (HIF-lα). The prepared population is used for recovery and stabilisation of the functional vasculature, stimulation of microgliacyte formation, and for stimulation of physiologic intra-retinal vascularisation of hypoxic retinal tissue in simultaneous suppression of abnormal preretinal vessel formation.
EFFECT: invention enables preparing the cells with vasculotrophic and neurotrophic activity which possess a considerable therapeutic potential in the intraocular introduction into a mammal suffering a degenerative ocular disease.
7 cl, 63 dwg, 2 tbl, 21 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, particularly a polypeptide and its homodimer which possess agonist activity with respect to a growth hormone receptor, a nucleic acid molecule coding them, a vector which involves said nucleic acid, a cell of expression of said polypeptide, a pharmaceutical composition and a method with the use of said polypeptides for treating growth hormone deficiency. The polypeptide has an amino acid sequence presented in SEQ ID NO: 11 or 12. The homodimer consists of two polypeptides consisting of SEQ ID NO: 11 or 12. The nucleic acid molecule consists of a nucleic acid sequence presented in SEQ ID NO: 4. The pharmaceutical composition to be used in treating growth hormone deficiency contains said polypeptide or homodimer and an excipient or a carrier. The method of treating growth hormone deficiency involves introducing an effective amount of said polypeptide or homodimer.
EFFECT: invention provides creating the polypeptide which possess agonist activity on growth hormone receptor, and it is effective in treating growth hormone deficiency.
17 cl, 9 dwg, 1 tbl, 1 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of molecular biology and biotechnology and can be used in medicine and in pharmaceutical industry. RNA molecule, capable of target-specific RNA interference, represents double-stranded RNA molecule, 23 nucleotides long, which has 3'-overhang from 1-5 nucleotides. It is obtained by joining two RNA strands and used for obtaining pharmaceutical composition. When introduced into multicellular eukaryotic organism or in a cell of multicellular eukaryotic organism, said RNA molecule ensures promotion of target-specific RNA interference and leads to reduction of target-gene expression level or to target-gene knockout. Method of promoting target-specific RNA interference by means of said RNA molecule is applied for determination or modulation of gene function. Cell, containing endogenic target nucleic acid, RNA molecule, capable of target-specific RNA interference, and exogenic target nucleic acid, is used in analytical procedures.
EFFECT: application of invention ensures target-gene silencing, mediated by target-specific RNA interference.
40 cl, 23 dwg, 3 ex
SUBSTANCE: obtained is protein complex, possessing GPCRα1L affinity to ligand, which includes GPCRα1A and polypeptide with sequence of amino acids SEQ ID NO:1. Binding of said G-protein-conjugated receptor with polypeptide alters ligand affinity of the receptor. Also claimed are methods of screening agonists or antagonists of G-protein-conjugated receptor with application of transformant, in which said altered G-protein-conjugated receptor is expressed.
EFFECT: carrying out analysis of many supposed G-protein-conjugated receptors with still unknown structure.
12 cl, 3 dwg, 2 tbl, 4 ex
SUBSTANCE: disclosed is a nanobody against tumour necrosis factor-alpha (TNF-alpha) which has 4 framework regions FR and 3 HCDR. Described are versions of a nanobody-based polypeptide, each capable of binding with TNF-alpha. One version of such polypeptides contains at least one nanobody directed against human serum albumin. Corresponding coding nucleic acids are described. The invention also discloses a host cell for expression a nanobody and a host cell for expressing a polypeptide, each containing a corresponding nucleic acid; a method of producing a nanobody and a method of producing a nanobody-based polypeptide, where each of the methods employs the corresponding cell. Use of the nanobody to obtain a medicinal agent and a pharmaceutical composition against TNF-alpha are also discribed.
EFFECT: use of the invention provides a nanobody against TNF-alpha with high affinity, stability and suitability for production in multivalent format and low immunising power, which can be used in medicine to prevent, treat and diagnose TNF-alpha activity-mediated diseases.
80 cl, 62 dwg, 47 tbl, 65 ex
SUBSTANCE: what is presented is a vector for independent expression of at least two nucleic acid molecules which codes the same or different polypeptides of closely related human papilloma virus (HPV) serotypes and naturally contains the sequences having an identity of max. 80% on sites of the length of min. 40 sequential nucleotides. The invention also refers to a host cell containing the presented vector construct for multiple expression of HPV polypeptides, as well as to pharmaceutical compositions for prevention and therapy of the conditions caused by papilloma virus infection containing the vector or the vector-transformed host cells.
EFFECT: if involved in the vector, said molecules are modified so that to reduce percentage of identity of the sequences and reducing size of homology sites to the number of sequential nucleotides max 8.
32 cl, 4 dwg, 5 ex
SUBSTANCE: present invention refers to immunology. What is contemplated is an antibody which specifically binds sphingosine-1-phosphate (S1P), and its antigen-binding fragment. There are presented a pharmaceutical composition, an antibody expression vector, a host cell, as well as a method of treating an S1P-related disease or disorder.
EFFECT: use of the invention can find further application in treating various S1P-related diseases.
17 cl, 26 ex, 11 tbl, 13 dwg
SUBSTANCE: invention relates to the field of biochemistry. Seeds of transgenic corn is described containing more than 4000 parts per million (ppm) of free lysine, where seeds of corn contain native DNA and one or more exogenous cassettes for expression in plants containing a DNA molecule encoding a dihydrodipicolinatsynthase resistant to inhibition by free L-lysine by the feedback principle, the DNA molecule, transcribed to produce RNA molecule, which suppresses lysinketoglutarate reductase/ sugar pindehydrogenase, and a DNA molecule encoding aspartatkinase resistant to the action of lysine-mediated feedback, where the DNA molecules are functionally linked to one or more promoter molecules in such a way that transcription of one or more RNA molecules occurs mainly in the endosperm of corn seed, which, optionally, the corn seed contains an exogenous gene, which confers resistance to herbicides. A method of obtaining corn flour is proposed, including obtaining of the said corn seed and seed processing to obtain flour. Flour is described obtained by this method. Use of the said seed is proposed to produce fine flour or coarse flour, animal feed, obtaining protein concentrate or isolate, and use of this seed by a human or an animal.
EFFECT: invention enables to obtain food products rich in lysine that do not require further addition of lysine.
11 cl, 2 tbl, 8 dwg, 2 ex
SUBSTANCE: invention refers to nucleic acid constructs used to produce recombinant parvoviral vectors in insect cells containing such constructs, and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferentially contains a first nucleotide sequence coding parvoviral Rep proteins wherein a translational initiation codon of the parvoviral Rep78 protein represents a suboptimal initiation codon which have an action of a partial exon skipping in expression in the insect cells. The insect cell additionally contains a second nucleotide sequence containing the nucleotide sequence with at least one parvoviral (AVV) inverse terminal redundancy (ITR), and a third nucleotide sequence containing sequences coding core parvoviral proteins.
EFFECT: method improvement.
30 cl, 6 dwg, 2 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention provides RANK-L targeted amino acid sequences, as well as to a compound or a construct, and particularly, proteins and polypeptides which comprise or substantially consist of one or more of these amino acid sequences. The invention also discloses nucleic acids encoding such amino acid sequences and polypeptides; methods for preparing such amino acid sequences and polypeptides; host cells expressing or able for expressing such amino acid sequences or polypeptides. There are also disclosed compositions and particularly, pharmaceutical compositions for preventing and/or treating bone diseases and disorders, which include such amino acid sequences, polypeptides, nucleic acids and/or host cells. What is shown is a possibility to use the amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions for the preventive, therapeutic or diagnostic purposes.
EFFECT: polyvalent construct of the invention has a half-life in human serum from 12 hours to 19 days.
32 cl, 30 dwg, 24 tbl, 17 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to immunology. There are presented versions of antibodies specifically bound with amino acid residues of murine 1446-1725 Notch1 or human 1446-1735 Notch1. What is disclosed is a coding polynucleotide, an expression vector based on the polynucleotide, a host cell for antibody expression. What is described is a method for producing the antibody with the use of the vector, as well as using the antibodies as a therapeutic agent or in a method of treating the disorders associated with higher signal transmission or higher Notch1 expression.
EFFECT: use of the invention provides the antibodies which reduces the Notch1 signal transmission that can find application in medicine for treating the disorders associated with higher Notch1 expression.
42 cl, 17 dwg, 5 tbl, 10 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention discloses CD19-binding agents representing an antibody or an antigen-binding fragment containing an amino acid sequence of a variable region of a heavy chain, and an amino acid sequence of a variable region of a light chain which are bound with human CD 19 with a dissociation constant equal or less 1x10 -7M. The amino acid sequences are presented in the description. There are disclosed nucleic acid coding the heavy and/or light chain of the antibody or the antigen-binding fragment and a ligand conjugate - a therapeutic agent, or its pharmaceutically acceptable salt or solvate for treating a CD19-associated disorder in a mammal specified in a group involving CD19-expressing cancer, chronic leukaemia, B-cell lymphoma, multiple myeloma and a number of the other oncological diseases. The conjugate for treating the disorder in the mammal is used in an effective amount.
EFFECT: use of the presented antibodies or conjugate enables higher survival rate of the patients with oncological diseases expressing CD19, as well as in treating immunopathological diseases.
18 cl, 26 dwg, 8 ex
SUBSTANCE: invention discloses an isolated antibody which selectively binds to the C-end of beta-amyloid (Abeta) and is humanised or fully human. The antibody can be a single-chain antibody (scFv), a Fab fragment or a P(ab')2 fragment. The antibody is capable of preventing oligomerisation of Abeta. The invention discloses a nucleic acid sequence which codes the disclosed antibody, a vector and a host cell for producing the antibody, as well as a pharmacological composition for treating neurological disorders, particularly Alzheimer's disease. The invention provides methods for diagnosis using labelled antibodies disclosed herein and treating neurological disorders associated with abnormal accumulation and/or deposition of Abeta in the central nervous system by administering a therapeutically effective amount of a polynucleotide, vector or host cell to a subject.
EFFECT: invention enables successful application of the disclosed antibodies for therapeutic purposes and clinical application since in contrast to mouse antibodies, said antibodies are low- or nonimmunogenic when used in humans.
25 cl, 23 dwg, 1 tbl, 17 ex
SUBSTANCE: there are described versions of humanized monoclonal anti-factor D antibodies and their functional fragments. There are offered: a coding nucleic acid, an expression vector, as well as a cell for preparing c the antibody containing the vector. What is described is a method for preparing the anti-factor D antibody by cell culture and expressed antibody purification. Also, there are offered: an antibody composition and use of the antibody for treating disorders mediated by a complement system.
EFFECT: higher clinical effectiveness in the diseases related to excessive or uncontrolled activation of the complement system.
32 cl, 9 dwg, 4 tbl, 6 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention discloses a human antibody or an antigen-binding antibody fragment which specifically binds human nerve growth factor (NGF) with KD in the order of 5 pM or less and does not cross-react with neurotrophin-3 (NT-3). The antibodies are effective in treating inflammatory pain, postoperative suture pain, neuropathic pain, fracture pain, osteoporotic fracture pain, postherpetic neuralgia, pain associated with osteoarthritis, rheumatoid arthritis, cancer, burns, joint pain in gout, as well as such diseases as hepatocellular carcinoma, breast cancer and hepatic cirrhosis. The invention discloses a nucleic acid coding a human antibody or its antibody-binding fragment, an expression vector containing the nucleic acid, and a method for producing the antibody or its antigen-binding fragment. The antibody or its fragment is used to create a pharmaceutical composition for treating the above diseases, as well as a drug preparation additionally containing IL-1 inhibitor, an antiepileptic drug, a cytokine antagonist and another neurotrophin.
EFFECT: specificity of human NGF binding by the antibody or its fragment according to the invention is 2-10 times higher than a binding ability of the antibody or its fragment in relation to rat's or mouse's NGF.
21 cl, 28 tbl, 13 ex
SUBSTANCE: disclosed is a binding protein which is specific to IL-13, having 6CDR sections (three light and three heavy chains). The invention discloses a structure of an antibody based on this protein and a conjugate based on the antibody. The invention describes versions of nucleic acid which codes the antibody or structure, and an expression vector, a replication vector and cells bearing such vectors. Described is a method of producing the protein by culturing cells and proteins obtained using said method. Described are compositions based on versions of the proteins.
EFFECT: invention can be used in medicine to treat and diagnose diseases associated with IL-13 activity which negatively affects health.
50 cl, 23 tbl, 2 ex
SUBSTANCE: there are described versions of polypeptides specifically binding human DR5 each of which contains CDR versions containing a high-limited variety of amino acid sequences. There are described versions of polypeptides fused with at least a portion of protein, viral envelopes specifically binding human DR5. There are offered: a coding nucleic acid, an expression vector, as well as a cell for polypeptide expression containing the vector. What is also described is a method for producing the polypeptide by cell culture and a method for selecting an antigen-binding variable domain using the polypeptide. Besides, there are presented compositions and methods for using for the purpose of treating a malignant tumour and conditions related to the immune system.
EFFECT: use of the invention can find application in medicine in treating and diagnosing the tumour diseases.
88 cl, 29 dwg, 1 tbl, 6 ex
SUBSTANCE: what is presented is an antibody neutralising endothelial cell infection by human cytomegalovirus (hCMV) characterised by the presence of three heavy chain CDR and three light-chain CDR. There are described: a nucleic acid for antibody expression containing coding NA and a cell based on such expressing NA. There are disclosed: a composition for preventing and treating hCMV based on the antibody and the use of the antibody or NA for preparing a drug for treating hCMV. The invention provides the antibodies the concentration of which are required for 50% neutralisation of hCMV in endothelial cells makes 0.003 mcg/ml or less that can find further application in methods of screening, as well as in diagnosing and treating the hCMV-mediated diseases.
EFFECT: higher effectiveness of the use of the antibodies.
13 cl, 5 dwg, 2 tbl, 3 ex
SUBSTANCE: present invention relates to immunology and biotechnology. Disclosed are: versions of an isolated molecule of a human IL-6 antibody, which contain VH and VL; nucleic acid which includes an antibody-coding nucleotide sequence; a cell for obtaining the antibody molecule which is transformed by said nucleic acid. Described is a method of obtaining the antibody by cell culturing, a composition and a method of obtaining a composition of the antibody molecule.
EFFECT: invention can be used in medicine for treating diseases associated with IL-6.
34 cl, 1 dwg, 11 tbl, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology. What is presented is a monoclonal antibody or a fragment thereof, which causes NK-cell induced lysis of human target cells carrying HLA-E on their surface, optionally conjugated with a detectable marker. The antibody or the fragment thereof are specific to NKG2A, have heavy and light chains, each of which contains 3 CDR and does not bind specifically: human NKG2C or NXG2E and Fc receptor. There are described: a pharmaceutical composition, a method for recovering the NK-mediated lysis of target cells, a conjugate, a set and a method for detection based on the use of the antibodies. What is disclosed is a composition for treating autoimmune and inflammatory diseases.
EFFECT: use of the invention can find application to stimulate the NK-cells leading to the lysis of dendritic cells, which contribute to the pathology of autoimmune and inflammatory diseases, that can find application in medicine.
36 cl, 4 dwg, 5 tbl, 12 ex