Fc-erythropoietin fused protein with improved pharmacokinetics
SUBSTANCE: invention concerns medicine and Fc-erythropoietin fused protein with improved pharmacokinetics. Invention claims novel sialylated Fc-EPO fused proteins preferably including modification pair in Fc part, as well as in EPO part, showing improved pharmacokinetics. Particularly, Fc-EPO proteins have longer half-life in blood serum and higher efficiency in vivo. Fc-EPO fused proteins synthesised in BHK cells show much longer half-life in blood serum and higher efficiency in vivo than similar Fc-EPO fused proteins obtained in other cell lines, such as NS/0 cells.
EFFECT: improved pharmacokinetic properties of erythropoietin.
23 cl, 14 ex, 6 tbl, 11 dwg
The text descriptions are given in facsimile form.
1. Highly purified sililirovany dimeric protein, which essentially consists of a dimeric Fc portion containing the CH2 and CH3 domain of human IgG2, hinge area human IgG, and human erythropoietin (EPO), where each chain dimeric Fc part is linked through its C-terminal end directly or through a link is REGO peptide with N-terminal end of the molecule of EPO,
this protein has the following properties:
(i) the EPO molecule comprises residues 15-28 sialic acid;
(ii) plot Gln-Phe-Asn-Ser sequence replaced by Gln-Ala-Gln-Ser within the CH2 domain of the specified IgG2, and
(iii) amino acid sequence plot Leu-Ser-Leu-Ser near the C-terminal end of the CH3 domain of the specified IgG2 replaced by Ala-Thr-Ala-Thr.
2. Dimeric Fc-EPO protein according to claim 1, which further C-terminal Lys residue CH3 domain is replaced by Ala.
3. Dimeric Fc-EPO protein according to claim 1, in which the hinged section is derived from human IgG1.
4. Dimeric Fc-EPO protein according to claim 3, wherein said hinge section IgG1 is modified by replacing the amino acid Cys residue within the sequence Pro-Lys-Ser-Cys-Asp-Lys articulated plot the Ser residue, forming, thus, the sequence Pro-Lys-Ser-Ser-Asp-Lys within the hinge area.
5. Dimeric Fc-EPO protein according to claim 1, in which eritropoyetina part includes at least one of the following amino acid substitutions:
(i) a residue other than cysteine, at position 29 of the molecule of EPO,
(ii) a residue other than cysteine, at position 33 of the molecule of EPO,
(iii) a cysteine residue at position 88 of the molecule EPO and
(iv) the cysteine residue at position 139 of the EPO molecule.
6. Dimeric Fc-EPO protein according to claim 5, in which amino acid residue, Otley is hydrated from Cys, is in position 33 of the EPO molecule instead of the original residue Cys, and Cys residue is located at position 88 of the EPO molecule instead of the original Trp residue, allowing, thus, EPO part within the fused protein to form Cys29-Cys88disulfide bonds.
7. Dimeric Fc-EPO protein according to claim 6, in which amino acid residue other than Cys, located at position 33 is a Pro.
8. Dimeric Fc-EPO protein according to claim 1, in which the EPO portion includes one or more mutations selected from the group:
9. Dimeric Fc-EPO protein of claim 1, wherein the linker peptide comprises a glycosylation site.
10. Dimeric Fc-EPO protein according to claim 9, in which the glycosylation site comprises the amino acid sequence Asn-Ala-Thr.
11. Dimeric Fc-EPO protein according to claim 1, in which the full IgG molecule, including a hinged section, derived from IgG2.
12. Dimeric Fc-EPO protein according to claim 1 which further includes CN domain.
13. Dimeric Fc-EPO protein according to claim 1, in which the protein contains 20-22 residue sialic acid.
14. Dimeric Fc-EPO protein according to claim 1, comprising the sequence:
NFLRGKLKLYTGEACRTGDR (SEQ ID NO: 14).
15. Dimeric Fc-EPO protein according to claim 1, comprising the sequence:
LRGKLKLYTGEACRTGDR (SEQ ID NO: 15).
16. A DNA molecule encoding a protein according to claim 1.
17. Pharmaceutical composition suitable for the treatment of disorders hematopoiesis or hematopoietic failure in a mammal, comprising an effective amount of Fc-EPO fused protein described in claim 1, optionally together with a pharmaceutically acceptable carrier, diluent or excipient.
18. The population of highly purified sililirovany Fc-EPO fused protein according to claim 1, which is acceptable for administration to a mammal, which is obtained by introducing a DNA molecule encoding the corresponding Fc-EPO protein in the cell KSS, expression, isolation and purification of a population of the corresponding Fc-EPO fused proteins, where this population has a longer half-life in serum compared with the population of the corresponding Fc-EPO if the s protein, synthesized in cells, NS/0, PerC6, or 293.
19. The population of purified Fc-EPO fused proteins on p where a specified population of fused proteins contains an average of 20-22 residues of sialic acid in purified Fc-EPO protein.
20. The population of purified Fc-EPO fused proteins on p or 19, where KSS cell is adapted for growth in an environment that does not contain protein, or in suspension.
21. A method of obtaining a population of highly sililirovany purified recombinant Fc-EPO fused protein according to claim 1, whereby the method includes the following steps:
(i) constructing a DNA molecule that encodes the corresponding Fc-EPO protein;
(ii) the transformation of the KSS cells using the selected DNA molecules in an environment that does not contain protein, or in suspension;
(iii) the expression of populations Fc-fused proteins encoded by the specified DNA molecule,
(iv) the collection, isolation and purification of a specified population of Fc-EPO fused proteins.
22. The method according to item 21, in which the specified synthesized population of fused proteins contains an average of 15 to 28 residues of sialic acid in purified Fc-EPO protein.
23. The method according to item 21, in which the specified synthesized population of fused proteins contains an average of 20-22 residues of sialic acid in purified Fc-EPO protein.
SUBSTANCE: there is provided fused protein, including immunoglobulin chain and the molecule IL-7 or its fragment, displaying activity typical to IL-7, that is modified in comparison with IL-7 of wild type, where modification in IL-7 represents amino-acid residues in positions 70 and 91, that are glycated, and amino-acid residue in the position 116 is nonglycated. There is proposed pharmaceutical composition, containing described protein. There is provided the method fro production of described fused protein, including: host cell transformation by DNA, coding specified fused protein IL-7, host cell culture and collection of fused protein IL-7.
EFFECT: invention can be applied for treatment of disorders, accompanied by immune deficiencies.
27 cl, 16 dwg, 1 tbl, 10 ex
SUBSTANCE: according to the invention there is provided protein fused with albumine comprising two or more tandem-oriented polypeptides GLP-1 or its fragments containing (7-36 A8G)), nucleic acid molecules coding albumine proteins according to the investigation as well as vectors containing the specified nucleic acids, host cells transformed by vectors comprising the specified nucleic acids, methods of albumine protein preparation according to the invention and their application methods. Moreover, the present invention refers to pharmaceutical composition containing fused proteins of albumine, treatment of diseases, disorders and conditions using fused proteins of albumine according to the invention.
EFFECT: improved therapeutic polypeptide stability.
34 cl, 81 ex, 11 dwg
SUBSTANCE: invention relates to medicines, particularly to the use of chimeric peptide VP-22_p16INK4a for epithelial and mesenchymal malignant neoplasms treatment. The claimed chimeric peptide VP-22_p16INK4a contains two amino acid sequences. The first sequence comprises inhibitor of cycline kinases as active fragment p16INK4a as therapeutic agent and the second sequence comprises peptide VP22 of herpes simplex virus as carrier agent to deliver cycline kinase inhibitor into target cells.
EFFECT: enlarging the application range of medicine.
SUBSTANCE: there is offered method for identification and/or verification of inhibitors of receptor tyrosine kinases that involves application of a new test system which represents a yeast host cell containing an expression vector including a nucleic acid sequence that encodes fused protein essentially consisting of a complete cytoplasmic part of analysed receptor tyrosine kinase and the dimerisation domain and, if necessary, in addition including anchoring sequence for fused protein in a membrane wherein expression of fused protein conduces to termination of cell proliferation. The method provides production of specified host cells being in contact with a candidate compound and identification of inhibitors of tested tyrosine kinase activity as a result of cultivation on the assumption that inhibition of tyrosine kinase activity with a candidate compound causes restoration of proliferation process.
EFFECT: prospected application of the invention is related to development of selective therapeutic, including anticancer, agents.
13 cl, 5 dwg, 2 ex
SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.
EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.
10 cl, 21 dwg, 11 ex
SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.
EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.
6 cl, 1 dwg, 4 tbl, 5 ex
FIELD: chemistry, biochemistry.
SUBSTANCE: current invention relates to the field of biotechnology and immunology. Proposed is an antibody, specific to the human ED-B. Antibody specified is a molecule in the form of either dimerizated mini-immunoglobulin or IgG1, whose variable region comes from the antibody L19. In case the mini-immunoglobulin variable region L19 is merged with εS2-CH4, then as in the case IgG1, the variable region L19 is merged with the constant domain of IgG1. Conjugates of antibodies with radioisotopes have been discovered. Described is the coding nucleic acid, carrying its host cell, capable of producing antibodies, and method of obtaining antibodies from cells. Discovered is a method of determining the degree of bonding of antibodies, also compositions based on antibodies. Described is the use of antibodies for preparing medicine for treating either damage related to angiogenesis, or for treating tumours. Utilisation of the invention provides antibodies, which possess high accumulating capacity to tumours, improved capability to bonding with radioactive labels and unexpectedly retains immunoreactivity in the plasma, in comparison to scFv L19. Antibody specified can be used in diagnostics and treatment of tumours.
EFFECT: obtaining antibodies which can be used in diagnostics and treatment of tumours.
22 cl, 13 dwg, 8 tbl
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention pertains to biotechnology and can be used in biomedicine for producing hyaluronan. Proposal is given of a method of producing hyaluronan, involving culturing Bacillus host cells in conditions, suitable for obtaining hyaluronan, and extraction of the target product from the culture medium. The Bacillus host cell contains a genetic structure, comprising a promoter, functionally active in the given cell, and encoding a region, consisting of a nucleotide sequence, encoding streptococcal hyaluronansynthase (hasA); sequence encoding UDP-glucose-6-dehydrogenase Bacillus (tuaD) or a similar enzyme of streptococcal origin (hasB), and a sequence, encoding bacterial or streptococcal UDP-glucose pyrophosphorylase (gtaB and hasC respectively). Use of the invented method provides for production of considerable quantities of hyaluronan with good examined, nonpathogenic cellular system.
EFFECT: obtaining considerable of hyaluronan with good examined, nonpathogenic cellular system.
15 cl, 45 dwg, 2 tbl, 20 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention pertains to genetic engineering, more specifically to chimeric polypeptides, containing an antagonist of growth hormone receptor. The invention can be used in medicine. The binding domain of the growth hormone is modified by substituting glycine amino acid residue in position 120 and is further modified in site 1, where at least one amino acid residue is substituted, which increases affinity of the growth hormone to its binding domain on the growth hormone receptor. The amino acid residue is then conjugated with the ligand-binding domain of the growth hormone receptor, through a peptide linker.
EFFECT: obtaining a highly effective antagonist of the growth hormone receptor with longer half-life, reduced immunogenesity and nontoxicity, compared to known mutant forms.
35 cl, 16 dwg, 1 tbl
SUBSTANCE: invention claims compositions which can include one or several mammary gland tumour proteins, their immunogenic parts or polynucleotides encoding such parts. Alternatively the therapeutic composition can include antigen-presenting cell expressing mammary gland tumour protein, or T-cell specific to cells expressing such protein. These compositions can be applied in prevention and treatment of such diseases as mammary gland cancer. Invention also claims diagnostic methods based on determination of mammary gland tumour protein or mRNA encoding such protein in sample.
EFFECT: use of peptides obtained from protein expressed from mammary gland by tumour in diagnostics and therapy of mammary gland cancer.
37 cl, 6 ex, 1 dwg
SUBSTANCE: invention relates to the bio-organic chemistry and relates to the novel erythropoietin conjugate, the method of production and its pharmaceutical composition. The hydroxyethylamylum and erythropoietin conjugate containing one or several hydroxyethylamylum molecules, is invented. Each hydroxyethylamylum molecule is conjugated with erythropoietin via hydrocarbon fragment. The said hydroxyethylamylum has the molecular mass from 1 to 300 kDa and shows the correlation of the C2:C6-substitution in the range of 2-20 by hydroxylethyl groups. The method for producing the hydroxyethylamylum and erythropoietin conjugate is invented; in accordance with the method, the sialic acid is not necessarily removed, partially or totally, with use of enzyme and/or chemical way, and the erythropoietin end saccharide link is oxidised not necessarily partially or totally to receive the erythropoietin which is able to react with the modified hydroxyethylamylum. Into hydroxyethylamylum, the free hydrazide, hydroxylamine, thyol or semicarbazide functional group is entered to receive the modified hydroxyethylamylum which is conjugated with erythropoietin which is able to react with the modified hydroxyethylamylum, and the hydroxyethylamylum and erythropoietin conjugate containing one or several hydroxyethylamylum molecules, is obtained. The pharmaceutical composition contains the hydroxyethylamylum and erythropoietin conjugate with the erythropoietin activity, and the pharmaceutically acceptable solvent, adjuvant and/or carrier.
EFFECT: hydroxyethylamylum and erythropoietin conjugate with erythropoietin activity.
19 cl, 4 tbl, 25 dwg, 20 ex
FIELD: biology, genetic engineering, biotechnology, medicine.
SUBSTANCE: invention relates to preparing glycosylated polypeptide (glycoprotein) as a component of human erythropoietin by using the technology of recombinant DNAs. This polypeptide shows ability to increase production of reticulocytes and erythrocytes, to enhance the level of hemoglobin synthesis and consumption of iron by marrow cells and characterized by the higher molecular mass as compared erythropoietin isolated from human urine. Invention describes variants DNA sequences encoding this polypeptide that comprise vector constructions with these sequences, a method for preparing transformed mammalian cell lines producing the recombinant human erythropoietin, and a method for its preparing and purification. Also, invention proposes pharmaceutical compositions comprising glycosylated polypeptide (glycoprotein) of erythropoietin as an active component. Applying this invention provides scaling the process for preparing active human erythropoietin useful for its using in medicine.
EFFECT: improved preparing method, valuable properties of polypeptide.
10 cl, 4 dwg, 21 tbl, 12 ex
FIELD: medicine, molecular biology, polypeptides.
SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.
EFFECT: valuable medicinal properties of polypeptide.
21 cl, 92 dwg, 14 tbl, 24 ex
SUBSTANCE: invention relates to crystalline forms of 1,2-pyrrolidinedicarboxamide, N1-(4-chlorophenyl)-N2-[2-fluoro-4-(2-oxo-1(2H)-pyridinyl)phenyl]-4-methoxy-, (2R,4R)-(9CI), as well as to methods of obtaining said crystalline forms and to their pharmaceutical compositions, which have inhibitory activity towards Xa factor.
EFFECT: high stability of new crystalline forms.
19 cl, 9 ex, 7 dwg, 5 tbl