The method of modification, the method of securing the binding protein with the antibody, pharmaceutical composition, method of diagnosis
(57) Abstract:The invention relates to medicine and relates to a method of modifying a protein such as an antibody, methods of securing binding protein antibody pharmaceutical compositions of modified proteins for the treatment of humans. The invention includes associated with lipid protein in which one or more acyl groups bound to protein via carbohydrate side chains and various covalent bonds. Lepidosirenidae antibodies can be used in therapeutic and diagnostic purposes. The advantage of the invention is the increased penetration of modified proteins in cells. 7 C. and 13 C.p. f-crystals, 4 Il., table 2. The invention provides methods of targeting protein, such as antibody, in the intracellular compartments of eukaryotic cells, methods of increasing uptake bodies proteins, pharmaceutical compositions of modified proteins for the treatment of human and ways of preparation of modified proteins. Modified proteins of the present invention include associated with lipid protein in which one or more acyl groups bound to protein via carbohydrate side chains and various covalent tie avannah proteins serves as a diagnostic and/or therapeutic agents for humans and animals. However, proteins, mainly very poor pass through vascular endothelial membrane, if any, are, and are not usually able to cross cell membranes to gain access to intracellular compartments. For example, it is possible to obtain antibodies against the purified intracellular proteins, such as transcription factors, intracellular enzymes and structural proteins, but such antibodies are usually not able to penetrate into intact cells and contact with intracellular antigen targets, while the cell membrane is not damaged.The methodology of monoclonal antibodies in the mid 1970's ushered in a new era in medicine. In the first time, researchers and clinicians have access to virtually unlimited amounts of standard antibodies capable of contact with a predetermined antigenic sites and with a variety of immunological effector functions. These proteins, known as "monoclonal antibodies" seemed very promising, for example, to remove harmful cells, microbial pathogens and viruses in vivo. Methods that make possible the development of specific monoclonal antibodies having binding specificity, napravleniyaner compartments, promised a veritable cornucopia of medicines magic bullets".Unfortunately, the development of suitable medicinal products based on monoclonal antibodies and polyclonal antisera, been seriously hampered by a number of shortcomings related to the chemical structure of natural antibodies. First, antibodies, mostly, are not able to penetrate into the cells, because the antibodies are not able to overcome the plasma membrane of cells, and usually only internalisers, if at all, as a consequence of inefficient endocytotic mechanisms. Second, antibodies, mostly, do not cross the choroid (for example, subendothelial basement membrane), which complicates the effective mainstreaming of antibodies in the organs and interstitial space. Therefore, it would be possible to develop drugs for the treatment of many serious diseases, if there was an effective way of specific delivery of biologically active molecules of immunoglobulins through capillary barriers and into the intracellular sites. For example, the life cycle of a retrovirus, such as HIV, includes intracellular replication, in which two or more encoded by the virus to be oppressed or blocked, if specific monoclonal antibodies against the encoded virus protein could easily penetrate into the intracellular sites where there is a replication of a retrovirus.Immunoliposome were developed as a potential target delivery system of different molecules contained in the liposome, in the target cell. Immunoliposome use antibodies as targeted agents, and acylated immunoglobulin attached to the lipid bilayer of liposomes to target the liposome to certain cell types which have foreign antigens that bind acylated immunoglobulin (immunoglobulin) immunoliposome (Connor and Huang (1985) J. Cell Biol. 101: 582; Huang, L. (1985) Biochemistry 24:29; Babbitt and others, (1984) Biochemistry 23: 3920; Connor and others (1984) Proc. Natl. Acad. Sci. (U. S. A.) 81:1715; Huang and others (1983) J. Biol. Chem. 258:14034; Shen and others (1982) Biochim. Biophys. Acta 689:31; Huang and others (1982) Biochim. Biophys. Acta 716:140; Huang and others (1981). J. Immunol. Methods 46:141; and Huang and others (1980) J. Biol. Chem. 255: 8015). Immunoliposome mainly contain immunoglobulins that are attached to the acyl substituents liposomal bilayer through cross-linking agent such as N-hydroxysuccinimide, and which, therefore, fixed to the lipid bilayer of liposomes. Therefore, the cross-linked is that knowingly convey a particular foreign antigen, by joining the outer cell antigen. While such methods can be used to target liposomes to specific cell types, immunoliposome have several significant disadvantages, which limit their use as a means of drug delivery agents, in particular for the delivery of proteins into the cell.Attempts were made to modify proteins to facilitate their transport across the capillary barriers and into the cells (EP 0329185), but still not reported on the creation of a fully satisfactory way. It was reported on the chemical modification of proteins, such as antibodies, through nonspecific "cationization", to facilitate transvascular and intracellular delivery of proteins (U. S. S. N. 07/693, 872). However, current methods of obtaining cationizing immunoglobulins lead to a significant loss of binding affinity (approximately 90%) cationizing immunoglobulin to its predetermined epitope, compared with decationizing immunoglobulin. In General, cationization includes attaching a diamine, such as putrescine or hexanediamine through carbodiimide Cline. These chemical modification of the primary amino acids, likely violate the secondary and tertiary structure of an antibody to a degree sufficient for the loss of binding affinity. Also these methods produce some degree of cationization residues of glutamic and aspartic acids located in different domains of immunoglobulin chains, which leads to significant loss of binding affinity and/or specificity.Chemical modification of small molecules has also been proposed as a means to enhance transport of small biologically active compounds. Felgner (W 091/17242) describes the formation of lipid complexes consisting of lipid vesicles and the prisoners in their biologically active substances. Felgner, etc. (W 091/16024) describe cationic lipid compounds that are allegedly suitable to enhance the transfer of small biologically active molecules in plants and animals. Liposomes and poly-nucleic acids have been proposed as methods of delivery of polynucleotides in cells. Liposomes often exhibit a narrow range of cell specificity, and when DNA covers them outside, it is often sensitive to the action of cellular nucleases. The latest polecat is:6982) and DNA cover these compounds. In addition, the combination of neutral and cationic lipids is seen as a way transfection of animal cells (Rose and others, (1991), Bio-Techniquer 10:520).Other approaches to enhance drug delivery, in particular through the blood-brain barrier, is used pharmacologically reasonable procedures, including transfer of drugs into an inert state or the conversion of hydrophilic drugs in the fat-soluble. Most approaches to translation into an inert state include the blocking of hydroxyl, carboxyl and primary amino groups of drugs, to make it more soluble and, thus, to facilitate its transport through the blood-brain barrier. Pardridge and Schimmel, U. S. Patent 4902505 disclose chimeric peptides for enhanced transport by receptor-mediated transcytosis are activated.Thus, in this area there is a need for methods to facilitate the transport of specific proteins, such as antibodies, through capillary barriers and inside cells and in the pharmaceutical compositions of such antibodies for the treatment of human and animal diseases that are treatable intracellular proteins and targeted agents, such as monoclonal antibodies.The present invention provides methods of cooking lepidosiren proteins, mainly through lepidosirenidae carbohydrate portion of the glycoprotein or glycopeptide. In General, the methods of the present invention is used for attachment of a lipid, such as lipomin, polypeptide, usually covalent bond lipid and carbohydrate part of the protein, and carbohydrate part in the General case of chemically oxidized and reacts with lipoamino, forming lepidosiren protein. The resulting lepidosiren protein, mainly, has a favorable pharmacokinetic characteristics, such as increased ability to overcome vascular barriers and achieve parenchymal glue Otsego of the invention lepidosirenidae proteins, such as antibodies directed against transcription factors (e.g., Fos, Jun, AP-1, OCT-1, NF-AT), facilitates intracellular localization lepidosirenidae protein (proteins).The invention also provides methods of cooking lepidosiren antibodies that effectively transported through the capillary barriers and internalisers in mammalian cells in vivo. The methods of the present invention relate to methods for the chemical joining of at least one lipid substituent (for example, lipoamino) to the carbohydrate portion of immunoglobulin with the aim of obtaining lepidosirenidae through the carbohydrate portion of the immunoglobulin, which lepidosiren immunoglobulin capable of intracellular localization. In alternative embodiments of the invention, at least one of a lipid substituent (for example, lipoamino) covalently linked to neugeborne part of a protein or polypeptide (for example, through the formation of amide linkages with the rest of aspartic or glutamic acid carboxyl substituent on the side chain or thioester connection with the cysteine residue). You can also attach a fatty acid to the residue of arginine or lysine using aminosalicylic side chain.
-CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH-(CIS and TRANS),
-COCH2, -CH(OH)-CH2and-CH2SO-using methods known in this field, and also described in the following references: Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids, Peptider, and Proteins", B. Weinstein, eds. , Marcel Dekker, New York, page 267 (1983); Spatola, A. F., Vega Data (March 1983), vol.1, Issue 3, "Peptide Backbone ModificCH2-CH2-); Spatola, A. F., and others, Life Sci. (1986) 38:1243 - 1249 (-CH2-S); Hann, M. M., J. Chem. Soc. Perkin Trans I (1982) 307-314 (-CH-CH-, CIS and TRANS); Almquist, R,G and others, J. Med. Chem. (1980) 23:1392 - 1398 (-COCH2-); Iennings-Wtite, C. and others, Tetrahedron Lett. (1982) 23:2533 (-COCH2); Szelke, M., and others , European Appln. EP 45665 (1982) CA: 97:39405 (1982) (-CH(OH)-CH2-); Holladay, M. W., and others, Tetrahedron Lett. (1983) 25: 4401 - 4404 (-C(OH)CH2-); and Hruby, V. J., Life Sci. (1982) 31:189 - 199 (-CH2-S-); each of these sources is included in this document as a reference. Particularly preferred ones communication is - CH2NH-. Such peptidomimetics may have significant advantages over polypeptide, including, for example, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, power, efficiency, and so on ), altered specificity (e.g., a wide spectrum of biological activities), reduced antigenicity, and others. Lepidosirenidae of peptidomimetics usually involves the covalent addition of one or more acyl chains directly or through a spacer (e.g., aminogroup), reiterferien items peptidomimetic that predicted quantitative data structure-activity and/or molecular modeling. Takurou (for example, receptor), which peptidomimetic bound, thereby producing a therapeutic effect. Lepidosirenidae of peptidomimetics should not significantly interfere with the desired biological or pharmacological action of peptidomimetics.
Fig. 1 shows a structural formula representing different lipoamide that can be used in the present invention. The right column gives examples lipoamino branched chain, the left column gives examples lipoamino with direct chain.Fig. 2 schematically represents (1) a glycosylated antibody, comprising a tetramer of immunoglobulin (two light chains, connected to two heavy chains) and (2) lepidosirenidae through carbohydrate immunoglobulins of the present invention. In the example shown lipoamide Deputy branched chain attached to the partially oxidized carbohydrate side chains of the tetramer immunoglobulin. These carbohydrate side chains can be located in areas CHVHCZand the and survival in vitro of cells, infected with HIV-1, compared with the absence of effect in native (i.e. delipidation) anti-Tat antibody. Fig. 4 shows that lepidosirenidae antibodies anti-Tat significantly inhibited the activity of chloramphenicol - acetyltransferase (approximately 75%), while native (delipidation) anti-Tat antibodies, lepidosirenidae anti-GP 120 antibodies or rs CD4 were significantly less effective in the inhibition of the activity of this enzyme in cells HZCD4 HAT.Detailed description
All technical and scientific terms used herein have the generally accepted meaning, unless it was given a different definition, and will be easily understood by experts in this field, which is intended for the present invention. Although any methods and materials similar or equivalent to those described herein, can be used in the practice or testing of the present invention, describes the preferred materials and methods. Listed below are the definitions of the present invention.In the form in which they are used here, the names of the amino acids and their abbreviations are common (Immunology-A Synthesis, 2nd edition, E. S. G "corresponds to" means, what polynucleotide sequence is homologous (i.e., is identical, but not strictly evolutionarily related) to all or part of the polynucleotide sequence, referenced, or that the polynucleotide sequence is identical to a polypeptide sequence that is referenced. In contrast, the term is complementary" means that the complementary sequence is homologous to all or part of the polynucleotide sequence referenced. For illustration, the nucleotide sequence "TATAC matches the" TATAS", referenced, and is complementary to the sequence "GDATA".The terms "substantial similarity" or "substantial identity" as they are used in this document to indicate a characteristic of the polypeptide sequence or in the sequence of the nucleic acid, in which the polypeptide sequence has at least 59% identical to the sequence with the sequence, referenced, and the sequence in the nucleic acid has at least 70% sequence identical to that blue 25% sequence referenced. The sequence is referenced, can be part of a larger sequence, such as a constant region domain of a constant region immunoglobulin gene; however, the sequence of which is referenced at least 18 nucleotides long in the case of polynucleotides, and at least 6 amino acid residues longer than in the case of polypeptides.The term "naturally occurring" is used here to object, which can be found in nature. For example, the polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by man in the laboratory is naturally occurring. Lipoprotein (for example, a naturally occurring protein containing isoprene or maritimelaw acid), which can be isolated from an organism found in nature, and was not made by man, is a naturally occurring lipoprotein."Sites of glycosylation" refers to amino acid residues that are recognized by the eukaryotic cell as plots accession residues sugar is in (N-connection), serine (O-coupling) and threonine (O-coupling). Amino acid sequence, which is usually the signal of the specific site join, denoted here as "a sequence of glycosylation site". The sequence of glycosylation site for glycosylation with N-connection - ASN-X-[- ASN-X-Tre, where X can be any of the conventional amino acids, except Proline. Predominantly sequence glycosylation site for glycosylation O-link - (tre or ser)-X-X-Pro, where X is any of the conventional amino acids. The sequence of recognition for glycosaminoglycans (specific type of sulfated sugar) - -ser - gli-X-gli-, where X is any other amino acid. The terms "N-link" and "link" refers to chemical groups, which serve as sites of connection between a sugar molecule and amino acid residue. N-linked sugars are attached through the amino group; O-linked sugars are attached via the hydroxyl group.However, not all sequences of the glycosylation site of the protein necessarily glycosylated; some proteins are secreted as a glycosylated, and deglycosylation forms, while others fully glycosylated the simulation, which is not glycosylated. Therefore, not all sequences glycosylation sites, which are present in the polypeptide, are necessarily sites of glycosylation, which really attached sugar. The initial N-glycosylation during biosynthesis involves "core carbohydrate" or "core oligosaccharide" (Proteins, Structures and Molecular Principles, (1984) Creighton (ed.), W. H. Freeman and Company, New York, incorporated herein by reference).Used here, the term "glycosylases cell" means a cell capable of glycosylate proteins, in particular eukaryotic cell, and can add N-linked core oligosaccharide containing at least one mannose residue, and/or can add to the O-linked sugar to at least one glycosylation site, at least one of the polypeptide expressed by the named cell, in particular, secreted protein. So, glycosylases cell has at least one enzymatic activity that catalyzes the joining of the sugar residue to the sequence glycosylases plot of the protein or polypeptide, and this cell is really glycosylase at least one expresser leccami. Other eucharistically cells such as insect cells or yeast, can be glycosylamine cells.Used here, the term "antibody" refers to a protein consisting of one or more polypeptides, mainly encoded by genes of the immunoglobulin superfamily (for example, see The Immunoglobulin Gene Superfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and T. H. Rabbitts, eds. , (1989) Academic Press: San Diego, CA, pp. 361-387, which is incorporated herein by reference). For example, but not limitation, the antibody can include a part of or the entire heavy chain, the part or the entire light chain, or may include only a portion or the entire heavy chain. Gene recognition immunoglobulin include the Kappa, lambda, alpha, gamma(IgG1, IgG2, IgG3, IgG4), Delta, Epsilon and mu-genes constant region, as well as countless genes for the variable regions of immunoglobulin. The "light chains" of antibodies (about 25 KD or 214 amino acids) are encoded by variable gene NH2-the end (about 110 amino acids) and a Kappa or lambda constant region gene at the COOH-end. "Heavy chains" of antibodies (about 50 KD or 446 amino acids) is also encoded variable region gene (about 116 amino acids) and one of the previously mentioned kondrusik list: fragments of immunoglobulins (e.g., Fab, F(ab)2, Fv), immunoglobulins, single chain, chimeric antibodies, humanized antibodies, primaryservername immunoglobulins and various combinations of light chain-heavy chain. Antibodies can be produced in glycosylases cells (for example, in human lymphocytes, the cells hybridoma, yeast cells, and so on), negligibility cells (e.g., E. coli) or synthesized chemically or produced by the broadcast system in vitro using polynucleotide matrix for the live broadcast.Used here, the term "lepidosirenidae antibody" means an antibody that is modified so that the obtained lipid a derivative (for example, covalent joining lipoamino, such as glycylcycline, drawroundedrectangle or diastatochromogenes) one or more carbohydrate fraction connected with immunoglobulin in the glycosylation site. Basically, lipid Deputy, such as lipomin, covalently connected via a naturally occurring carbohydrate part in naturally occurring glycosylation site. However, it is possible to obtain antibodies with altered posledovatelnoy chain immunoglobulin) and/or the changed nature of glycosylation (e.g., by expression of encoding the immunoglobulin polynucleotides in glycosylases cells other than lymphocytes or lymphocytes of other species). Lipid substituents may be attached to one or more naturally occurring or not naturally occurring carbohydrate side chain of the immunoglobulin. When antibody is produced by direct polypeptide synthesis or by biosynthesis in negligibility cells (for example, a sample library of phages), basically you attach carbohydrate Deputy by chemical or enzymatic modifications for subsequent lepidosirenidae (in contrast, the carbohydrate can be lepidosirenidae before joining to the immunoglobulin).Used here, the term "lepidosiren protein" refers to a protein (including multimeric proteins, glycoproteins and polypeptides of different lengths), which was modified by the addition of a lipid (e.g., lipoamino), mainly through the carbohydrate part. Lepidosiren protein is prepared by obtaining such a derived protein, which lepidosiren protein differs from a naturally occurring associated with lipids, proteins and lipoproteins. For proteins, obladayushhie significantly reduce the biological activity (e.g., at least 15% of native biological activity should be stored in lepidosiren protein). Lepidosirenidae peptidomimetics should save at least 25 to 95% of the pharmacological activity of the corresponding delipidation of peptidomimetics."Alkyl" refers to a fully saturated aliphatic group which may be straight, branched or cyclic. Alkyl groups include, examples of which may include: methyl, ethyl, cyclopropyl, cyclopropylmethyl, sec-butyl, heptyl, and dodecyl. All of the above groups may be either unsubstituted, or substituted reiterferien substituents, for example halogen; C1-C4alkoxy, C1-C4aryloxy; formyl; alkylenedioxy; benzyloxy; phenyl or benzyl, each of which is optional substituted from 1 to 3 substituents selected from halogen, C1-C4alkoxy or C1-C4acyloxy. The term "nenterprise" characterizes the substituents as not having a harmful effect on any reactions that will be performed in accordance with the procedure of the present invention. If the molecule contains more than one alkyl group, each of them can independently researched the mu bivalent radical, containing only carbon and hydrogen, and which may be branched or straight chain. This term is further illustrated by such examples as methylene, ethylene, n-propylene, t-butylene, i-pentile, n-heptylene and the like. All of the above radicals can be unsubstituted or substituted by one or more reiterferien substituents, for example halogen; C1-C4alkoxy, C1-C4aryloxy; formyl; alkylenedioxy; benzyloxy; phenyl or benzyl, each of which is optional substituted from 1 to 3 substituents selected from the following group: halogen, C1-C4alkoxy or C1-C4acyloxy. The term "nenterprise" characterizes deputies, as not having an adverse impact on any reactions that must be performed in accordance with the procedure of the present invention. If the molecule contains more than one alkalinous groups, each of them can independently chosen from the group alkylene", unless otherwise specified."Aryl", denoted by Arincludes monocyclic or condensed carbocyclic aromatic group of 6 to 20 carbon atoms. Aryl groups include here as a researcher and, for example, selected from the following groups: lower alkyl, lower alkenyl, lower quinil, lower alkoxy, lower alkylthio, lower alkylsulfonyl, lower alkylsulfonyl, dialkylamino, halogen, hydroxy, phenyl, phenyloxy, benzyl, benzoyl and nitro. Each Deputy may be optional substituted incremental reiterferien substituents."Amino" refers to the group-NH2."Alkylaryl" refers to the group -(CHR1)-CO-, in which R1is additionally marked position. R1can be hydrogen, alkyl or amino group. Preferably R1is an amino group.Description of the preferred embodiments of the invention
In accordance with the present invention created new ways to facilitate chemically modified proteins, such as antibodies, passing through the capillary barriers and into the cells. In General, the methods include covalent joining of at least one nenterprise lipid substituent (for example, glycylcycline, glycylglycine, glycylcycline, drawroundedrectangle and gazilliotrilliobillionth) to the reactive site of the molecule which can be attached to proteins to obtain lepidosiren proteins, such as lepidosirenidae antibodies of the present invention. For example, but not limiting, examples of lipids that can be attached to proteins of interest, to obtain lepidosirenidae protein: lipoamide, lipopolymer, fatty acids (e.g. stearic acid, oleic acid, etc.). In General, the lipid must be attached by covalent linkage to the carbohydrate associated with the protein (for example, the carbohydrate side chains of glycoproteins). It is preferable to use a naturally occurring carbohydrate side chains to attach lipoamino, although can be created and new glycosylation sites in the polypeptide by genetic manipulation of nature with coded polynucleotides and the expression of the coding polynucleotide in glycosylase cell to obtain a glycosylated polypeptide.Glycosylated proteins can be lepidosirenidae to enhance its transsoudata transport, absorption body and intracellular penetration, including penetration of the nucleus of the cell. In General, glycosylated polypeptide such as an antibody, are oxidized by chemical oxidant (for example, periodate) obtained for ntnui (amide or kidney, respectively) due lipoamino and protein.In a typical case, the oxidation of the carbohydrate side chain is a partial oxidation, resulting in the formation of at least one reactive carboxyl or aldehyde group, although, generally, methods of chemical oxidation to produce a number of molecules that are partially oxidized, and a number of molecules that are not oxidized or oxidized completely. However, to be lipidation reaction with lipoamino, the protein must be oxidized to obtain at least one additional aldehyde groups, which can react with lipoamino, despite the fact that it is possible to get lepidosirenidae proteins by linking with additional carboxyl groups. Additional carboxyl or aldehyde group of the oxidized glycoprotein is carboxyl or aldehyde group with the carbonyl carbon derived from oxidized oligosaccharide, and which is covalently linked to protein, both directly and through the insert (e.g., non-oxidized portion of the N - or O-linked carbohydrate side chain). Preferably, N-linked or O - linked carbohydrate chain should be okislityelnoye for further reaction with lipoamino. Often glycoproteins having one or more complex N-linked oligosaccharides having, for example, branched (mannose)3(N-acetylglucosamine) rod, partially oxidize limited by reaction with a suitable oxidant, mainly periodata. Linked oligosaccharides containing N-acetylglucosamine (NAG), mannose, galactose, fucose (6-deoxyglucose), N-acetylneuraminic acid (sialic acid), glucose, N-acetylmuramic acid, N-atsetilgalaktozamin, xylose, or a combination of these sugars can be oxidized and react with lipoamino to get lepidosiren proteins, in particular lepidosiren through carbohydrate. Glycoproteins containing oligosaccharides of monosaccharide units, other than those listed above as examples, including non-naturally occurring monosaccharides can also be oxidized and covalently attached to lipoamino to get lepidosirenidae protein.Lipoamide are molecules having at least one acyl group and at least one free amine (i.e., primary or secondary amine). It is assumed that in the practice of the present invention can also be used lipoamide, which are tertiary amines, aimery lipoamino, possessing primary amine, shown in Fig. 1. For example, the method of the present invention allows to obtain lepidosirenidae proteins by reaction of peptide with lipoamino having a straight chain of the following formula:
where R is a disubstituted alkyl (alkylene), preferably, methylene (-CH2-); 1,4-disubstituted cyclohexyl; disubstituted aryl (Allen), preferably 1,4-disubstituted phenyl (phenylene); aminogroup formula(CHR1)-CO-NH-, where R1is hydrogen or an amino group; alkylaryl, preferably aminosilanes alkylsulphonyl; or phosphate fluids, preferably of the formula-CH2-O-PO2-O-. n is a whole number, usually from 1 to 50, preferably from 5 to 30, most preferably from 0 to 25, usually from 15 to 20. In General, the selection is at the discretion of practice, which should be guided by the following consideration: if the molecule you need to lipodisolve is large (i.e. more protein 10 kDa), preferably at least 8 to 12 or more, to increase the hydrophobicity of the resulting lepidosirenidae protein; if the molecule you need to lepidosirenidae, small (for example, Oligopeptide), typical of the molecule.In the practice of the present invention can be applied also lipoamide branched chain, which include, for example, lipoamide the following formula:
< / BR>where R' is trehzameshchenny alkyl, preferably - CH2CH< or 1,2,4-trehzameshchenny cyclohexyl; trehzameshchenny aryl, preferably 1, 2, 4-trehzameshchenny phenyl; aminogroup formula(CHR1)-CO - N<, where R1is hydrogen or an amino group; aminogroup formula-CHR2-NH-CH<, where R2is hydrogen or amino group, or aminogroups formula-CH2-N< or phosphate fluids, preferably of the formula-CH2-CH2-O-PO2-O-CH2-CH(CO2-)2m and n independently are elected and are integers, typically from 1 to 50, preferably from 5 to 30, more preferably from 10 to 25, usually from 15 to 20. In General, selected at the discretion of the practice that is guided by the following considerations: if the molecule you need to lipodisolve is large (i.e. more protein 10 kDa), preferably, and/or were at least 8-12 and more, to increase the hydrophobicity of the resulting lepidosirenidae protein; if the molecule you need to lepidosirenidae, small (for example, Oligopeptide), typically ranges from 2 DPO essence, any glycoprotein can be lepidosirenidae according to the method of the present invention by reaction between lipoamino and oxidized carbohydrate side chain. Fig. 2 schematically depicts a glycosylated antibody and lepidosirenidae through carbon antibody of the present invention, respectively. Deglycosylated proteins can be konjugierte with lipids via a suitable cross linking agent (e.g., via carbodiimide communication).In accordance with the present invention, new lepidosirenidae antibodies capable of specific contact with a predetermined intracellular epitopes with strong affinity. These antibodies can easily penetrate into the intracellular compartment and have a binding affinity of at least about 1 106M-1preferably 1 to 107M-1to 1 108M-1more preferably at least 1 of 109M-1or more. In the typical case lepidosirenidae antibodies have lipid Deputy attached to a naturally occurring carbohydrate side chain of the original chain of immunoglobulin, which is the antibody that is specific reactive with intracellular, transmembrane or extracellular what the situation carbohydrate residues by lepidosirenidae resulted in a significant loss of the affinity of antibodies for their antigens (Rodwell and others, (1986) Proc. Natl. Acad. Sci. (U. S. A) 83:2632). Lepidosirenidae antibodies in General maintain a strong affinity for their antigens, and their avidity can easily be measured using known assays of binding of the antigen-antibody. These antibodies can be quite economical to produce in large quantities and used, for example, for the treatment of various human diseases using a variety of techniques.The main structural unit of antibodies is some form of immunoglobulin. This form is a tetramer and consists of two identical pairs of chains of immunoglobulin, each pair has one light and one heavy chain. In each pair different parts of the light and heavy chains together are responsible for binding to the antigen and a constant region are responsible for the effector functions of antibodies. In addition to antibodies, the immunoglobulins may exist in many different forms, such as Fv, Fab, and (Fab')2and also in the form of bifunctional hybrid antibodies, fused proteins, and in other forms (e.g., Lanzavecchia and others, Eur. J. Immunol. 17, 105 (1987)) and in the form of single chains (e.g., Huston and others, Proc. Natl. Acad. Sci. U. S. A., 85, 5879-5883 (1988) and Bird and others, Science, 242, 423-426 (1988)). (Cm. Hood. and others, "Immunology", Benjamin, N. Y., 2nd ed. (1984) and Hunkapiller and Necheloveka, cells hybridoma, yeast and so on), negligibility cells (e.g., E. coli), or synthesized chemically or produced vi vitro translation systems using polynucleotide matrix for the live broadcast. One of the sources of hybridoma cell lines and encoding the immunoglobulin polynucleotide is an American collection of tissue cultures (ATSS), Rockville, MD. Methods the expression of xenogenic proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known to specialists in this field and are described further in Maniatis, etc. , Molecular Cloning: A Laboratory Manual (1989), 2nd edition. Cold Spring Harbor, N. Y; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, CA; Merrifield, J. (1969) J. Am. Chem. Soc. 91: 501; Chaiker J. M. (1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser and others, (1989) Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Ann. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which is incorporated herein by reference). Antibodies that develop in negligibility cells, can be attached to the lipid via a bifunctional Poperechnaya agent or, preferably, excision of glycosylated in glycosylase system such as peeled Oasi document by reference). In another embodiment, polynucleotide encoding the antibodies can be isolated by screening of libraries of expression of prokaryotic cells, such as libraries of fragments of antibodies and subsequently expressed in glycosylases cells for production of glycosylated antibodies. In accordance with these methods can be obtained glycosylated antibody with naturally occurring or unnaturally occurring nature of glycosylation. Such glycosylated antibodies can be lepidosirenidae in accordance with the methods of the present invention.It is shown that glycosylation of immunoglobulins significantly affects their effector functions, structural stability and the rate of secretion of antibody producing cells (Leatherbarrow, etc. , Md. Immunol. 22:507 (1985)). Carbohydrate groups responsible for these properties, mainly attached to the constant (C) regions of antibodies. For example, glycosylation of IgG to asparagine 297 in the domain CH2 to achieve full capacity of IgG in relation to activation of the classical complement-dependent cytolysis (Tao and Morrison, J. Immunol. 143:2595 (1989)). Glycosylation lgM for asparagine 402 in the domain CH3, it is necessary to obtain Dashkov glycosylation sites at positions 162 and 419 in domains CH1 and CH3 IgA antibodies leads to intracellular degradation and at least 90% suppression of secretion (Taylor and Wall, Mol. Cell. Biol. 8: 4197 (1988)).It was also considered glycosylation of immunoglobulins in the variable region (V). Sox and Hood. Proc. Natl. Acad. Sci. U. S. A. 66: 975 (1970) report that about 20% of human antibodies glycosylated in the region. Suggest that glycosylation domain is due to the random occurrence of the signal N-linked glycosylation of ASN-XAA-ser-tre in the sequence field and does not play a significant role for the function of immunoglobulins.So, basically, it is preferable that lepidosiren were exposed to antibodies with the natural character of glycosylation. If the sites of glycosylation built into the antibody, preferably a new glycosylation site was introduced at a constant region or variable region of the frame area, which is less likely to have an adverse impact on antigennegative activity of antibodies. In General, the preferred option is the integration of new sites of glycosylation of the antibody to the constant region.In another embodiment, the polypeptide is to form with the lipid substituent, can be obtained, and these fragments will have one or more activity of an antibody (for example, antigen-binding activity). These polypeptide fragments can be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by site-directed mutagenesis in desirable areas expressing vectors containing sequences encoding immunoglobulin proteins, for example, after CH1to obtain Fab fragments or after the hinge region to produce (Fab')2the fragments. Antibodies with one chain can be obtained by connecting the VZand VHDNA linker (see Hustor and others, CIT. Bird and others, CIT.). As well as, like many other genes, genes associated with encoding immunoglobulins contain separate functional areas, each of which has one or more distinct biological activities, the genes can be fused to functional areas with other genes that have new properties. Polynucleotide sequences to produce immunoglobulins of the present invention is able, ultimately, to Express the desired antibodies can be sformirovatb (for example, V, J, D and C plots), as well as using many different techniques. The connection of suitable synthetic and genomic sequences is currently the most common method, however, can also be used cDNA sequence (see European Patent Publication 0239400 and L. Reichmann and others, Nature, 332,323-327 (1988)).Immunoglobulins and/or DNA sequences encoding immunoglobulin chains can be obtained, for example, by a hybridoma clones that can be obtained using techniques well known in the art (Kohler and Milstein (1976) Eur. J. Immunol. 6: 511, incorporated herein by reference), or can be obtained from various sources ("ATCC Catalog of Cell Lines and Hybridomas", ATCC, Rockville, MD, incorporated herein by reference). DNA sequences encoding immunoglobulin chains can be obtained by standard cloning methods well known in the art and described in various publications, for example, Maniatis, etc., Molecular Cloning: A Laboratory Manual, 2nd ed. , (1989), Cold Spring Harbor, N. J., and Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc. , San Diego, CA, which is incorporated herein by reference.As mentioned above, posledovatelnuju connected to the sequence control expression. These expressing vectors is typically replicated in the body of the host as AMISOM, and as a part of the chromosomal DNA of the host. In the General case, expressing the vectors will contain selection markers, e.g., resistance to tetracycline or resistance to G418 in order to make possible the detection of those cells that are transformed with the desired DNA sequences (see, for example, U. S. Patent 4,704,362).E. coli is one prokaryotic hosts, suitable in particular for cloning the DNA sequences of the present invention. Other microorganisms-hosts suitable for these purposes include bacilli, such as Bacillus Subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, you can also create expressing vectors that contain sequences control expression that is compatible with the host-cell (e.g., Replicator). In addition, there will be present a lot of well-known promoters, such as the lactose promoter system, a tryptophan promoter system-galactosidase promoter system, or a promoter system from phage lambda. Promoters will be contrasty binding site of the ribosome and the like, to initiate and complete transcription and translation. Proteins, such as antibodies that are expressed deglycosylation cells, excision can be glycosylamine in glycosylase system (Mueckler and Lodish, CIT.).Other microorganisms, such as yeast, can also be used for expression. Saccharomycer is preferred glycosylase the host-cell in the presence of suitable vectors having the sequence control expression, such as promoters, including 3-phosphoglycerate or other glycolytic enzymes, the port Replicator terminal sequence and the other, if so desired.In addition to microorganisms, cell cultures, mammalian tissue can also be used for expression and production of polypeptides of the present invention (see, Winnacker, "From Genes to Clones", VCH Publishers, N. Y., N. Y. (1987)).Eukaryotic cells are generally preferred since created many beautiful lines of master cells that can secrete intact immunoglobulins, and include CHO cell lines, various COS cell lines, HeLa cells, preferably myeloid cell lines, etc., and transformirovaniya B-cells or the such as the Replicator, the promoter amplifier (Gueen and others, Immunol. Rev., 89, 49-68 (1986)), and the necessary processing sites, such as sites of binding of ribosomes, the sites of splicing PHK, sites and polyadenylation sequence, termination of transcription. Preferred sequences control the expression of promoters are selected from immunoglobulin genes, SV40, adenovirus, cytomegalovirus virus, bovine papilloma etc.The vectors containing the DNA segments of interest (e.g., sequences encoding the light and heavy chain, and a sequence controlling the expression), can be introduced into the cell-the owner with the help of a well-known in the art methods, which vary depending on the type of host cell. For example, prokaryotic cells are usually used transfection by calcium chloride, while for other host cells can be used the treatment with calcium phosphate or electroporation (see Maniatis and other, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, (1982)).Being expressed, the whole antibodies, their dimers, individual light and heavy chains, or other forms of immunoglobulins of the present invention, can be selected in accordance with afia, the electrophoresis gel and the like (see R. Scopes, "Protein Purification", Springer-Verlag, N. Y. (1982)). Preferred substantially purified immunoglobulins with homogeneity at least 90% to 95%, for pharmaceutical purposes the most preferred homogeneity 98-99% or more. Being purified, partially or to complete homogeneity as desired, the polypeptides can then be applied for therapeutic purposes (including in vitro use) or for the development and production of analytical procedures, immunofluorescent staining, etc. (see Immunological Methods, Vols. 1 and 11, Lefkovits and Pernis, eds. Academic Precc, New York, N. Y. (1979 and 1981).In the methods of the present invention is preferably used intact immunoglobulins or their binding fragments. In the typical case lepidosirenidae antibodies are IgM isotypes or IgG person, but other constant region mammals can be applied if required. Can be obtained lepidosirenidae antibody classes IgA, IgG, IgM, IgE and IgD. Preferably, lepidosirenidae antibodies of the present invention are antibodies to human, mouse, cattle, horses, pigs or primates, the most preferred human antibodies or mouse. The present invention can be applied to the s antibodies, humanized antibodies, primaryservername antibodies, Fv fragments, conjugates with a toxin-antibody conjugates isotope-antibody and conjugates tomographic agent-antibody. For imaging in vivo lepidosirenidae antibodies mark the diagnostic label, enter the patient and determine their localization in different periods of time after injection. Various methods of labeling antibodies, diagnostic agents (e.g., Tc99other radio, radiopaque agents, roentgenographically dyes are well known to specialists in this field.Proteins and oligopeptides (i.e., a polypeptide comprising 2 to 50 amino acid residues connected by peptide bonds, other than immunoglobulins, you can lepidosirenidae according to the methods of the present invention. Naturally occurring glycoproteins (for example, glutamyltranspeptidase, thrombomodulin, proteins transport glucose) are the preferred substrates for lepidosirenidae through carbohydrate, although essentially any polypeptide can be lepidosirenidae covalent joining through cross linking agent (for example, N-hydroxysuccinic) to a suitable amino acid side chains. In other embodiments, Onen to neugeborne part of a protein or polypeptide (for example, the formation of amide linkages with the rest of ASP or Glu carboxyl substituent on the side chain or thioester connection with the remainder of the CIS). You can also attach a fatty acid to arg or Lys residues via an amine substituents on the side chain. Examples deglycosylated proteins that can be lepidosirenidae to enhance transvascular or intracellular transport, include, but are not limited to the following list: c-for c-myc, c-src, NF-AT and HMG CoA reductase. Naturally occurring lipoproteins, such as native proteins, undergoes physiological farnesiana, geranylgeranylation and palletirovanie are natural products and are not called in this document "lepidosirenidae protein".Lepidosirenidae antibodies and pharmaceutical compositions based on them are particularly useful for parenteral administration, i.e., subcutaneous, intramuscular or intravenous. Compositions for parenteral administration include often the solution of an antibody or a cocktail of him, dissolved in an acceptable carrier, preferably water. You can use a variety of aqueous carriers, such as water, buffered water, 0.4% sodium chloride solution, is to elisavet standard, well-known sterilization methods. These compositions may contain pharmaceutically acceptable additive substance, if it is required to approximate physiological conditions, such as pH adjustment and buffering agents; agents that reduce the toxicity and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, albumin human, and so on, the Concentration of antibody in these formulations can vary widely, i.e. from less than 0.5%, usually at least 1% to 15-20% by weight and is selected based on the volume of the liquid, viscosity and so on, in accordance with a particular chosen method of administration to the patient.Thus, a typical pharmaceutical composition for injection containing 1 ml of sterile water with a buffer and 1-10 mg lepidosirenidae immunoglobulin. A typical composition for intravenous administration contains 250 ml of sterile ringer's solution, and 150 mg of antibody. Actual methods of preparing the compositions for parenteral administration are well known to specialists in this field and are described in detail, for example, in Remington''s Pharmaceutical Science, 15th edition, Mack Publishing Company, Easton, Pennsylvania (1980), incorporated herein by reference.Lepidosirenidae white in a suitable medium immediately before use. These techniques proved effective against the standard of immunoglobulins, so you can apply known in the field of methods of freeze drying and subsequent recovery. Experts clear that lyophilization and recovery can lead to various degrees of loss of activity (for example, with regard to the standard of globulins, it is known that IgM lose activity more than IgG) and that the levels of use should be adjusted to compensate for the loss of activity.Compositions containing these lepidosirenidae proteins (e.g. antibodies), or a cocktail of them can be used as a prophylactic and/or therapeutic purposes. For therapeutic purposes the composition is introduced to the patient in a quantity sufficient to cure or at least suspension of the disease and its complications. Quantity adequate to complete these tasks is defined here as "therapeutically effective dose". Amounts effective for this purpose will depend on the severity of the disease and the General condition of the patient's immune system, but generally range from about 1 to about 200 mg of antibody per dose, the most commonly used dose of from 5 to 25 mg. Well the s States, that is life-threatening or potentially life threatening situations.In the preventive purposes compositions containing the present antibodies or a cocktail of them, entered the patient who is not in a morbid state, to enhance its resilience. This number is defined here as a "prophylactically effective dose." For these purposes, the exact amount also depends on the patient's state of health and General level of immunity, but generally range from 0.1 to 25 mg per dose.Single or multiple injection the patient of these songs are performed with dose levels and pattern of use, as determined by the attending physician. In any event, the pharmaceutical formulations will provide a number lepidosiren proteins and/or antibodies(a), sufficient for effective treatment of the patient.For diagnostic purposes lepidosirenidae antibodies can be labeled and its. Its antibodies can be used in combination with other labeled antibodies (second antibodies) that react with lepidosirenidae antibodies, such as antibodies that are specific against the constant regions of immunoglobulins che is the number of labels, such as radionuclides, enzymes, enzyme substrates, cofactors of enzymes, enzyme inhibitors, Legendy (particularly haptens), radiopaque agents, chelates metals and so on Many kinds of topographic applications are available and well known to specialists in this field. For example, but not limitation, antibodies that bind tumor antigens (e.g., antibodies against carcinoembryonic antigens) can be lepidosiren and connected with a radiopaque agent or material for nuclear magnetic resonance, is introduced to the patient and determined to establish places of localization of tumor or metastatic lesions.Lepidosirenidae immunoglobulins of the present invention can be used for diagnosis and treatment. To illustrate, but not limit, we will use for the treatment of cancer, autoimmune diseases, or viral infections. For the treatment of cancer antibodies will be specific to contact antigens that Express certain cancer cells, such as c-myc gene product and other well-known professionals. Preferably, lepidosirenidae antibody will be contacted with the mutant protein, such as c-ras oncore is 3, 59 or 61 protein (for example, replacement of serine at position 12 p21ras). For the treatment of autoimmune disease antibodies in the typical case should contact the chief regulatory protein, which primarily Express activated T cells, such as NF-AT, and many other intracellular proteins, are well known in the art (for example, see Fundamental Immunology, 2nd ed., W. E. Paul. ed., Raven Press: New York, N. Y. incorporated herein by reference). For the treatment of viral diseases antibodies in the typical case should contact the protein, which Express a cell infected with a particular virus, such as encoded by virus polymerase and HIV-1 Tat, and many other viral proteins, are well known in the art (see , for example, Virology, 2nd ed., B. N. Fields, and others , eds. , (1990), Raven Press: New York, N. Y. incorporated herein by reference).Can also be compiled sets to use a specific lepidosiren antibodies to protect, or determine the cellular activity or for detection of intracellular protein in certain cells or to diagnose diseases. So, you can create a composition of the present invention, usually in a lyophilized form in a container or road is about type. Lepidosirenidae antibodies, which can be conjugated with a label or toxin, or conjugatively include in the kits with buffers, such as Tris, phosphate buffer, carbonate buffer, and so on , stabilizers, biocidal agents, inert proteins, such as serum albumin or similar, and with instructions for use of kit. Mostly, these materials are present in amounts less than 5% by weight of active antibody, and, usually, in the amount of 0.001% by weight, again based on the concentration of antibodies. It is often desirable the inclusion of inert filler for cultivation of active ingredients in amounts of from about 1 to 99% by weight of the entire composition. If applicable, additional antibodies capable of contact lepidosirenidae antibodies, they must be in separate vessels. These second antibodies may be conjugated with a label and to be in the same formulation as described above formulation for the core antibodies, as well as yourself can be lepidosirenidae.Lepidosirenidae antibodies of the present invention are also suitable for use in advanced diagnostic methods and methods of purification and separation of proteins. For example, the I or sensitive to proteolysis) or prone to aggregation (for example, -amyloid protein), which makes the selection and/or diagnostic determination difficult. Lepidosirenidae antibodies are able to penetrate cells and to communicate with specific intracellular antigens on target; this binding of the antigen-antibody can stabilize the antigen target and block the enzymes involved in the cleavage of the target antigen (e.g., protease, ubiquitin-binding enzymes, glycosidase), facilitating the identification and/or selection of the target antigen.In one embodiment of the invention, lepidosirenidae antibody, which is specific associated with intracellular target antigen comes into contact with living cells containing intracellular antigen-target under physiological conditions (e.g., cell culture, in terms of the body), and incubation occurs within a time period appropriate for binding (for example, from 10 minutes to several hours). Lepidosirenidae antibody specifically binds to a target antigen, forming a complex of the antigen-antibody, which is less susceptible to degradation and/or aggregation of the antigen target. In a typical case, the cells are then fixed, make permeable, and define the complex is sustained fashion labeled antibody, which specifically binds to lepidosiren antibody. Examples of preferred labels connected with additional antibodies are: FITC, rhodamine, conjugates of horseradish peroxidase, alkaline phosphatase conjugates, conjugates --galactosidase, biotinyl, radioisotopes and the like. In some embodiments of the invention, the secondary antibody can be lepidosiren and stages of fixation and/or increasing the permeability can be omitted and replaced with a thorough laundering of the cell sample to remove non-specific staining. Is also possible to directly use lepidosiren and labeled primary antibodies without the use of additional, secondary antibodies. Labeled protein And can replace the secondary antibody to determine primary (lepidosiren) antibodies.Lepidosirenidae antibodies can also be used for cellular therapy, for example to associate a predefined intracellular target antigen and modification of the biochemical properties of the target antigen. For example, multimeric proteins, such as heteropolymeric proteins (e.g. transcription factors, G-protein) or homodimeric proteins (Napoli other activity, requiring intracellular interactions that can be blocked lepidosiren antibody, which is specific associated with one or more subunits and prevents the functional interaction between subunits. For example, lepidosirenidae Fos antibody that binds to part of the Fos (for example, with the "zipper" leucine), which is required for binding to Jun for the formation of transcriptionally active transcription factor AP-1 (Fos/Jun heterodimer), can block the formation of functional AP-1 and to inhibit AP-1-mediated gene transcription. For example lepidosirenidae anti-ras antibody may contact the ras epitope that is required for its function signal transduction (for example, GTP-binding site/GDF, part of the ras that binds extension protein, such as GAP or similar), thus modifying the activity of intracellular ras in living cells.The following examples are offered to illustrate but not to limit.EXPERIMENTAL EXAMPLES
Preparation lepidosirenidae bovine IgG
Was obtained glycylcycline by the addition of a glycine residue to dioctadecyl, with the e links. 1 equivalent of benzyloxycarbonyl-glycyl-p-NITROPHENOL and 1.1 equivalents of triethylamine in CH2-Cl2reacted for 5 hours, then added H2, 10% Pd/C in CH2Cl2/ethanol, and the reaction was continued for another 1 hour.Two mg of bovine IgG (Sigma) was dissolved in 400 μl of 300 mm NaHCO3in a 1.5 ml Eppendorf vial. Fifty ál of freshly prepared NaIO4(42 mg/ml in water) was added to the bottle, wrap it in aluminum foil and gently attractively 90 min at room temperature. The reaction mixture is then loaded into the column PD-10 (Pharmacia), previously equilibrated with 10 mm Na2CO3(fraction 1) and from the column was washed with 500 µl fractions. Fraction 7 (between 3 ml and 3.5 ml) contained approximately 1.6 mg bovine IgG, which was measured using analysis of Bradford.The solution glycylcycline in dimethyl sulfoxide was prepared as follows: 5 mg of lipid was placed in 1 ml of dimethyl sulfoxide was mixed vortex motion of the fluid by rotation of the vial, intensely for several minutes. Under these conditions, the lipid is not completely dissolved. Fifty μl of this solution was carefully selected so as not got undissolved lipid, and was added to 350 μl of fra is Orono was shaken for 20 hours at room temperature.One hundred μl of a solution of NaBH4(10 mg/ml in water) was added at the next stage. After one hour, 40 μl of a solution of ethanolamine (15 μl in 1 ml of P2O) was added to the mixture. After another hour, the reaction mixture was loaded onto a column PD-10, pre-equilibrated with 100 mm HEPES buffer, pH 8.5. The fraction containing lepidosiren IgG (between 3 and 3.5 ml) was collected and stored in ice.Tagging14C-acetic anhydride
Used14C-acetic anhydride (500 MX, Amersham) in benzene (10 of 106imp. in min/l). Two 5 ál aliquots were added to 500 ál fractions containing lepidosiren IgG, with 10 min intervals in an Eppendorf vial. The reaction mixture was left in ice. 500 μl of a solution of native IgG (800 μg in 100 mm HEPES, pH 8,6) was treated in the same way.After 30 min, the vials were heated to 20-25oC, 14C-labeled IgG was separated from free14C-acetate on column PD-10, balanced saline solution with phosphate buffer. Incorporated radioactivity was approximately 10 106imp./min to 500 mcg.The study of organ absorption
Mouse male Swiss albino, (20 g) were used in this study. Intravenously (via tail vein) BB is the logical solution with phosphate buffer (approximately 400,000 decays/min). After 30 min or 3 h, mice were killed, blood was collected in tubes containing ethylenediaminetetraacetic acid (edtc), brain (excluding the stem and the cerebellum, spleen, one kidney and one lobe of the liver was crushed. Organs homogenized in 1 ml of 10 mm TRS buffer, pH 7.4 and 500 μl aliquots were counted in a scintillation counter Beckman. The concentration of protein in these homogenates was determined by analysis of Bradford (Kumasi blue). The blood was centrifuged and counted 20 µl fractions of plasma. Table 1 shows absorption14C the brain, liver, spleen and kidney, marked private by dividing the radioactivity of 1 µg of protein body on radioactivity 1 µl of plasma (data expressed in µl/µg protein).The control group consisted of 4 and 6 mice at 30 min and 3 h, respectively. Groups of mice treated with lepidosirenidae IgG, everywhere was 6 individuals. Data are the average of the mean square error of the mean.Example 2
Inhibition of cytotoxicity of HIV-1 lepidosiren anti-Tat antibodies
Monoclonal antibody that is specific associated with the Tat protein of HIV-1, was lipidsoluble according to the procedure described in example 1, supra, including oxidation periodata carbohydrate portion of the antibody, in the following the explicit part); antibody was washed from the final column PD-10 saline phosphate buffer.Sup T1 cells were kept in 24-hole plates (100,000 cells per ml, in 2 ml of modified RPM1 1640 medium). The granulosa cells: (1) without additional processing (dual control), (2) in the presence of native anti-Tat antibodies (15 μg/ml) or (3) in the presence of lepidosiren anti-Tat antibodies (11,7 µg/ml), during the first five days of the experiment. At the end of the first day in one hole control cells were added to HIV-1 W and in wells with cultures containing cells treated with native anti-Tat antibody and lepidosiren anti-Tat antibody. Viable cells were counted daily. Untreated HIV-infected cells were grown to a density of approximately 500,000 cells per ml, and their number started to decrease after approximately eight days, due to the cytotoxic effect of the virus.Uninfected cells were grown to a density of approximately 1,000,000 cells per ml. Effects on infected cells native anti-Tat antibodies did not protect cells from the cytotoxic effect of the virus. On the contrary, lepidosirenidae anti-Tat antibodies provided almost complete protection of the cells from the cytopathic effect of the virus. This of estaline in Fig. 3.In another experiment, cells were kept as described above in the previous example, without any impact and without infection with HIV-1 and without treatment, with the influence of native anti-Tat antibodies (1 μg/ml) and infected with, or exposed lepidosirenidae anti-Tat antibodies (1 μg/ml) and infected. In the last three versions of the virus was added at the end of the first day of cultivation. In the last two versions of native or lepidosirenidae antibodies were present from the first to the seventh day.The data presented in table 2, show that at that time, as native anti-Tat antibodies were negligible impact, if provided at all, the number of viable cells and the activity of reverse transcriptase, lepidosirenidae antibodies created significant protection of cells in culture from the cytopathic effect of the virus and a significant reduction in the activity of reverse transcriptase. The latter fact suggests that lepidosirenidae antibodies inhibited intracellular replication of HIV-1.HIV-1-infected SupT1 cells were treated daily with anti-Tat antibodies in native and lepidosirenidae forms or rsCD4 (all proteins used when the concentration is 10 days after infection. The number of cells and the activity of the reverse transcriptase () in culture medium was determined every day, starting from the second day after infection. Until the tenth day, native anti-Tat antibodies had no significant effect on the number of cells, and activity, while lepidosirenidae anti-Tat antibodies increased cell survival compared with untreated these antibodies, infected cells by approximately 70%, and reduced activity FROM approximately the same amount. The cultivation was continued for three days without further addition of antibodies. The effect lepidosiren anti-Tat antibodies lasted three days, which indicates the accumulation lepidosiren antibodies in cells in quantities sufficient to ensure maintenance of the protective effect against viral infection/replication. The magnitude of impacts lepidosiren anti-Tat antibodies on cell survival and activity were very similar to those observed with the same dose rsCD4. Increasing the concentration lepidosiren antibodies to 10 mcg/ml did not cause a further reduction in activity FROM.Example 3
The ability lepidosiren anti-Tat antibodies to inhibit transcriptional polynucleotides, expressing CD4, membrane receptor, mediating HIV-1 infection, and also containing the HIV-1 long terminal repeat (LTR sequence) and induce the transcription of associated gene (chloramphenicol, acetyltransferase, HAT). These cells (HLCD4 HAT) - sensitive HIV-1 infection, which produces a functional Tat protein; the binding of newly synthesized Tat HIV-1 LTR sequences leads to transcription of a linked HUT gene. Thus, the magnitude of expression of the HAT is approximately proportional to the severity of HIV-1 infection and activity of Tat protein in the cells.Cell culture HeLa (3 of 105cells/ml in modified according to the method of Dulbecco environment Needle) was subjected to the same concentrations (1 or 10 μg/ml) of different antibodies (native or lepidosiren form) or recombinant soluble CD4 (rsCD4) within one hour long and were washed before the addition of HIV-containing culture supernatant fluid (100 μl). After 24 hours, cells were collected and the expression of the HUT was measured by the method of Ho and others (1984). Each experiment was performed in four replicates and for four different time periods. Fig. 4 shows that lepidosirenidae anti-Tat antibody significant is the body, lepidosirenidae anti-gp120 antibodies or rsCD4 were significantly less effective in the inhibition of the activity of the HUT. These data show that lepidosirenidae anti-Tat antibodies were capable of specific contact their intracellular targets, Tat, and to inhibit activity of the target, as an activator of transcription of LTR sequences.Moreover, data showing the passage lepidosiren anti-Tat antibodies into HeLa cells show that the transport mechanism probably does not require the formation of endosomes, as it was reported that HeLa cells do not possess or have low faguoqitirute ability.Example 4
Preparation lipidation of immunoglobulins, reactive with intracellular protein
Glycylcycline was obtained by attaching a glycine residue to dioctadecyl, according to the method described Behr and others (1989) Proc. Natl. Acad. Sci. (U. S. A. ) 86:6982, incorporated herein by reference. 1 equivalent of benzyloxycarbonyl-glycyl-p-NITROPHENOL and 1.1 equivalents of triethylamine in CH2Cl2reacted for 5 hours, and then thereto was added H2, 10% Pd/C in CH2Cl2/ethanol, and the reaction was continued for another 1 hour.Ig against c-myc man
Glycosylated immunoglobulins mouse that specific contact intracellular enzyme HMG CoA reductase, were prepared by separately culturing hybridoma cell line A9 (ATCC CRL 1811) modified by way of Dulbecco environment Needle with 4.5 g/l glucose, 5% horse serum and 2.5% fetal bovine serum, as described (Goldstein and others , (1983) J. BioL Chem. 258:8450, incorporated herein by reference), and secreted monoclonal antibodies was collected and purified by standard methods, well known in this field.Approximately 2 mg of each purified monoclonal antibody was dissolved in 400 μl of 300 mm NaHCO3in a 1.5 ml Eppendorf vial. Fifty μl of a freshly prepared solution NaIO4(42 mg/ml in water) was added to the solution, placates was loaded into the column PD-10 (Pharmacia), pre-equilibrated to 10 mm PA2CO3(fraction 1), and it was suirable 500 μl fractions. Collected fractions containing at least about 500 μg IgG, measured through analysis of Bradford.Prepare a solution of glycylcycline in DMSO (5 mg lipid in 1 ml DMSO, was intensively mixed for several minutes with rotation of the vial). Under these conditions, the lipid is not completely dissolved. Carefully selected 50 μl of this solution was added to 350 μl of IgG fractions obtained as described above, in the Eppendorf vial. The flask was wrapped in aluminum foil, and the mixture is gently shaken at room temperature for 20 hours.Then he added one hundred μl of a solution of NaBH4(10 mg/ml in water). An hour later there was added 40 μl of a solution of ethanolamine (15 μl in 1 ml of water). After another hour, the reaction mixture was loaded onto a column PD-10, pre-balanced saline solution with phosphate buffer. The fraction containing lepidosiren IgG against HMG CoA reductase (between 3 and 3.5 ml) was collected and stored in ice.Example 5
Preparation lipidation of immunoglobulins, reactive with the transmembrane protein
Glycylcycline received through the.S.A.) 86:6982, incorporated herein by reference. One equivalent of benzyloxycarbonyl-glycyl-C-NITROPHENOL and 1.1 equivalent of CH2Cl2reacted for 5 hours, then was added N2, 10% Pd/C in CH2Cl2/ethanol, and the reaction continued for another hour.Anti-Ras immunoglobulin
Glycosylated mouse immunoglobulins that are specific associated with oncogenic ras protein, were prepared by separately culturing hybridoma cell line 142-24E5 (ATCC HB 8679; U. S. Pats. 5015571 and 5030565, incorporated herein by reference) modified by way of Dulbecco environment Needle with 4.5 g/l glucose, 2 mm L-glutamine, 1 mm sodium pyruvate, non-core amino acids, 1 x TOGETHER with vitamins, 0.1 mm gipoksantina, to 0.032 mm thymidine, 0.05 mg/ml gentamicin and 10% fetal bovine serum; and hybridoma cell line MX (ATCC HB 9158) in Iscove's modified by way of Dulbecco environment Needle with 1% L - glutamine and HT and 10% fetal bovine serum under special conditions (U. S. Patent 4820631, incorporated herein by reference); monoclonal antibodies, hybridoma secreted by the cells, was collected and purified using standard techniques, well known in this field.
4(42 mg/ml in water), the flask was wrapped in aluminum foil and gently shaken for 90 min at room temperature. The reaction mixture is then loaded into the column PD-10 (Pharmacia), previously equilibrated with 10 mm Na2CO3(fraction 1), and collected fractions of 500 µl. The fraction or fractions containing at least approximately 500 ál of IgG, as measured by analysis of Bradford, was collected from the column.Prepare a solution of glycylcycline in DMSO (5 mg lipid in 1 ml DMSO, was intensively stirred by rotation of the bottle in a few minutes). Under these conditions, the lipid was dissolved incompletely. Carefully selected 50 μl of this solution was added to 350 μl of purified IgG fractions obtained as described above, in the Eppendorf vial. The flask was wrapped in aluminum foil and the mixture gently shaken for 20 hours at room temperature.Then there was added 100 μl of a solution of NaBH4(10 mg/ml in water). An hour later, there was added 40 μl of a solution of ethanolamine (15 ml in 1 ml water). After another hour, the reaction mixture was loaded onto a column PD-10, pre-balanced saline solution with phosphate buffer. The faction with the mouse to turn, as mentioned in the above examples may be obtained from the American Collection Tissue culture, Rockville, MD (ATCC Cell Lines and Hybridomas (1992), 7th edition, incorporated herein by reference).Example 6
Lepidosirenidae transmembrane enzyme
The enzyme gamma-glutamyltranspeptidase (GGT: EC-2.3, 2.2) is a common enzyme that catalyzes the cleavage of glutathione and other-glutamyl compounds through hydrolysis-glutamyl part or its transfer to a suitable acceptor. GGT is a heterodimeric glycoprotein that is synthesized in the form of protein-precursor, which in turn glycosylases and splits into two subunits of the Mature enzyme. GGT is attached to the cell membrane via the N-terminal part of the heavy chain. The active site of the enzyme is located on the extracellular part of the molecule that is abundant glycosylated.GGT was isolated separately from the kidneys of rats and cell culture the human hepatoma, according to the procedure described previously (Barouki and others, (1984) J. Biol. Chem. 259:7970; Curthoys and Hughey (1979) Enzyme 24:383; Matsuda and others, (1983) J. Biochem. 93: 1427; Taniguchi and others, (1985) J. Natl. Cancer Inst. 75:841; Tate and Meister (1985) Methods Enzymol 113:400; and Toya and others, (1983) Ann. N. Y. Acad. Sci 417:86, kotoryy and man was dissolved in 400 μl of 300 mm NaHCO3in a 1.5 ml Eppendorf vial. Added 50 μl of a freshly prepared solution NaIO4(42 mg/ml in water), the flask was wrapped in aluminum foil and gently shaken for 60 min at room temperature. Then the reaction mixture was loaded onto a column PD-10 (Pharmacia), previously equilibrated with 10 mm Na2CO3(fraction 1), and it was suirable 500 µg faction. Collected fractions containing at least about 100 μg GGT measured through analysis of Bradford.Prepare a solution of glycylcycline in DMSO (5 mg lipid in 1 ml of DMSO, were thoroughly mixed for several minutes with rotation of the vial). Under these conditions, the lipid is not completely dissolved. Carefully selected 50 μl of this solution was added to 350 μl of purified GGT fractions, obtained as described above, in Eppendorf vials. The flask was wrapped in aluminum foil, the mixture was gently shaken for 20 hours at room temperature.Then added 100 μl of a solution of NaBH4(10 mg/ml in water). An hour later added 40 μl of a solution of ethanolamine (15 μl in 1 ml water). After another hour, the reaction mixture was loaded onto a column PD-10, pre-balanced saline solution with phosphate buffer. The fraction containing the T of human and rat were investigated on the activity-glutamyltranspeptidase by standard techniques (Tate and Meister (1983), CIT., incorporated herein by reference) and determined the specific activity lepidosirenidae GGT person and lepidosirenidae GGT rats.Lepidosirenidae GGT humans and rats were labelled with moderowaniem125I using chloramine T, and approximately 50 micrograms of radioactive lepidosirenidae GGT was administered administered intraperitoneally to rats. After 24 hours, rats were killed and selected tissue samples for autoradiography to determine the nature of the distribution lepidosirenidae GGT in various organs.Example 7
Lepidosirenidae antiactin antibodies and intracellular staining using immune labels
In order to demonstrate that lepidosirenidae antibodies can penetrate living cells and bind with intracellular targets, antiactin antibody was lipidsoluble and determined its ability to penetrate in the fibroblast culture Swiss ZTZ and contact with actin, a protein of the cytoskeleton. As control was used native (delipidation) antechinomys antibodies.Protein-A-purified antechinomys polyclonal rabbit antibodies were lipidsoluble in accordance with the following procedure: Lipoamino, pisidiidae/recovery borohydride. The antibodies were dissolved in 0.8 ml of 300 mm NaHCO3to a concentration of about 0.2 - 1.0 mg/ml was Added to 50 μl of a freshly prepared aqueous solution NaIO4(42 mg/ml), the vials were wrapped in aluminum foil and gently shaken for 90 minutes at room temperature. The reaction mixture was then purified on a column PD-10 (Pharmacia, Piscataway, NJ), equilibrated and elyuirovaniya 10 mm NaCO3. Fifty μl of 10 mg/ml solution of glycylcycline in benzene was added to the fraction containing antibodies (for example as defined by A280monitoring, analysis of Bradford), then the reaction mixture was incubated at room temperature for 20 hours when lightly shaken. Then added 100 μl of a freshly prepared aqueous solution of NaBH4(10 mg/ml) and left at room temperature for one hour, then added 50 μl of a solution of ethanolamine (15 μl ethanolamine in 1 ml of water). After another hour, at room temperature obtained lepidosirenidae antibody was purified by chromatography on a column PD-10, balanced saline solution with phosphate buffer.Enzyme-linked immunosorbent assay (ELISA)
Lepidosirenidae antechinomys and lepidosirenidae anti-Tat antibodies (supra) inimi) antechinomys and anti-Tat antibodies using research ELISA. Lepidosirenidae as antiactin and anti-Tat antibodies did not cause a measurable loss of affinity of the antibodies to their specific antigens, compared with native (delipidation) antibodies.Intracellular staining using immune labels
To demonstrate that lepidosirenidae antechinomys antibodies are able to bind with intracellular actin in living cells, lepidosirenidae antechinomys antibodies or native antechinomys antibodies are contacted with the cell culture Swiss ZTZ within hours, the cells are then extended washed to remove unbound antiactin antibodies. Then the cells were fixed, raised their permeability and antechinomys antibodies were determined using a secondary antibody with a fluorescent label. While in cells pre-incubated with native (delipidation) antibodies were not determined specific staining in cells pre-incubated with lepidosirenidae antechinomys antibodies obvious was specific staining of actin. The nature of the staining observed in the case lepidosiren antiactin antibodies applied to fixation was similar t the e capturing and increase permeability. These data indicate that lepidosirenidae antechinomys antibodies were able to achieve intracellular actin and contact them and that they, however, could be easily distinguished and contact labeled secondary antibodies, indicating their structural and functional intactness.Although the present invention has been described in detail for its greater clarity and understanding, it is obvious that under the present claim can practice certain changes and modifications. 1. The method of obtaining lepidosirenidae antibodies, characterized in that carry out oxidation of carbon in the antibody to obtain oxidized antibodies, and then perform the join lipid Deputy lipoamino to oxidized antibody in appropriate circumstances, through covalent binding to obtain lepidosirenidae antibodies and selection lepidosirenidae antibodies.2. The method according to p. 1, characterized in that as lipoamino use lipoamino with a straight chain of the following formula:
NH2- R - (CH2)n- CH3< / BR>where R is selected from the group consisting of disubstituted of alkyl (alkylene); 1,4-desaldata hydrogen or amino group; alkylcarboxylic and phosphate diapir; n = 1 to 50.3. The method according to p. 1, characterized in that as lipoamino use lipoamide branched chain of the following formula:
< / BR>where R' is trehzameshchenny alkyl; trehzameshchenny aryl; aminogroup formula(CHR1-CO-N<, where R1is hydrogen or an amino group; aminogroup formula-CHR2-NH-CH<, where R2is hydrogen or amino group or aminogroups formula-CH2-N< or phosphate fluids; m = 1 to 50; n = 1 to 50; m and n are chosen independently.4. The method according to p. 3, characterized in that the branched lipoamino use glycylcycline.5. The method according to p. 1, characterized in that the antibodies used antibody encoded by the genome of the superfamily of immunoglobulins.6. The method according to p. 5, characterized in that the gene of the immunoglobulin superfamily, use the gene encoding the heavy chain.7. The method according to p. 1, characterized in that as antibodies, using a monoclonal antibody.8. The method according to p. 1, characterized in that get lepidosirenidae antibody, comprising at least one residue lipoamino attached to the carbohydrate side CE is m, it includes contacting cells with lepidosiren antibody, comprising at least one residue lipoamino covalently attached to the carbohydrate side-chain antibodies, with the indicated antibody specific associated with intracellular protein.10. The method according to p. 9, characterized in that lepidosirenidae antibody comprises at least one residue lipoamino attached to the carbohydrate side chain of the immunoglobulin.11. The method according to p. 10, characterized in that as lipoamino use glycylcycline.12. The method according to p. 9, characterized in that lepidosirenidae antibody is administered to an animal in vivo.13. The method according to p. 12, characterized in that used lepidosirenidae antibody is absorbed by the body to a greater extent than the corresponding naturally occurring antibody having the same amino acid sequence and the same character glycosylation.14. The pharmaceutical composition inhibiting the cytotoxicity of HIV-1 infected cells, characterized in that it contains a therapeutically effective dose lepidosirenidae anti-Tat antibodies, comprising at least one residue of lipomin, covalently PR the position on p. 14, characterized in that it contains the antibody, lepidosirenidae through the carbohydrate part.16. The composition according to p. 14, characterized in that lepidosirenidae anti-Tat-antibody is an immunoglobulin, lepidosiren through the carbohydrate part.17. The composition for diagnosis of diseases associated with expression of the target antigens, characterized in that it contains lepidosirenidae antibody, which binds to the target antigen, and liberitarian antibody contains at least one residue lipoamino covalently attached to the carbohydrate side-chain antibodies, and lepidosirenidae antibody further comprises a diagnostic agent.18. Pharmaceutically acceptable composition, characterized in that it includes immunoglobulin, lepidosiren through the carbohydrate part and a filler.19. The way to diagnose pathological conditions of the disease associated with the expression of the target antigen, characterized in that it includes, as one of the stages of the appointment the patient lepidosirenidae antibody, which binds to the target antigen, containing the diagnostic agent, and lepidosirenidae antibody includes, in addition, mensitaly diagnostic agent.20. Lepidosirenidae antibody, characterized in that it comprises at least one residue lipoamino covalently attached to the carbohydrate side-chain antibodies.