Human monoclonal antibodies to receptor of epidermal growth factor (egfr), method of their preparation and usage, hybridome, transfectome, transgene animal, expression vector
FIELD: medicine; pharmacology.
SUBSTANCE: allocated human monoclonal antibodies which specifically bind a receptor of the epidermal growth factor (EGFR), and also corresponding compositions on the basis of antibodies and a biospecific molecule are described. Human antibodies can be received with use of the transgenic mouse capable to formation of set of isotypes of human monoclonal antibodies by recombination V-D-J and switching of isotypes. The pharmaceutical compositions containing human antibodies for treatment or prevention of diseases, mediated by expression EGFR, the transgenic animals distinct from a human, the specified expressing antibodies, hybridomes and transfectomes which produce human antibodies are also presented. Ways of therapy and diagnostics of the diseases mediated by expression EGFR, with use of human antibodies or their antigen-binding of fragments, and also methods of growth suppression of the cells expressing EGFR, and an induction of cytolysis of the specified cells are described.
EFFECT: invention allows obtaining therapeutic and diagnostic preparations of antibodies with improved properties.
53 cl, 22 dwg, 4 tbl, 11 ex
The text descriptions are given in facsimile form.
1. Selected human monoclonal antibody that binds to human receptor for epidermal growth factor (EGFR), selected from the group including IgGI antibodies, IgA, IgE, IgM, IgG4, and IgD, and when this antibody contains: frame segments of the human heavy chain CDR1 plot of the human heavy chain CDR2 area of human heavy chain and CDR3 of the area of the human heavy chain, which is a CDR3 area of human heavy the th chain monoclonal antibodies 2F8, presented at Fig and 22; and a frame segments of the human light chain, CDR1 area of human light chain CDR2 area of human light chain and CDR3 area of human light chain, which is a CDR3 area of human light chain monoclonal antibodies 2F8, presented at Fig and 22.
2. Selected human monoclonal antibody according to claim 1, characterized in that it is represented by IgGI antibody.
3. The human antibody according to claim 1, characterized in that it inhibits the binding of the ligand EGFR with human EGFR.
4. The human antibody according to claim 3, characterized in that the EGFR ligand represented by EGF or TGF-α.
5. The human antibody according to claim 1, characterized in that it binds to human EGFR with an equilibrium constant of Association (KAndat least approximately 108M-1.
6. The human antibody according to claim 1, characterized in that it binds to human EGFR with an equilibrium constant of Association (KAndat least approximately 109M-1.
7. The human antibody according to claim 3, characterized in that it blocks the binding of the ligand with human EGFR EGFR of at least approximately 50%.
8. The human antibody according to claim 2, characterized in that it comprises a variable region from an IgGl heavy chain and the variable region of the Kappa light CE is I.
9. The human antibody according to claim 1, characterized in that it is encoded by nucleic acids of the human heavy chain and human Kappa-light chain containing in their variable regions of the nucleotide sequence represented in SEQ ID NO:1 and SEQ ID NO:3, respectively, and conservative modifications of these sequences.
10. The human antibody according to claim 1, characterized in that it has a variable region heavy chain and Kappa light chains, which contain the amino acid sequence represented in SEQ ID NO:2 and SEQ ID NO:4, respectively, and conservative modifications of these sequences.
11. The human antibody according to claim 1, characterized in that it has a variable region heavy chain and Kappa light chains, which contain the amino acid sequence represented in SEQ ID NO:2 and SEQ ID NO:4, respectively.
12. The human antibody according to claim 1, characterized in that it binds to EGFR and inhibits induced by EGF or TGF-α autophosphorylation of EGFR.
13. The human antibody according to claim 1, characterized in that it binds and inhibits the growth of cells expressing EGFR.
14. The human antibody according to item 13, characterized in that it binds to the cell, which is a tumor cell selected from the group comprising cell bladder, clickomania gland, cell, colon cell tumors of the kidney, cell, ovarian cell, prostate cell renal cell tumors, squamous cell and nemiscau cell lung.
15. The human antibody according to item 13, characterized in that it binds to the cell that is selected from the group consisting of cells of the synovial fibroblast and keratinocyte.
16. The human antibody according to claim 1, characterized in that it binds to cells expressing EGFR and induces lysis of the cells in the presence of human effector cells.
17. The human antibody according to claim 1, characterized in that it binds to cells expressing EGFR, but does not induce complement-mediated lysis of the cells.
18. The selected human antibody according to claim 1, characterized in that it is a Fab fragment or single-chain antibody.
19. The selected human antibody according to claim 1, characterized in that it is produced by hybridomas, which includes In-cell obtained from a transgenic animal other than human, and having a genome that contains the transgene, human heavy chain and human transgene light chain, merged with immortalizing cell.
20. The selected human antibody according to claim 1, characterized in that it is produced by transfective containing nucleic acids encoding a human heavy is EPI and a human light chain.
21. Selected human monoclonal antibody, which binds to the epitope on EGFR, a specific antibody according to item 11.
22. Hybridoma containing a cell obtained from a transgenic animal other than human, and having a genome that contains the transgene, human heavy chain and human transgene light chain, merged with immortalizing cell, while hybridoma produces detectable quantity of the monoclonal antibody according to claim 1 or an antigen-binding part.
23. Transfactual containing nucleic acids encoding a human heavy chain and human light chain, while transfactual produces detectable quantity of the monoclonal antibody according to claim 1 or an antigen-binding part.
24. Transfactual according to item 23, characterized in that it contains a nucleic acid encoding a human heavy chain and human light chain, which in their variable regions contain nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:3, respectively, or conservative modifications.
25. Transgenic animal other than human, which expresses the antibody according to claim 1, in this transgenic animal other than human, has a genome containing the transgene of human heavy chain and human transgene light chain.
26. The way the floor is for the antibody according to claim 1, characterized in that carry out the immunization of non-human transgenic animal having a genome containing the transgene of human heavy chain and human transgene light chain, using EGFR or cells expressing EGFR, therefore the production of antibodies by b-cells of the animal, isolated b cells of the animal and carry out the fusion of b cells with myeloma cells with the formation of immortalized cells hybridoma that secrete antibody.
27. Bespecifically molecule containing human antibody according to claim 1 and a second binding specificity against human antigen-presenting cells.
28. Bespecifically molecule containing human antibody according to claim 1 and a second binding specificity against human Fc receptor.
29. Bespecifically molecule on p, characterized in that the said Fc receptor is a human receptor FcγRI or human Fc receptorα.
30. Bespecifically molecule on p, characterized in that it binds the Fc-receptor on the center, which is different from the binding site of the receptor and immunoglobulin.
31. Pharmaceutical composition for treatment or prevention of diseases mediated by the expression of EGFR containing human antibody according to claim 1 and pharmaceutically acceptable wear the ü.
32. Pharmaceutical composition for treatment or prevention of diseases mediated by EGFR expression containing a combination of two or more human antibodies according to claim 1, or antigen-binding fragments, where each of the above antibodies or antigen-binding fragments binds a single epitope of EGFR.
33. Pharmaceutical composition for treatment or prevention of diseases mediated by the expression of EGFR containing human antibody according to claim 1 and a chemotherapeutic agent.
34. Immunotoxin containing human antibody according to claim 1, associated with the cytotoxic agent.
35. The method of suppressing the growth of cells expressing EGFR, characterized in that exercise contacting cells with an effective amount of the antibody of claim 1 that inhibits binding of a ligand of EGFR with human EGFR, and thus inhibit the growth of cells expressing EGFR.
36. The method according to p, wherein the cell is chosen from the group comprising cell, bladder cell, breast cancer cell, colon cell, kidney cell, ovarian, prostate cell, squamous cell, nemelka cell lung cell synovial fibroblast and keratinocyte.
37. Method of induction of cytolysis of cells expressing EGFR, characterized in that exercise contacting cells expressing EGF, with the antibody according to claim 1 in the presence of effector cells with the provision of cytolysis of cells expressing EGFR.
38. The method according to clause 37, wherein the cell is chosen from the group comprising cell, bladder cell, breast cancer cell, colon cell, kidney cell, ovarian, prostate cell, squamous cell, nemelka cell lung cell synovial fibroblast and keratinocyte.
39. The method of treatment or prophylaxis of a disease mediated by expression of EGFR, wherein the subject is administered a pharmaceutical composition according to any one of p-33 in amounts effective for the treatment or prophylaxis of a disease mediated by expression of EGFR.
40. The method according to § 39, wherein the disease is a cancer.
41. The method according to § 39, wherein the disease is an autoimmune disease.
42. The method according to § 39, wherein the subject is administered a human antibody conjugated with a binding specificity for an Fc receptor.
43. The method according to § 39, wherein the subject is administered a human antibody conjugated to a cytotoxin.
44. The method according to § 39, characterized in that it further carry out joint introduction of therapeutic agent.
45. The method according to item 44, wherein therapeutic agent is chosen from the group, ostoja of doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, hlorambuzila and cyclophosphamideinduced.
46. The method according to p, wherein the disease is a cancer selected from the group including bladder cancer, breast cancer, colon cancer, kidney cancer, ovarian cancer, prostate cancer, renal cell cancer, head and neck cancer.
47. The method according to paragraph 41, wherein the disease includes hyperproliferative epithelium.
48. The method according to § 39, wherein the disease presents inflammatory arthritis.
49. The method of detecting the presence of EGFR antigen or cells expressing EGFR in the sample, characterized in that carry out the contacting of the sample with the antibody of claim 1 under conditions ensuring formation of a complex between the antibody or a part of it and EGFR, and make the detection of complex formation.
50. The expression vector containing the nucleotide sequence encoding the variable region of the light chain and/or heavy chain of the human monoclonal antibody according to any one of claims 1 to 21.
51. The expression vector according to item 50, characterized in that it further comprises a nucleotide sequence encoding a constant region of light chain and/or heavy chain of human monoclonal antibody that binds EGFR.
52. Expression of vecto is, containing the nucleotide sequence encoding the variable region of the heavy chain and light chain, which contain amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and conservative modifications of those sequences.
53. Transfactual containing the expression vector according to item 50.
SUBSTANCE: invention concerns medical microbiology. The method of chronic urogenital gonococcal infection course forecast involves seeding of accompanying fungi of Candida genus in case of gonococcus detection, and persistence factors of accompanying microorganisms is evaluated. If titration of fungi of Candida genus gives not less than 102 colony-forming cells per millilitre and antilysozyme activity evolves simultaneously in the quantity not less than 1.3 mcg/ml per optical density unit, and anticomplementary activity not less than 1.5·106 antilytic complement units, then chronic character of urogenital infection is confirmed diagnosed.
EFFECT: increased accuracy of chronic urogenital gonococcal infection course forecast.
2 ex, 3 tbl
SUBSTANCE: method comprises determination of IgG-antibodies level in blood plasma before and after treatment. For this purpose content of specific IgG-antibodies to herpes simplex virus of I and II type in patient's blood is determined before treatment and on 7th, 14th, and 21st days after treatment at the remission stage; also the degree of IgG-antibodies' avidity and level of circulating immune complexes (CIC) is evaluated. If the content of specific IgG-antibodies increases no less than 27% in 7 days after treatment, in 14 days - no less than 33%, and in 21 days - no less than 60% compared to initial level; if degree of specific IgG-antibodies avidity compared to initial level increases no less than 25% in 7 days, in 14 days - no less than 38%, and degree of avidity subsequently increases, or remains constant, or reduces no more than 6% in 21 days compared to previous period (14 days); and if CIC content increases no less than 27% in 7 days compared to initial value, in 14 days - no less than 36%, and content of CIC reduces no more than 23% in 21 days compared to previous period (14 days) in cases with initial content of CIC $ 150 arbitrary units, or content of CIC reduces no more than 37% in 7 days compared to initial value, in 14 days - no more than 43%; in 21 days increases no less than 6% compared to previous period (14 days) in cases with initial content of CIC > 150 arbitrary units - then conclusions can be drawn that the treatment was effective and prognosis for clinical course is favourable.
EFFECT: increase of valuation objectivity and opportunity to apply the method for treatment at remission stage.
FIELD: medicine, pharmacology.
SUBSTANCE: method comprise salt-water extraction from raw material using Evans-Cook extraction liquid, mites cultivation during from 3 to 4 months, at temperature 25±2°C and relative air humidity 73±3%, on house dust and bristle. As initial basis, the mix of house dust mites Dermatophagoides farina or Dermatophagoides pternissinus culture and culture medium, including excrements, cast off skins, and food debris, is used. Salt-water extraction from raw material is performed during three days, after mixing it and homogenization with glass powder; the maintained incubation temperature is 2° to 10°C; the extraction material being shaken for 30 minutes three times a day with 1.5 hour intervals. Extraction completed, the supernatant liquor is decanted and centrifuged at 5000 to 6000 rpm for 30-40 minutes, then it is filtered through paper filter; in consequence sterilization through filtering, 3 to 4 months mother solution stabilisation, and bottling are carried out; the finished preparation is produced.
EFFECT: invention provides producing high effective treatment allergen in simplified process technology.
SUBSTANCE: invention relates to medicine, namely to gastroenterology and can be used in clinical practice for diagnostics of gastritis in children and timely prescription of adequate therapy. Immunologic test of empty stomach portion of gastric juice is carried out and content of antiinflammation cytokine IL1β in it is determined. With index values from 28.6 pg/ml and higher gastritis is diagnosed. The diagnostic technique is accurate, simpler and more sparing, which allows its wide use in children.
EFFECT: more accurate and sparing method of gastritis diagnostics.
1 tbl, 4 ex
FIELD: medicine; veterinary science; microbiology.
SUBSTANCE: invention includes homogenisation of diagnostic material, added with radioactive ferromagnetic microparticle suspensions with surface antibodies to corresponding bacterial infectious agent, incubation at room temperature during 60 min, ferromagnetic microparticle deposition in constant magnetic field during 30 min, ferromagnetic microparticle introduction to produced deposit using magnetic probe-sampler with interchangeable nonmagnetic tip with surface antibodies similar to those registered on surface ferromagnetic microparticles, placement of tip in cleaning tank filled with phosphate-salt buffer and containing constant magnet at the bottom, evaluation of washed tip radio-activity by means of radioactive radiation recorder.
EFFECT: invention provides bacterial cell isolation from diagnostic material with simultaneous identification.
FIELD: medicine; paediatrics; allergology.
SUBSTANCE: newborn umbilical blood is tested for absolute amount of mononuclear leukocyte, synthesising interferon-gamma (INFγ) and not having activation marker CD69 (cell phenotype - INF γ+ CD69-). In case value thereof is equal or lower than 5.3 kl/ml newborns atopic dermatitis is forecasted.
EFFECT: method provides advance detection of newborns atopic allergic diseases development and early preventive actions for prevention of their development.
SUBSTANCE: invention refers to virology, namely, to methods of immunosorbent production. Method of immunosorbent production virus-specific antibody binding is offered. Method includes inorganic sorbent-carrier incubation with virus-containing liquid. Ultradisperse oxygen-containing graphite is used for inorganic sorbent-carrier as foamed particles of the stratified graphite, containing oxygen in amount 12-14 mass % per 73-76 mass % of carbon. Specific surface of graphite is 1500-2000 m3/g, particle size is 25-50 mcm. Graphite is pre-boiled in distilled water. Produced suspension of sorbent-carrier at graphite content not less than 2 mass % is incubated with virus-containing liquid at temperature 5-35°C. Virus-containing liquid contains, e.g. influenza virus.
EFFECT: produced immunosorbent for virus-specific antibody binding has high sorptive power, enables to bind completely virus-specific antibodies of immune serum.
2 cl, 5 tbl
SUBSTANCE: invention concerns obstetrics and gynecology, and can be used for forecasting of effective procreation restoration after immunocytotherapy for women suffering from miscarriage at early stages. For method realisation, traditional treatment with following immunocytotherapy out of pregnancy, peripheral venous blood of women with miscarriage at early stages in the anamnesis is tested for relative content of CD69+ lymphocytes. If value of this indicator it is less or equal 6.2% effective procreation restoration is forecasted.
EFFECT: offered method is easy-to-perform and enables high accuracy, specificity and sensitivity of forecasted effective procreation restoration, provides selection of correct management tactics for women with miscarriage at early stages in the anamnesis that decreases rate of given pathology.
1 tbl, 3 dwg
FIELD: medicine; paediatrics.
SUBSTANCE: peripheral blood slides are analysed for intensity of neurophillic and monocytic autorosetting through light microscopy. Increased amount of monocytic rosettes to within 4.7±1.3% and neurophillic - to within 9.4±2.0% indicates pharyngeal tonsil hypertrophy. In case of reduced amount of monocytic rosettes to within 3.1±0.1% and neurophillic - to within 3.1±0.1%, chronic adenoid is diagnosed.
EFFECT: method application enables to perform differential diagnostics of pharyngeal tonsil hypertrophy and chronic adenoid.
SUBSTANCE: proposed purpose of non-invasive evaluation of hepatic fibrosis for patients suffering from chronic viral hepatitis is achieved with assessed parameters of transforming growth factor-I, epidermal growth factor in serum accompanied with calculation of regeneration index.
EFFECT: application of proposed method enables to evaluate fibrosis severity accompanied with various aetiological form of chronic viral hepatitis without resorting to puncture liver biopsy.
1 tbl, 5 ex
SUBSTANCE: obtaining monoclonal antibodies immunoreactive with the nucleocapsid protein of the hepatitis virus C-4G5, and the development of a method of diagnostics based on the double layer version of immunoenzymometric assay for the detection of the nucleocapsid (core protein) of the hepatitis C virus using a combination of linear and conformational core protein epitope monoclonal antibodies 27, 1F9, and 4G5: MCA 1F9: linear epitope with amino-acid residues (AAR) 81-85, MCA 27, and 4G5: conformational epitopes at site 1-150 of the core protein AAR. The combination of monoclonal antibodies 27, 1F9, and 4G5, immunoreactive with the hepatitis C virus nucleocapsid protein may be used both for the capture (capture antibodies) and for the detection (detection antibodies) of the core protein. The double layer variant of the immunoenzymometric assay developed for the detection of the nucleocapsid of the hepatitis C virus using a combination of monoclonal antibodies including MCA 4G5 has high sensitivity and allows to detect core proteins in blood of both asymptomatic HCV-infected donors and patients suffering from acute and chronic hepatitis C. The advantages of the immunoenzymometric assay of the core protein may be useful for determining the viral load during IF therapy.
EFFECT: allows to diagnose hepatitis C with high probability at early stages of disease and is efficient for detection of HCV of at least three genotypes most actual for Russia.
3 cl, 3 tbl, 4 ex
FIELD: medicine, peptides, biochemistry, pharmacy.
SUBSTANCE: invention relates to modification of glycosylation of proteins for preparing polypeptides with improved therapeutic indices including antibodies with enhanced antibody-dependent cellular cytotoxicity. For preparing indicated polypeptides cell-host is used that is modified with nucleic acid encoding enzyme β-1,4-N-acetylglucosaminyltransferase III (GnTIII). Prepared polypeptide represents, in particular, IgG or its fragment. Invention discloses a method for preparing polypeptide and antibodies or its fragment and a fusion protein prepared by indicated method. Invention describes a pharmaceutical composition used for increasing Fc-mediated cellular cytotoxicity and comprising antibody and carrier, and its using in cancer treatment, and a method for treatment of disease associated with elevated amount or production of B cells using indicated antibody, in particular, against CD20, and representing antibody IDEC-C2B8 in the preferable variant. Invention provides preparing polypeptide and antibody possessing the enhanced Fc-mediated cellular cytotoxicity that decrease the content of B cells in a patient body.
EFFECT: improved preparing method, valuable medicinal properties of polypeptide and antibodies.
38 cl, 21 dwg, 4 ex
FIELD: biotechnology, immunology.
SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.
EFFECT: valuable biological and medicinal properties of antibodies.
53 cl, 4 tbl, 10 dwg, 2 ex
FIELD: biotechnology, immunology, molecular biology, pharmacy.
SUBSTANCE: invention describes variants of MCP-1-binding molecules. One of MCP-1-binding molecule comprises at least one variable region of immunoglobulin (VH) heavy chain comprising of hypervariable sites CDR1, CDR2 and CDR3 while other molecules comprises both light and heavy chains. Invention proposes DNA constructs encoding indicated MCP-1-binding molecules and expressing vector carrying at least one of these DNA constructs. Invention describes a method for preparing MCP-1-binding molecule. Invention discloses a method for treatment of disease or disorder mediated by MCP-1 or eotaxine-1 based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode. Invention describes a pharmaceutical composition based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode and used in treatment of disease or disorder mediated by MCP-1 or eotaxine-1 in a patient. MCP-1-binding molecules inhibit binding MCP-1 with its receptor. The full immobilized antibody is highly specific as far as it binds human recombinant MCP-1 with value KD = (43 ± 2.9) x 1012 and can be used in medicine.
EFFECT: valuable medicinal properties of antibodies, improved method of treatment.
13 cl, 5 dwg, 4 tbl, 2 ex
FIELD: immunology, medicine.
SUBSTANCE: claimed recombinant antibody (Ab) has at least constant regions in heavy and light chains representing human Ab regions. Said At inhibits bonding of integrine recognizing RGD and SVVYGLR sequences to integrine or fragment thereof. Also disclosed are nucleotide sequences (NS) encoding heavy and light chains of recombinant Ab as well as expression vectors containing respective NS. Described are host cell for Ab production, transformed with two vectors for expression of Ab heavy and light chains and method for abovementioned host cell application to produce recombinant Ab. Ab of present invention is useful in diagnosis and treatment of autoimmune diseases, rheumatism and rheumatoid arthritis.
EFFECT: therapeutic methods of increased efficiency.
45 cl, 14 tbl, 28 ex
FIELD: microbiological and immunological methods.
SUBSTANCE: invention is intended for use to study structural and functional arrangement of nucleolus and mechanisms of action of pharmacological preparations on human cells. Mouse hybridoma cell strain, called A3, is obtained through fusing mouse splenocytes immunized by coarse fraction of nuclei of human cell line RAMOS with cells of mouse myeloma line P30X63-Ag8.653. Resulting hybridoma secretes monoclonal antibodies against antigen, called A3 antigen, localized inhuman cell nucleoli irrespective of their tissue or line origin. In cases of different cell fixation ways. A3 antigen is revealed as incorporated in discrete (typically several tens) foci located exclusively in the zone of nucleoli. Unique property of A3 antigen is its high sensitivity to the action of various protein synthesis inhibitors, e.g. emetine, anisomicyne, cycloheximide, and puromicyne. During incubation of cells in presence of above-listed substances, A3 antigen migrates from nucleoli to numerous foci located in nuclear nucleoplasma. This A3 antigen migration precedes apoptotic death of cells. Monoclonal antibodies A3 produced by the strain A3 are recommended to reveal nucleoli and to estimate total level of protein synthesis in human cells by cell biology techniques. Antibodies can be used to reveal possible contamination of human cell cultures with another-species cells as well as to investigate biological activity of known and novel protein synthesis inhibitors, including pharmacological preparations in human in vitro cells.
EFFECT: expanded microbiological and immunological possibilities.
2 cl, 6 dwg
FIELD: biotechnology, in particular tumor immunotherapy.
SUBSTANCE: invention relates to monovalent and divalent single-stranded (diantibody) Fv-fragments (scFv) of antibodies obtained by using of extracted RNA producing of hybridoma Mab CB/ior-CEA.1. Antibodies fragments according to the invention have predetermined amino acid sequence (such as defined in sequence list), are specific to human CEA, have CEA affinity constant of (5.0±0.4)x10-9 l/mol for monovalent fragment and (2.8±0.3)x10-10 l/mol for diantibody. Disclosed is application of abovementioned fragments in pharmaceutical compositions for treatment of human CEA-expressing tumors and determination of tumor localization in vivo. Described is modified cell expressing said antibody fragment, as well as multicellular genetically modified organism such as transgenic plant. ScFv fragments of monovalent antibody and diantibody have no Fc-domains and have smaller molecule size than mice Mab and as a result fragments are better penetrate in tissue in vivo and have less immunogenic action in living body.
EFFECT: antibody fragments with improved specificity to human cancer-embryonic antigen.
12 cl, 6 dwg, 8 tbl, 9 ex
FIELD: biotechnology, hybridoma technology.
SUBSTANCE: hybridoma T2/S-6E11 is prepared by fusion of murine myeloma strain Sp-2/0 and murine BALB/c splenocytes immunized with the TOPC virus, strain CoD, purified preparation inactivated with concentrate formaldehyde solution and by using polyethylene glycol-1000 Da and the following cloning by method of limited dilution. Prepared hybridoma T2/S-6E11 produces monoclonal antibodies (MCAb) to epitopes of the abovementioned pathogen. Specificity of prepared MCAb is shown by absence of cross-reactions in IFA-test with Hantaan and Ebol viruses and with noninfected substrates accumulated by TOPC virus. IFA-test system based on MCAb provides carrying out the specific detection of TOPC virus in analyzed samples. The sensitivity of this test-system based on MCAb is estimated to be 2.5 x 103 plaque-forming units (virus) in cubic centimeter (PFU/cm3). Using the invention in immunological researches in creature of diagnosticum used for detection of TOPC virus in samples provides enhancing the specificity of analysis in detection of coronaviruses.
EFFECT: valuable properties of strain.
3 tbl, 1 ex
FIELD: biotechnology and virology.
SUBSTANCE: strain of hybrid culturing Rattus norvegicus 122H9 cells is disclosed. Said strain is obtained by fusion of rat myeloma cells 210RC.Y3-Ag1.2.3 with rat spleen cells LOU, immunized with purified ectromelia virus of K-1 strain. Said strain is producer of cross-reactive neutralizing monoclonal antibodies against pathogenic for human orthopoxviruses.
EFFECT: vaccines for prophylaxis and therapy of diseases associated with pathogenic for human orthopoxviruses.
2 tbl, 5 ex
SUBSTANCE: invention relates to immunoenzyme analysis and can be used for assay of von Willebrand factor. Method involves immunoenzyme analysis wherein monoclonal antibody 5C3 is used as an immobilizing antibody, and a mixture of biotin-labeled monoclonal antibodies 2H2 and 7D12 is used as a detecting antibody. Also, invention relates to monoclonal antibodies produced by the strain of hybridoma cultured cells Mus musculus L. and directed against von Willebrand factor, and to strains of hybrid cultured cells Mus musculus L. producing indicated monoclonal antibodies. Invention provides the development of highly sensitive method for assay of von Willebrand factor.
EFFECT: improved method for analysis.
9 cl, 1 tbl, 2 dwg, 3 ex
FIELD: biotechnology, immunology, medicine, oncology.
SUBSTANCE: strain of hybrid cultured mammalian cells Mus musculus VKPM H-98 is prepared by the hybridoma technology method. This strain is a producer of monoclonal antibodies possessing individual specificity to hypoglycosidated and deglycosidated isoforms of tumor-associated human antigen Muc I. Productivity of the strain and specificity of produced antibodies is estimated based on the immunoenzyme assay using some markers of specificity: natural purified antigen Muc I isolated from human milk; VNTR22-polypeptide; synthetic monomeric polypeptide (TR1); deglycosidated antigen Muc I (de-Muc I) prepared by chemical oxidation of natural Muc I; hypoglycosidated antigen Muc I (o-Muc I) prepared by periodate oxidation of natural Muc I. Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of antigen Muc I and allows assaying its concentrations in human serum blood in carrying out the early diagnosis of tumors. Invention can be used in preparing monoclonal antibodies to tumor-associated human antigen Muc I.
EFFECT: valuable properties of strain.
2 dwg, 3 ex