The ligand specifically binds the trunk of the receptor for polymeric immunoglobulins (pigr) of a cell, the method of introduction of the ligand into the cell and the method for attaching the ligand to the cell expressing the receptor for polymeric immunoglobulins

 

(57) Abstract:

The invention relates to biotechnology and concerns of the ligand and its specific ways of linking with the barrel of the receptor for polymeric immunoglobulins (pIgR) of a cell, provided that the ligand practically does not bind secretory component pIgR in physiological conditions. The ligand may be directed to a cell into the cell or to pass through it and may contain biologically active substances. This invention can be used for transport of therapeutic and diagnostic compositions on the epithelial cells of the mucous membranes inside these cells or through them. 3 C. and 25 C. p. F.-ly.

The invention mainly relates to a ligand and methods of specific binding of the ligand to the section of the trunk of the receptor for polymeric immunoglobulins for internalization into a cell or transport through it.

Art

One of the most pressing issues facing the pharmaceutical and biopharmaceutical industry, is the delivery of therapeutic agents through various semi-permeable membrane in the body. In particular, when it comes to macromolecules, effective from the standpoint of cost and cartonnage substances. In turn, this problem dictated by economic feasibility of production of a medicinal product. Thus, the search for alternative delivery systems often competes with finding themselves new drugs.

Methods of gene transfer can be considered as an example of the delivery of macromolecular drug. These methods can be divided into three categories: physical (e.g., electroporation, direct gene transfer and particle bombardment), chemical (e.g., proteinoid, microemulsions, and liposomes) and biological (e.g., vectors based on viruses and is caused by receptors absorption). Among the biological methods of transfer is caused by the receptors absorption is the most promising approach. The direction of the ligand on absorbed by endocytosis receptor becomes a means of transfer (transportation) of the ligand into the cell. However, one disadvantage of the systems associated with the receptor, is a common dependence on intravenous method of administration, which significantly limits their application.

The mucosal epithelial cells line the number of easily accessible tissues, such as tissues, having the school target for drug delivery. See, for example, article Fercol et al. (J. Clin. Invest. 92: 2394-2400 (1993)); Fercol et al. (J. Clin. Invest. 95: 493-502 (1995)). In the work Breitfeld et al. (J. Cell Biology 109:475-486 (1989) described retrograde (like feedback) transport of antibodies from the cavity to basolateral surface of epithelial cells, although carried out at very low levels. This study found that after advancing through the cell, the antibody binds to the secretory component of the receptor for polymeric immunoglobulins (pIgR). Regarding the transport level with basolateral surface to the apical Breitfeld et al. reported that less than 5% of transport was retrograde in nature. Nominal level oppositely directed transport minimizes the use of secretory component as a means for delivery of biologically active compositions in the cell. Moreover, due to the predominance in the cavity of the split pIgR binding ligand split pIgR, but not with intact pIgR cell surface would reduce the possibility of the use of oppositely directed transport of pIgR as a mechanism of drug delivery.

Accordingly, in the prior art, it is strongly required tools for porting lne equipment needed funds for the delivery of macromolecules to the cell, lining the gastrointestinal tract or the respiratory tract, in this cell, or to pass through it. This invention is for these or other advantages.

The invention

According to one aspect this invention is directed to a ligand that specifically binds to the stem receptor polymeric immunoglobulin pIgR cells under this condition that the ligand practically does not bind secretory component pIgR in physiological conditions. In a typical case, the antibody specifically binds only the trunk. In one embodiment, the ligand is an antibody, preferably a human antibody. In another embodiment, the ligand is a fragment of the variable segment of the recombinant antibodies of the same chain. In yet another embodiment, the ligand binds to the extracellular epitope on the site of the first 33 amino acids, which in the cell membrane are proximal to the cleavage site of the receptor.

The ligand may contain a linking component for linking with the barrel and a biologically active component, such as a nucleic acid, a protein, a radioisotope, lipid and hydrocarbon. Biologically active components contain against the ante implementation of the biologically active component is introduced nucleic acid, encoding the cystic fibrosis transmembrane conductance regulator wild type. In a preferred embodiment, the cell presents epithelial cell, most preferably epithelial cells of a mammal.

In another aspect this invention is directed to a method of introduction of the ligand into the cell expressing the receptor for polymeric immunoglobulins, by attaching a ligand to the trunk of the receptor for polymeric immunoglobulins cells, provided that the ligand practically does not bind secretory component pIgR in physiological conditions. In one embodiment, the ligand is attached to the trunk on the apical surface of the cell, while in other embodiments, implementation of the ligand undergoes transcytosis are activated on basolateral the cell surface and is released from the barrel basolateral surface.

In another aspect, the invention relates to a method of attaching a ligand to the cell expressing the receptor for polymeric immunoglobulins, which provides for the stage of binding of the ligand with the barrel of the receptor, provided that the ligand practically does not bind secretory component pIgR in physiological conditions. In one embodiment of the invention may be understood with reference to various aspects of the present invention.

Among the various applications in vitro and in vivo, the invention can be used for transport of therapeutic and diagnostic compositions on the epithelial cells of the mucous membranes inside these cells or through them. Thus, the invention is highly effective and convenient means for the transfer of nucleic acids or proteins in epithelial cells.

Information confirming the possibility of carrying out the invention

This invention is directed to a ligand that specifically binds the receptor for polymeric immunoglobulins (pIgR) of a cell, provided that the ligand practically does not bind secretory component pIgR in physiological conditions. The invention is, inter alia, methods of joining, and the introduction of the ligand into the cell expressing the PlgR.

After transport to the apical surface of epithelial cells a large part of the pIgR is cleaved to release secretory component. We have surprisingly and unexpectedly found that cleavage of intact pIgR in the cavity retains the intact residual extracellularly (extracellular) plot pIgR (i.e., "trunk") and moreover that the barrel retains the ability to bind, dismantles ligand, associated with the trunk pIgR, partly represents the invention as described and claimed here.

This invention has application as a means for transport of therapeutic or diagnostic compositions to cells expressing pIgR, inside these cells (endocytosis) and through them (transcytosis are activated). Thus the invention can be used for transport of biologically active compositions, such as proteins, nucleic acids or defined labels, specifically to cells expressing pIgR. The invention also represents a tool for tagging and recognition of epithelial cells from a mixed cell population for studies of pathologies. Further, because the expression of pIgR in carcinomas reduced relative to that of the cells of normal epithelium, tagging pIgR has application as a diagnostic aid in ways endoscopy or radiology. In addition, the binding of therapeutic ligands with pIgR can be used to increase the period of their stay in the cavity of different ways and increase their effectiveness.

Definition

Up until here is not defined otherwise, all technical and scientific terms used in the context of Zas invention. Dictionaries Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons (New York)and Hale and Marham (1991) The Harper Collins Dictionary of Biology, Harper Perennial, NY offer specialist vocabulary is the basis of many of the terms used in this invention. Amino acids can be determined either their commonly known three letter symbols or one-letter symbols recommended by Biochemical nomenclature Commission of the IUPAC-IUB. The nucleotides in this way can be defined generally accepted single-letter designations. For the purposes of this invention below defines the following terms.

The terms "ligand" or "linking structure (portion) of ligand" refers to all molecules capable of specific binding of the receptor polymer immunoglobulin (pIgR). Ligands include, but are not limited to antibodies, proteins, peptides, nucleic acids, lipids and carbohydrates.

The term "biologically active component" means a compound that in vivo directly causes or inhibits the increase or decrease of cellular transcription, translation, receptor binding, active or passive transport, cellular signal, signal transduction, cell division, cell different is ü, apoptosis, protein degradation, promoting proteins (e.g., secretion), the stability of proteins or phosphorylation. Biologically active components also contain diagnostic compositions, which allow assessment of the above events.

The terms "bind(et) specifically" or "specifically bind(et) or associated (attached) or linking (connecting)" means the preferred Association of the ligand (fully or partially) with a cell or tissue bearing a particular molecule is a target or a marker, but not to cells or tissues lacking this molecule is the target. It is understood of course that there may be a certain degree of non-specific interaction between the molecule and the cell or tissue-namereny. Nevertheless, specific binding can be detected and selected, as it is determined by the specific recognition of a target molecule. Typically, specific binding results in a much stronger Association between the delivered molecule and cells, the carrier molecule is a target than between related molecules and cells that are missing molecule-target. Specific binding typically results in increase Balsillie more than 100 times the number of bound ligand (per unit time) to a cell or tissue, the carrier molecule is a target compared to a cell or tissue, which are not molecule-target or marker.

The term "barrel" means extracellularly (extracellular) component of the receptor for polymeric immunoglobulins (pIgR), the corresponding section of the pIgR, which binds to the cell after cleavage segment pIgR, forming secretory component. The barrel is present regardless of whether the segment pIgR, which corresponds to the secretory component, derived or not derived from pIgR.

The term "pIgR" or "the receptor for polymeric immunoglobulin" refers to the receptor, which is expressed in the epithelial cells of mucous membranes, including epithelial cells of the respiratory tract, glandular cells of the submucosal membranes, cells of the intestine, the epithelium of the nose, the epithelium of the mammary gland, oral mucosa, urinary and genital tracts, and co tissues and is involved in the transcytosis are activated dimeric immunoglobulin A (dIgA) and/or IgM pentamers from basolateral to the apical surface.

The term "practically does not bind" means that not more than 15% of the ligand, a specific binding molecule-target, binds a specific molecule namesign. More pre the equipment less than 1%.

The term "secretory component" means extracellularly part of the pIgR, which is normally cleaved after completion of the process of transcytosis are activated from basolateral to the apical surface. In the typical case of secretory component contains dimeric binding fragment pIgR, namely IgA (dIgA). Secretory component is usually allocated in the cavity together with or without associated dIgA.

The term "physiological conditions" means the extracellular environment, with conditions (such as temperature, pH or osmolarity), which provide nutrients or growth of interest in cells.

The term "antibody" means an immunoglobulin molecule obtained during the generation of the humoral response in vitro or in vivo, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (for example, bespecifically antibodies and recombinant Fv fragments from a single chain (scFv). The term "antibody" also includes antigen binding forms of antibodies (e.g., Fab, F(AB)2).

The term "humanitariannet antibody" means an antibody that contains the monogenist in humans.

The term "the cystic fibrosis transmembrane conductance regulator wild-type" means functional (active) form of cystic fibrosis transmembrane conductance regulator (CFTR). Cm. article'riordan et al., Science 245:1066-1073 (1989).

The term "apical surface" means that surface of cells, which occurs transcytosis are activated intact pIgR after endocytosis with basolateral surface. Mainly the apical surface of the cells adjacent to the cavity, in which the intact pIgR is cleaved to release secretory component.

The term "basolateral surface" means that surface of cells, which after synthesis in the endoplasmic reticulum and transit through the Golgi apparatus is delivered intact pIgR.

The term "surface of the barrel" means extracellularly section of the trunk.

The term "released (selected)" means interference in the specific Association of the ligand (in whole or in part) with his molecule-target.

The term "subject to transcytosis are activated or transcytosis are activated" means the transfer of one of the plasma membrane of cells to another by intracellular. Typically, the transcytosis are activated happens to basolateral on apical Noah membrane" means at or near the cell membrane.

The term "extracellularly (extracellular)" means an area extending outward from the double lipid layer surrounding the cell.

Identification of pIgR

Nucleic acid sequence and amino acids of the receptor for polymeric immunoglobulins have been identified in many taxonomically different species. Cm. work Piskurich et al. Journal of Immunology 154:1735-1747 (1995). Identification of pIgR from other species can be performed by any well-known in the art. For example, there may be constructed a probe nucleic acid to pIgR using published sequences pIgR. The probe typically should be created based on a conservative site pIgR. Hybridization of the probe with the genomic library or cDNA can be used to identify pIgR in unstudied species. The specialist should be understood that the sequence of the nucleic acid probe pIgR should be in most cases a sequence that is closest to the mean of the analyzed sounding. Extensive material on hybridization of nucleic acids is presented in the monograph Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with nucleic acid probes, part 1, Chapter 2 "Overview when the native approach pIgR or its peptide fragments (e.g., secretory component) can be used for screening expressing libraries. See, for example, article Fercol et al., J. Clin. Invest. 95:493-502 (1995). Data and other methods well known in the art, can be detected, for example, the guides Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2 ed.), volumes 1-3 and Current Protocols in Molecular Biology, Ed. by F. M. Ausubel et al., Current Protocols, Union, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994 Supplement) (Ausubel). Evidence that nucleic acid (or protein) is identical to that encodes pIgR can be accomplished by using approaches such as design of antibodies to the alleged pIgR protein and confirmation of the ability of antibodies to bind protein, which are inherent properties of pIgR (for example, the presence on the surface of epithelial cells, the binding of dimeric IgA or IgM pentamers, etc.,).

Identification of trunk

The barrel can be identified by many well-known specialists of ways. Was identified putative consensus heptapeptide sequence that are identical with the site of cleavage of the pIgR and determines the thus identified aminoil identified as located directly before this alleged plot cleavage. Cm. article Piskurich et al. Journal of Immunology 154: 1735-1747 (1995). The splitting of the consensus site releases secretory component and determines its carboxy-end. The carboxyl end of the secretory component can be changed by the events of secondary cleavage (for example, using ectopeptidases or endopeptidase activity), which can lead to secondary carboxy-ends. However, the amide bond, which initially determines the amino-end of the barrel and carboxy-late secretory component may be detected by comparative sequence analysis and identification of fissionable consensus sequence.

Methods of analysis of sequences for comparison are known in the prior art. Optimal comparative sequence analysis can be performed using the algorithm of the local homology proposed by Smith and Waterman (1981) Adv. Appl. Math., 2:482; algorithm comparative analysis of the homology of the sequences of Needleman and Wunsch (1970) J. Mol. Biol. 48:443; way of finding analogies proposed by Pearson and Lipman (1988) the OEWG. Natl. Sci. USA 85:2444; by computerized application of these algorithms (including, but not limited to CLUSTAL in the PC/Gene Intelli75 Science Dr. Madison, Wisconsin, USA); the program CLUSTAL described in detail in the publications Higgins and Sharp (1988) Gene, 73:237-244, and Higgins and Sharp (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Research 16, 10881-90; Huang et al. (1992) Computer Applications in the Biosciences 8, 155-65 and Pearson et al. (1994) Methods in Molecular Biology 24, 307-31. Comparative analysis is also often carried out by research and comparison "manually".

In another approach, the secretory component can be separated from liquids in the apical cavity (for example, from milk or bile) and defined well-known in the art methods of sequencing, amino acid sequence, such as degradation by Edman or mass spectrometry, as described in article Eiffert et al., Hoppe-Seyler's Z. Physiol Chem. 365:1489-1495 (1984). Among the various secondary carboxyl ends, certain events secondary cleavage can be identified carboxy-terminal amino acid, which is adjacent to the site of cleavage.

Peptides that correspond to the trunk pIgR selected species include:

Mouse: Glu-Arg-Glu-Ile-Gln-Asn-Val-Arg-Asp-Gln-Ala-Gln-Glu-Asn-Arg-Ala-Ser - Gly-Asp-Ala-Gly-Ser-Ala-Asp-Gly-Gln-Ser-Arg-Ser-Ser-Ser-Ser-Lys(SEQ ID No 2)

Rat: Glu-Arg-Glu-Ile-Gln-Asn-Ala-Gly-Asp-Gln-Ala-Gln-Glu-Asn-Arg-Ala-Ser - Gly-Asn-Ala-Gly-Ser-Ala-Gly-Gly-Gln-Ser-Gly-Ser-Ser-Lys (SEQ ID No 3)

Human: Glu-Lys-Ala-Val-Ala-Asp-Thr-Arg-Asp-Gln-Ala-Asp-Gly-Ser-Arg-Ala-Sr-Lys (SEQ ID No 5)

Rabbit: Leu-Ala-Glu-Val-Ala-Val-Gln-Ser-Ala-Glu-Asp-Pro-Ala-Ser-Gly-Asp-Pro-Ala-Ser-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg-Ser-Lys (SEQ ID No 6).

Cells expressing pIgR

Although this invention broadly relates to eukaryotic cells, pIgR-expressing cells, corresponding to the present invention are preferably mammalian cells and more preferably epithelial mammalian cells that normally secrete IgA. Mammalian cells can be transliterowany nucleic acid that encodes a pIgR, isolated, synthesized or otherwise collected from one or more desired species. Methods of transfection and gene expression in mammalian cells are known in the prior art. Transduction of these cells with viral vectors may include, for example, incubation of the virus with the cells of the host range of the viruses in the conditions and the concentrations needed to cause infection. See, for example, manual Methods in Enzymology, volume 185, Academic Press, Inc. San Diego, CA (Ed. by D. V. Goeddel) (1990) or M. Krieger, Gene Transfer and Expression - A Laboratory Manual, Stockton Press, New York, NY (1990) and cited here for reference.

Cell culture, which can be used in this invention, including the rotary Freshney (Culture of Animal Cells, a Manual of Basic Technique, 3rd edition, Wiley-Liss, New York (1994)) and the following links give you basic instructions for culturing cells. Nucleic acid sequences encoding pIgR from the desired view can be expressed in a number of eukaryotic host cells, including yeast and various cells of higher eukaryotes, such as cell lines, COS, CHO and HeLa cell lines myeloma, and MDCK cells isolated from carcinoma of the colon, such as SASO. The gene of the recombinant protein is functionally connected with suitable for each host sequences control expression. For eukaryotic cells, the sequence control will include a promoter and preferably enhancer allocated, for example, from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may also include donor and acceptor splice sequences.

Ligand binding to trunk

Specific stem-binding ligand is not critical to this invention and can be used in a variety of ligands. Most of the methods of design and selection of ligands such as nucleic acids, proteins or peptides (denoted overall termi; WO 90/15070; WO 92/10092; WO 96/11878) with the desired characteristics of specific binding are well known in the prior art. Preferably the ligands, corresponding to the present invention, will be under physiological conditions to link the trunk, almost no binding of secretory component pIgR. More preferably, when the ligands, corresponding to the present invention specifically bind only the trunk under physiological conditions. Usually physiological conditions for various different fabrics. However, examples of physiological conditions are the conditions encountered in the gastrointestinal tract or the respiratory tract of mammals, including, but not limited to man. To link to extracellularly the ligand-binding site ("epitope"), contained in the first 6, 9, 12, 15, 18, 21, 24, 27, 30 or 33 membrane proximal amino acids of the barrel, can be selected any ligand.

For use as ligands in this invention can be engineered antibodies, including polyclonal, monoclonal or recombinant antibody Fv of the same chain. Ways to obtain polyclonal and monoclonal antibodies are known in the art. See, for example, the publication Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY Acre given in the context of the invention; Goding (1986) Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York, NY and Kohler and Milstein (1975) Nature 256:495-497; see Huse et al. (1989) Science 246:1275-1281, and Ward et al. (1989) Nature 341: 544-546; Birch and Lennox, Monoclonal Antibodies: Principles and Applications, Wiley-Liss, New York, New York (1995).

Other suitable methods for obtaining ligands in the form of antibodies or peptides include the selection of libraries of recombinant antibodies/peptides in phage or similar vectors. High-affinity against the trunk of the antibodies or peptides can be quickly selected using phage reproduction for the expression of recombinant Fv fragments from a single chain (scFv), or peptide ligands on the surface of phage. Briefly, genes encoding surface protein of the phage, change so that it was possible to perform the insertion of the gene of the antibody or peptide, which is expressed in the form of a fused protein on the surface of phage carrying this gene. Possible selective enrichment of phage expressing the desired antibody or peptide ligand, and its secretion using the affinity/avidity against the trunk. DNA encoding the ligand, Packed in the same phage that allows you to isolate the gene encoding the ligand. A number of such methods are described in detail in the literature and well known chnology 14: 309-314 (1996), U.S. patent No 4642334, 4816397, 4816567, 4704692, WO 86/01533; WO 88/09344, WO 89/00999, WO 90/02809; WO 90/04036, EP 0324162, EP 0239400.

In chemical peptide synthesis method, called "Separation, Linkage and Recombination" ("Divide, Couple and Recombine" (DCR) was used to obtain a combinatorial peptide libraries. Cm. article Furka et al. Int. J. Pept. Protein Res. 37: 487-493 (1991) and Houghten et al. Nature 354: 84-86 (1991). Alternatively, DCR peptide mixtures were also prepared by direct binding of Monomeric mixtures. Cm. U.S. patent Rutter et al. No 5010175. It was proposed that the application of these methods for the preparation of mixtures of other linear polymers, such as peptide". Cm. the publication of Simon et al. Proc. Natl. Acad. Sci. USA 89:9367-9371 (1991). During the synthesis of oligonucleotides "degenerated" or "unstable" mixture of oligonucleotide products can be obtained, for example, by applying an equimolar mixtures of monomers at certain stages of the synthesis of the oligonucleotide to the polymer. Cm. the work of Atkinson and Smith in "Oligonucleotide Synthesis. A Practical Approach," 1984, IRL Press, Oxford, Ed. by M. Gait, C. 35-81. These methods of synthesis of peptides or oligonucleotides represent a significant number of connections for testing, which can easily be identified if they are active.

Preferred is the I number of autologous (own) sequences in the ligand. Accordingly, chimeric antibodies having nextagent variable plots are preferred. Especially preferably the use of antibodies that have xenogenic parts are missing or somethingoranother sections defining complementarity, as in humanized antibodies.

Linking and testing of ligands

Binding (i.e., the joining of the ligand to the trunk pIgR may occur before or simultaneously with the cleavage of secretory component, and the ligand can be associated with basolateral or apical surface. Thus, endocytosis of the ligand can occur basolateral or apical, or the ligand may be an object from the apical transcytosis are activated to basolateral or from basolateral to the apical surface. The fate of the ligand or of any of its elements will be different depending on its physical-chemical characteristics. Accordingly, the properties of the ligand can be selected or designed to implement the desired function on the cell surface, in endosome or after transcytosis are activated. For example, varying the sensitivity of the ligand to the media, causing proteolysis or restore, you can define a distribution linked, interest is managing specific binding to the cell after joining or transcytosis are activated or alternative released into the environment. Thus, the properties of any of the various elements of the ligand, including linking component, the biologically active component or the linker can be designed or chosen to provide different degrees of affinity, stability, or activity against various intracellular departments or surface cells as it is desirable.

A. Testing the binding of the ligand ex vivo

To assess binding of the ligand with the barrel in vitro convenient to measure endocytosis or transcytosis are activated the bound ligand in the epithelial cells of mammals. The term "endocytosis" mainly refers to the phenomenon of absorption of the material by the cell, e.g., by phagocytosis or pinocytosis. Mediated receptor endocytosis is an effective agent for inducing the cell to absorb the material that binds to a receptor on the cell surface. Cm. the work of Wu and Wu (1987) J. Biol.Chem. 262:4429-4432; Wagner et al. (1990) Proc. Natl. Sci. USA 87:3410-3414 and EP-A1 0388758. To assess the binding can be used any number of known methods for the analysis of endocytosis. For example, analyses of binding, transcytosis are activated and internalized described in detail in the article Breitfeld and augment Fab, with the barrel on the apical surface of the cells of the kidney of the dog Madin-Darby (MDCK) at 4oC, heated to 37oWith for a short time (0-10 min) and cooling of the cells again tooC. Methods of pIgR expression in MDCK cells known in the prior art (article Breitfeld et al. Methods in Cell Biology 32: 329-337 (1989)). Remaining on the surface of the Fab remove and destroy them at pH 2.3. Intracellular Fab is Fab, which keep the Association with the cells after the destruction, while associated with the surface are Fab Fab, which are removed by acid washing. Controls for nonspecific binding include the use of preimmune Fab and/or MDCK cells not transfected with the pIgR.

The transcytosis are activated can be easily estimated, allowing the MDCK cells to bind Fab on the apical surface at 4oWith, and they heat up to 37oWith 0-240 min and then determining the amount of Fab, delivered in basolateral environment. Data basolateral delivered Fab compared to the amount of Fab, which keep the Association with the cells (intracellular or destroyed by the acid), and Fab, coming back into the apical medium. Alternatively, the transcytosis are activated can be estimated using continuous processing of Fab cells in the apical medium and measuring the accumulation of Fab in the base of one group of the ligand. In both ways degradation Fab can be assessed by analyzing subjected to transcytosis are activated Fab using SDS-PAGE (polyacrylamide gel electrophoresis using sodium dodecyl sulfate) and the use of sensing protivoyuznyh antibody in Western-blot. Nonspecific transport (for example, due to liquid-phase endocytosis and transcytosis are activated or paracellular leakage between the cells) can be controlled through the use of MDCK cells not transfected with the pIgR and/or preimmune Fab.

C. Testing the binding of the ligand in vivo

To assess transcytosis are activated in vivo convenient to use does not contain pathogens experimental animals such as rats Sprague-Dawley. Labeled ligand (e.g. antibody labeled with radioactive iodine) may be inserted into the nostrils. As will be clear to the experts, "label" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence or chemiluminescence method. The transcytosis are activated from the apical to the basal surface can easily be determined by measuring the delivery of the ligand in the system kovoor the Jena analysis of ligand-polyacrylamide gel electrophoresis using sodium dodecyl sulfate.

Biologically active component

Biologically active component of the ligand can be covalently or ecovalence associated with ligand. For example, to link different isotopes can be used chelators, or nucleic acid may be associated with ligand hydrogen bonds. Biologically active components may also be present in the emulsions, proteinoid or liposomes. Specialists will be clear that such patterns can be covalently linked to the ligand via derivatizing polar head groups or entire proteins of the membrane. Connecting the components of the ligand can also contain biologically active component.

Biologically active components contain any number of compounds known in the art as anti-inflammatory agents, cytokines, anti-infective agents, activators or inhibitors of enzymes, allosteric modifiers or antibiotics. Thus, the biologically active component includes compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioactive isotopes, lipids, hydrocarbons or recombinant viruses. The term "nucleic acid" means deoxyribonu is mainly encompasses known analogues of natural nucleotides. Nucleic acids include antisense nucleic acids, derivateservlet oligonucleotides for covalent cross-linking with single-stranded or double-DNA, triplex forming oligonucleotides or nucleic acids encoding proteins or peptides. Nucleic acids also include compositions for gene therapy, such as a nucleic acid encoding the cystic fibrosis transmembrane conductance regulator wild-type.

Attaching biologically active compositions to the ligand

The method for attaching biologically active component to the ligand will vary in accordance with the chemical structure of the component. Basically ligands will contain a number of functional groups available for reaction with a suitable functional group on the biologically active molecule to bind through her agent. Alternative ligand and/or biologically active component can be derivationally so that they have or attach additional reactive functional groups. The derivatization may involve attaching any of a number of linker molecules such what postulantes or non-covalent binding of a ligand and a biologically active component. Suitable linkers are well known in the art and include, but are not limited to linkers branched or unbranched carbon chain, heterocyclic carbon linkers or peptide linkers. Cm. for example, the monograph Birch and Lennox, Monoclonal Antibodies: Principles and Applications, Chapter 4, Wiley-Liss, New York, New York (1995); U.S. Patent No 5218112, 5090914; Hermanson Bioconjugate Techniques, Academic Press, San Diego, CA (1996).

When both molecules are polypeptides, the linkers may be associated with individual amino acids by the side groups (e.g., disulfide bonds with cysteine). To obtain the desired conjugate can be used bifunctional linker having one group reactive with a group of specific biologically active component, and another group reactive with the ligand. Alternative derivatization may involve chemical treatment of the component, for example, glycol cleavage of the sugar structure of a glycoprotein antibody with periodate with formation of free aldehyde groups. The free aldehyde groups of the antibodies can react with free amine or hydrazine powered groups agent, linking thus the agent (See. U.S. patent No 4671958). How Popo 4659839). There are many ways and linker molecules for attachment of various compounds, including chelates of radionuclide metals, toxins and drugs to proteins such as antibodies. See, for example, European Patent application No 188256, U.S. Patent No 4671958, 4659839,4414148, 4699784, 4680338, 4569789 and 4589071 and article Borlinghaus et al., Cancer Res. 47:4071-4075 (1987)).

In some cases, it is desirable release of conjugated molecules, when it comes to plot the target. As a consequence, can be used in the conjugates, containing links, which are near the site of the target. The splitting due to the release of the biologically active component of the antibodies may be due to the activity of enzymes or conditions, subjected to conjugate or inside the target cells, or near the site of the target. When the plot is when the target is a tumor, can be used as linker that is cleaved under the conditions existing in the tumor area (for example, under the influence associated with tumor enzymes or acidic pH). The spacer of the CIS-amanitowoc acid is used to release the biologically active components in the endosomes. In this way the splitting of disulphide bonds is Kerov. Cm. U.S. patent No 4618492, 4545225 and 4625014. Mechanisms of release of agent from these linker groups include, for example, irradiation photolabile communication and acid catalyzed hydrolysis. In U.S. Patent No 5141648 described immunoconjugate containing linkers specific chemical structure in which the bond is cleaved in vivo, thus releasing the associated connection (radiotherapy agent, drug, toxin, etc). Believe that the linker susceptible to cleavage at slightly acidic pH, is cleaved during transport to the cytoplasm of target cells, thus releasing the biologically active compound within the target cells. U.S. patent No 4671958 includes a description immunoconjugates containing linkers, which are in the area of the target in vivo under the action of proteolytic enzymes of the complement system of the patient. In a large number of methods that have been described for connection of a number of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other components to the ligands, the expert is able to determine a suitable method for attaching the component to the ligand corresponding to this invention.

Output liganda or any part of endosome.

For efficient transfection can be used complex poly-L-lysine/nucleic acid associated with kiganda that specifically attaches to the trunk (see article Fercol et al., J. Clin. Invest., 92:2394-2400 (1993) and J. Clin. Invest., 95:493-502 (1995).

In another approach, poly-L-lysine can be associated by genetic fusion or chemical linkers with a ligand that specifically interacts with the barrel of pIgR. In turn, this complex may be associated with defective adenovirus. Curiel et al. demonstrated that "Nude" (deproteinisation) DNA electrostatically associated with poly-L-lysine or poly-L-lysine-transferrin, which were associated with defective mutants of adenovirus, can be delivered into cells by transfection efficiency approaching 90%. Conjugate adenovirus-poly-L-lysine-DNA binds to normal adenovirus receptor and then internalized caused by receptor endocytosis. This approach was used to obtain not less than 1000-fold increased expression of gene therapy vectors. Such are the properties of the herpes viruses. Cm. work Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850-8854; Gotten et al. (1992) Proc. Natl. Acad. Sci. USA 89:6094-6098; Curi is so (1992) Am. J. Respir. Cell Mol. Biol. 6:247-252, and Harris et al. (1993)Am. J. Respir. Cell Mol. Biol. 9:441-447; Gao et al. (1993) Hum. Gene Ther. 4:17-24; Curiel et al. Patent application USA SN 07/768039.

In another approach using influenza a hydrophobic peptide in the hemagglutinin can act as a fusion peptide at low pH, causing the fusion of the virus with the membrane of endosome and bringing the virus into the cytoplasm. This peptide was used in the complexes of transferrin/peptide/poly-L-lysine/DNA for gene transfer using transferring receptor, and it substantially increased the efficiency of expression. Cm. article Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89:7934-7938 (1992). This peptide may be engineering methods introduced in the ligand with the purpose of transport of the ligand or its fragment of endosome.

The following approach proposes the use of ricin a Chain of ricin And the ability to exit endosome and in the cytosol. Cm. article Beaumell et al. J. Biol. Chem. 268: 23661-23669 (1993). The ligand corresponding to this invention, can be associated with ricin And by genetic fusion or chemical linkers.

Although this invention has been described in detail by means of illustrations and examples for purposes of clarity of understanding, it is obvious that the definition is Example 1

Example 1 describe the method of obtaining and analysis of chicken antibodies and Fab fragments that specifically attached to the section of the trunk rabbit pIgR.

Connecting with a membrane segment rabbit pIgR begins with a valine residue at position 630. The sequence of twenty-four extracellularly residues rabbit pIgR, which is before communicating with the membrane segment is the following: 607-AspProAlaSerGlySerArgAlaSerValAspalaserseralaserglyglnserglyseralalys-629 (SEQ ID No 7).

Synthesized two peptides (Measurement Dynamics, Inc. , La Jolla, CA), representing extracellularly proximal membrane fragments from 16 and 23 amino acids pIgR. C-terminal cysteine added for conjugation. Peptide sequence (in single letter code) are: DPA SGS RAS VDA SSA SGQ SGS AKC (SEQ ID No 8) for the primary peptide and peptide represented by suppositionally: ASV DAS SAS GQS GSA CC (SEQ ID No 9). Half of each sample peptide kongugiruut with hemocyanin from the marine mollusk-saucer (keyhole limpet family Fissurellidae) KLH (manufactured by Measurement Dynamics, Inc.).

KLH-conjugated peptides send in Lampire Biological Laboratories (Pipersville, PA) for chicken antibodies from two chickens for each peptide. After immunization of chickens according to standard methods by Spezia complete Freund adjuvant in the design of stimulation, intramuscular injection of 0.5 mg of peptide with incomplete suspension Freund in week 1; intramuscular injection of 0.25 mg of peptide with incomplete suspension Freund at week 2; break week 3; intramuscular injection of 0.25 mg of peptide with incomplete suspension Freund at week 4; break week 5 and the selection of blood for analysis at week 6. Daily egg collection begins approximately at week 6 and selected monthly blood tests. Eggs take on a monthly basis.

When receiving the yolks of eggs thoroughly separated from proteins and stored at 4oWith 50-80 ml primary buffer (0.01 M phosphate, pH 7.5, 0.1 M NaCl, 0,01% azide) on egg yolk until processing for extraction chicken antibody (IgY"). IgY extracted from batches stored egg

the yolks through a series of deposition processes using PEG (polyethylene glycol), followed by a series of sediment ammonium sulfate in accordance with the method Polson et al., described in the journal of Immunol. Commun. 9:475 (1980)). In a few words, hard PEG (polyethylene glycol mol. mass 8000) add to the yolks in the main buffer to 3.5 wt.% PEG on the volume of the diluted egg yolk and stirred at room temperature until dissolution. The solution is centrifuged at 14000 g for 10 min at 20oAnd decanted through a funnel, the soda is the resultant filtrate to obtain a final concentration of PEG 12% with the order of deposition of the IgY. After sedimentation of the precipitate by centrifugation at 14000 g for 10 min at 20oTo the residue is dissolved in 60 ml of basic buffer/yolk and add an equal volume of 24% PEG in the main buffer to improve sediment. The precipitate twice centrifuged at 14000 g for 10 min at 20oFor complete removal of residual PEG solution. Precipitation was dissolved in 30 ml of basic buffer/egg yolk and precipitated protein in 50% saturated (NH4)2SO4by slowly adding an equal volume (NH4)2SO4and stirring overnight at 4oC. the Precipitate centrifuged at 14000 g for 10 min at 4oC and the precipitate washed with an equal volume of cold 50% aqueous solution of (NH4)2SO4. The precipitate centrifuged again at 14000 g for 10 min at 4oC, dissolved in PBS (phosphate buffered saline without calcium or magnesium, pH 7.5 and extensive cialiswhat in PBS to completely remove (NH4)2SO4. The purity of the drug IgY confirmed using SDS-PAGE (approximately 90-95%), and the number of IgY is determined by absorption measurement at 280 nm using an extinction coefficient of 1.3.

Affinity purification of IgY chicken, which injected primary PE the ical Company), which provides specific binding with sulfhydryl groups, such as C-terminal to the cysteine of the peptide. In accordance with the manufacturer's instructions prepare the column with 3 ml of 3 mg of peptide. Briefly, the column with 3 ml balance 6 column volumes (hereinafter: the volume) of 50 mm Tris, 5 mm EDTA (ethylenediaminetetraacetic acid), pH 8.5, and then applied to a column of 3 mg primary peptide SEQ ID No 8 in 3 ml of 50 mm Tris, 5 mm EDTA, pH 8.5 for mixing at room temperature for 15 minutes Gel column and peptide incubated for 30 min without mixing. Buffer peptide is removed from the gel and retain for further testing to confirm the effectiveness of binding using the Ellman reagent (DTNB (5-5'-dithiobis(2-nitrobenzoic acid) Pierce Chemical Company), which defines the sulfhydryl group. Primary peptide SEQ ID No 8 contains no aromatic group and cannot be determined spectrophotometrically or by standard methods of protein analysis, such as the method of analysis according to Bradford. Using the Ellman reagent according to the manufacturer's instructions to compare an aliquot of peptide solution before and after binding gel confirms 100% binding efficiency. The number of the deposits using a 3 ml solution of cysteine in 50 mm Tris, 5 mm EDTA, pH 8.5 by stirring for 15 min at room temperature followed by curing for 30 minutes, without stirring. Column drain and washed with 16 volumes of 1 M NaCl, then a 16 volumes of degassed 0.05% of sodium azide.

IgY get by affinity purification on this related peptide SULFOLINK gel in accordance with a modified version of the method Rosol et al. (Veterinary Immunology and Immunopathology, 35:321-337, 1993). Column at room temperature, washed with 10 volumes of PBS. IgY recycle on the column for 2 hours. Then the column was washed with 10 volumes of PBS and 10 volumes of phosphate buffered saline (PBS) with 0.5 M NaCI. Peptide-specific IgY elute 500 mm glycine at pH 2.5 and neutralized 1 M Tris, pH of 9.5. UV-spectrophotometer and a graphical device used in the washing and elution of the protein from the column. Samples with signal OD (optical density) 280 nm, concentrate in the device centriprep 30 (Amicon) to a volume of 500-600 ml.

The Fab fragments ("fragments Yab") is prepared from the obtained affinity purification of IgY, incubated with immobilized pepsin (Pierce Chemical Company) according to the manufacturer's instructions and the modified method Akita and Nakai (Journal of Immunological Methods. 162:155-164, 1993). Pepsinum Mara on the basis of sodium acetate. Obtained by affinity purification of IgY incubated with immobilized pepsin at 37=VOS and stirred for 5 hours. Odnokolernyh Tris-HC pH 8.0 added to obtain a final pH of 7.5. Pepsinum the mixture is centrifuged at 1000 g for 5 min and the supernatant, containing fragments, is applied to the filter METHOD 10 (Amicon) to remove small fragments of the Fc. Complete cleavage confirmed using SDS-PAGE.

Chicken serum from positive blood and IgY extracted from parties United egg yolks, analyzed by ELISA (enzyme-linked immunosorbent assay) to confirm recognition of the peptide. Obtained by affinity purification of IgY and the Fab fragments'("Yab") are tested for the ability to recognize intact pIgR using Western blotting. Cell lysates prepared from cells of the kidney of the dog Madin-Darby (MDCK) cells and MDCK transferout rabbit pIgR ("pWe") in accordance with the method described Breitfeld et al. (Methods in Cell Biology 32:329-337 (1989)), using 10% lytic NP40 buffer containing 1 μg/ml of protease inhibitors and fluoride phenylmethylsulfonyl (PMSF). Lysates of cells maintained in 10% gel under reducing conditions and transferred to PVDF membrane (polyvinyldifluoride) (Millipore, Bedford, MA). Mouse monoclonal Ana for positive control, and IgY isolated from preimmune yolks, used as a negative control. As secondary antibodies using HRP-conjugated rabbit protivoyuznye IgY (Jackson Immunochemicals) and HRP-conjugated rabbit protivorechivy (Biorad). IgY from hens who injected the native peptide, and IgY from hens who injected the peptide represented by suppositionally, recognize intact pIgR, but IgY from one of the chickens, which is injected antibody in the form of suppositionally not recognize it. Research immunofluorescence assay fragments IgY and Yab (from chickens, which is injected primary peptide) using MDCK cells and pWe grown on cover slides, fixed in 4% paraformaldehyde and treated with saponin for permeability show that pIgR-transfetsirovannyh cells more specifically stained (FITC-conjugated antibodies rabbit against chicken and mice, obtained from Jackson Immunochemicals). LIS for cells (modification of method M. "Hahne" et al., described in the Journal of Cell Biology, 121:655-64, 1993) fixed, processed for the permeability of the cells indicates that the fragment Yab painted 5 times stronger cells pWE than MDCK cells. These results demonstrate the successful obtaining polyclonal antitussive selection of a fragment of a single chain variable segment of human recombinant antibodies (scFv) using a play on phages.

For selection of scFv by playing on the phage requires soluble biotinylated antigen or antigen immobilized on a solid carrier. Because of scFv selected using the play on phages, are often low-affinity binding agents and because the use of soluble antigen can provide a selection of high-affinity scFv (article R. Schier et al. J. Mol. Biol. 255:28-43, 1996), choose selection approach with soluble antigen. Primary peptide trunk pIgR described in Example 1, which corresponds to 23 amino acids proximal to the membrane extracellular plot rabbit pIgR, kongugiruut with Biotin via the sulfhydryl group of the cysteine residue, using Biotin-VMS ((1-biotinamide-4-(4'[maleimidomethyl] cyclohexane-carboxamido)butane) Pierce Chemical Company, Rockford, IL) based on the method described in the manufacturer's instructions. In order to ensure that the peptide is not diminished for sulfhydryl groups, it first restores 1% sodium borohydride in 0.1 M Tris, 5 mm EDTA, pH 8.0. the pH of the solution reduced to 5.0 by adding 1 N. HCl. When the hissing of the solution, add 1 M Tris to obtain a pH of 7.0. 8.5 mm solution of Biotin-VMSS prepared by dissolving biotinylated and incubated overnight at 4oC. Biotinylated peptide was separated from free Biotin by using HPLC (high performance liquid chromatography) on a C18 column with a gradient of CH3CN, changing in the range from 10 to 50%, for 30 min with UV detection at 215 nm. The exact peak corresponding biotinylating the peptide identified by mass-spectrometry using electrospray and LSIMS (liquid secondary ion mass spectrometry).

Biotinylated primary peptide incubated with ragovoy library that encodes a large number of different human scFv (approximately 1010). This phage library is prepared, as previously described in the works of J. D. Marks et al. J. Mol. Biol. 222:581-97, 1991; J. D. Marks et al. Bio/Technology 10: 779-783, 1992; J. D. Marks et al. Bio/Technology 11:1145-1149, 1993; A. D. Griffits et al. EMBO J. 12:725-734, 1993). The overall process of the four cycles of selection, accumulation phagemid and expansion in the strain-suppressor TG-1 Eschericha coli conduct, as described by Marks et al. (J. Mol. Biol. 222: 581-97, 1991) with the following modifications. It is known that the phage library contains several agents that bind streptavidin, so the first three cycles of selection include preliminary treatment, providing two incubation of phage for 30 minutes with agrochemie 1 hour. To link biotinylating peptide with attached phage solution of peptide and phage incubated with avidinii magnetic particles in the first and third cycles for 15 and 5 minutes, respectively, and with streptavidin magnetic particles in the second and fourth cycles for 10 and 5 minutes respectively. Accumulated phage from the fourth cycle of selection infect supressory strain NW Eschericha coli and induce with IPTG individual pagemedia clones to form a soluble scFv fragments, as described by Marks et al. (J. Mol. Biol. 222:581-97 (1991)).

Bacterial supernatant from individual clones analyzed in relation to the expression of soluble scFv fragments using the dot-blot and binding with biotinylated primary peptide ELISA method (see Finnern et al. , Clin. Exp. Immunol., 102:566-574, 1995). The ELISA method, however, modify as follows: 96-well plates to micrometrology (lmmulon-4) cover Avidya (10 μg/ml in phosphate buffered saline (PBS) overnight atoC, washed three times in PBS, blocked with 2% milk in PBS and bound with biotinylated primary peptide (5 μg/ml in PBS). A solution of TMB (3,3',5,5'-tetramethylbenzidine) (Kirkegaard and is before determining the color reaction using reader tablets ELISA at a wavelength of 450 nm. Dot-blotting shows that 66% of the 96 selected colonies NW infected with phage accumulated in the fourth breeding cycle, produce scFv. Method ELISA shows that 43 of 96 colonies produce scFv that binds the peptide.

The differences are all positive clones set of PCR(polymerase chain reaction)-screening. The insertion of scFv heavy and light chain first amplified using the primers LMB3 and fd-Seq1 (see Marks et al., J. Mol. Biol. 222: 581-97 (1991)), and then digested with restriction enzyme BstN1. Clones with different samples of fingerprints DNA is sequenced using the set to cycle sequencing SequiTherm Long-Read (Epicentre Technologies) and apparatus Licor. Identify five unique sequences.

To obtain large quantities of purified scFv for further characterization and application of five unique scFv subcloning in the expression vector pUC119 Sfi-NotmycHis, which introduces hexaglycine tag in the end of the scFv (see Schier et al. J. Mol. Biol. 255:28-43, 1996).

Example 3

Example 3 describe the directed delivery of the cystic fibrosis gene regulator transposability (CFTR) wild-type mammalian cells expressing pIgR, using a range of methods described in the RA reference.

The Fab fragment that is reactive toward the section of the trunk pIgR, receive and purified by the methods described in Example 1. Fab is associated with poly(L-lysine) (mol. weight of 20,000 Da) using heterobifunctional cross-linking reagent N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) in accordance with the method described in Fercol et al. (1993).

A plasmid containing the CFTR gene, are ligated with early promoter of cytomegalovirus and injected into the vector RSV (see Thomas et al., J. Biol. Chem., 268: 3313-3320 (1993)). Complexes of Fab-polylysine-DNA is prepared by mixing plasmid DNA with Fab-polylysine in 3M NaCl.

The complex is administered by dissolving it in 0.1 ml phosphate buffered saline solution and placing in the nostrils does not contain the pathogen rats Sprague-Dawley (250-300 g) under weak inhalation anesthesia using Metofane respiratory anesthesia. The introduction of introduce 100 μl of the plasmid in PBS directly into the nostrils of rats, which are manually held in position on the back. Rats kept in this position until they are able to give the solution. This way, as shown, is effective for application of the sample on the mucous membrane of the nose. Cm. work Shahin et al. Infection and Immunity 60:1482-1488 (1992); Gizurarson et al., Vaccine 10:101-106 (1992). Transcription Tran is measures 4

Example 4 describe means of targeted delivery in vivo exogenous proteins into cells expressing pIgR.

An effective way for proteins from endosomes, use complex protein-Fab associated with adenovirus. This method is used with a number of receptor systems, to deliver not less than 1000-fold increased expression. Cm. article Curiel et al. Am. J. Respir. Cell Mol. Biol. 6: 247-252 (1992); Curiel et al. The OEWG. Natl. Acad. Sci. USA 88: 8850-8854 (1991); Gao et al. Hum. Gene Ther. 4:17-24 (1993), each of which is incorporated in this application by reference. Linking is performed by biotinidase adenovirus and Fab/polylysine with subsequent cross-linking with Avidya. The resulting complex is injected as described in Example 3.

Example 5

Example 5 describes the transcytosis are activated antibodies that specifically associated with the barrel, from the apical to basolateral the cell membrane MDCK (dog kidney Madin Darby) containing pIgR.

Fragments Yab, reactive with pIgR, prepared as described in Example 1. In fragments Yab against the trunk of pIgR injected radioactive iodine method using monochloride iodine proposed Goldsein et al. (see Meth. Enzymol. 96: 241-249, 1983). Radioterminal IgA is used as the control surface of MDCK cells or pWe, grown polarized way for 4 days on the liners for cell cultures with a diameter of 12 mm with a pore size of 0.4 μm (Transwells, Costar). Cm. work Breitfeld et al. in Methods in Cell Biology 32:329-337 (1989). Fragments Yab with a radioisotope label is added to the cells during pre-incubation for 2 h with a protease inhibitor-leupeptin (50 µl/ml) or without it. After 20 min apical uptake at 37oWith unbound ligand with a radioisotope label quickly washed three times with MEM (minimal primary environment)/S (bovine serum albumin) (one washing for 5 min and two shorter washing). Apical and basolateral environment collect and change points in time 7, 15, 30, 60 and 120 min with the aim of quantifying the counter-radiation (Beckman Instruments, Palo Alto, CA). Liners for cell cultures cut out for 120 minutes for the quantitative determination of the counter-radiation and to calculate the total initial uptake of radioactivity. Background absorption of MDCK cells is subtracted from the absorption cell pWe for counting specific recycling and transcytosis are activated ligands with a radioisotope label. The results show that 13-18% of fragments Yab are with apical transcytosis are activated on basolateral pavli and patents, mentioned in this description, included in the description by reference to the same extent as if each individual publication or patent was specifically and individually indicated that they incorporated herein by reference.

The list of sequences is given at the end of the description.

1. The ligand specifically binds the trunk of the receptor for polymeric immunoglobulins (pIgR) of a cell, characterized in that it is an antibody selected from the group including monoclonal, polyclonal, chimeric, heteroconjugate antibody-based Ig recombinant Fv fragments from the same chain and antigennegative forms of antibodies, virtually no binding of secretory component pIgR in physiological conditions.

2. The ligand under item 1, characterized in that it is a humanized antibody.

3. The ligand under item 1, characterized in that it is a recombinant fragment of the variable segment of the same chain antibodies.

4. The ligand under item 1, characterized in that it binds extracellularly epitope on the site of the first 33 amino acids, which are proximal to the cell membrane relative to the cleavage site of the receptor.

5. The ligand under item 1, otlichuy-Ala-Gly-Ser-Ala-Asp-Gly-Gln-Ser-Arg-Ser-Ser-Ser-Ser--Lys (SEQ ID No 2)

Rat: Glu-Arg-Glu-lle-Gln-Asn-Ala-Gly-Asp-Gln-Ala-Gln-Glu-Asn-Arg-Ala-Ser-Gly-Asn-Ala-Gly-Ser-Ala-Gly-Gly-Gln-Ser-Gly-Ser-Ser-Lys(SEQ ID No 3)

Human: Glu-Lys-Ala-Val-Ala-Asp-Thr-Arg-Asp-Gln-Ala-Asp-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg (SEQ ID No 4)

Bullish: Glu-Ser-Val-Lys-Asp-Ala-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Ala-Asp-Pro-Gly-Arg-Pro-Thr-Gly-Tyr-Ser-Gly-Ser-Ser-Lys (SEQ ID No 5)

Rabbit: Leu-Ala-Glu-Val-Ala-Val-Gln-Ser-Ala-Glu-Asp-Pro-Ala-Ser-Gly-Asp-Pro-Ala-Ser-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg-Ser-Lys (SEQ ID No 6).

6. The ligand under item 1, characterized in that it has a binding component for selective binding with the barrel of the receptor or biologically active component and a biologically active component is selected from the group consisting of a nucleic acid, a protein, a radioisotope, lipids and carbohydrates.

7. The ligand under item 6, wherein the nucleic acid encodes the cystic fibrosis transmembrane conductance regulator wild-type.

8. The ligand under item 1, characterized in that it has a binding component for selective binding with the barrel of the receptor or biologically active component and a biologically active component is selected from the group consisting of anti-inflammatory agents, antisense oligonucleotides, antibiotics and anti-infective agents.

9. The ligand under item 1, Otho binds the peptide, having the sequence Ala-Asp-Ala-Ala-Pro-Asp-Glu-Lys-Val-Leu-Asp-Ser-Gly-Phe-Arg-Glu-lle-Glu-Asn-Lys-Ala-lle-Gln-Asp-Pro-Arg-Leu-Phe-Ala-Glu-Glu-Lys-Ala-Val-Ala-Asp-Thr-Arg-Asp-Gln-Ala-Asp-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg (SEQ ID No 10).

11. The method of introduction of the ligand under item 1 in the cell expressing the receptor for polymeric immunoglobulins, namely, that attach the ligand to the trunk of the receptor for polymeric immunoglobulins cells, provided that the ligand practically does not bind secretory component pIgR in physiological conditions.

12. The method according to p. 11, characterized in that as a ligand antibody choose.

13. The method according to p. 11, characterized in that the ligand is chosen humanitariannet antibody.

14. The method according to p. 12, wherein the ligand is a recombinant fragment of the variable segment of the same chain antibodies.

15. The method according to p. 11, characterized in that the ligand binds extracellularly epitope on the site of the first 33 amino acids, which are proximal to the cell membrane relative to the cleavage site of the receptor.

16. The method according to p. 11, characterized in that the ligand binds to a peptide selected from the group:

Mouse: Glu-Arg-Glu-lle-Gln-Asn-Val-Arg-Asp-Gln-Ala-Gln-Glu-Asn-Arg-Ala-Ser-Gly-Asp-AlaSer-Gly-Ser-Ser-Lys (SEQ ID No 3)

Human: Glu-Lys-Ala-Val-Ala-Asp-Thr-Arg-Asp-Gln-Ala-Asp-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg (SEQ ID No 4)

Bullish: Glu-Ser-Val-Lys-Asp-Ala-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Ala-Asp-Pro-Gly-Arg-Pro-Thr-Gly-Tyr-Ser-Gly-Ser-Ser-Lys (SEQ ID No 5)

Rabbit: Leu-Ala-Glu-Val-Ala-Val-Gln-Ser-Ala-Glu-Asp-Pro-Ala-Ser-Gly-Asp-Pro-Ala-Ser-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg-Ser-Lys (SEQ ID No 6).

17. The method according to p. 11, characterized in that the ligand is chosen ligand having a binding component for selective binding with the barrel of the receptor or biologically active component and a biologically active component is selected from the group consisting of a nucleic acid, a protein, a radioisotope, lipid and hydrocarbon.

18. The method according to p. 17, wherein the nucleic acid encodes the cystic fibrosis transmembrane conductance regulator wild-type.

19. The method according to p. 11, characterized in that as a cell select the cell of the mammal.

20. The method according to p. 19, characterized in that the cell is an epithelial cell.

21. The method according to p. 11, characterized in that the ligand is attached to the trunk on the apical surface of the cell.

22. The method according to p. 21, characterized in that the ligand via transcytosis are activated to move on basolateral surface to which ernesti cells.

24. The method according to p. 11, characterized in that the ligand is attached to the trunk on basolateral the cell surface.

25. The method according to p. 11, wherein the ligand specifically binds only the trunk.

26. The method according to p. 11, characterized in that the ligand binds the peptide, having the sequence Ala-Asp-Ala-Ala-Pro-Asp-Glu-Lys-Val-Leu-Asp-Ser-Gly-Phe-Arg-Glu-lle-Glu-Asn-Lys-Ala-lle-Gln-Asp-Pro-Arg-Leu-Phe-Ala-Glu-Glu-Lys-Ala-Val-Ala-Asp-Thr-Arg-Asp-Gln-Ala-Asp-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg (SEQ ID No 11).

27. The method for attaching the ligand under item 1 to the cell expressing the receptor for polymeric immunoglobulins, namely, that the ligand is associated with the barrel of the receptor, provided that the ligand practically does not bind secretory component pIgR in physiological conditions.

28. The method according to p. 27, characterized in that after binding the ligand enters the cell by endocytosis.

 

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SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

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EFFECT: valuable medicinal properties of composition.

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EFFECT: immune serum with increased specific activity.

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4 cl, 5 ex

FIELD: pharmaceutics.

SUBSTANCE: the present innovation deals with cryoprotective ointment containing recombinant interferon-α2. The suggested cryoprotective ointment contains recombinant interferon-α2, glycerol, polyethylene glycol 300-6000, polyglucin, buffered 0.02%-Trilon B solution at pH of 5.5-7.0 and ointment foundation at a certain content of components per 1.0 g ointment. Additionally, cryoprotective ointment could contain glycine 3,7-bis(dimethylamino)phenothiazonium chloride, dry immunoglobulin preparation or dry immunoglobulin preparation for enteral application. Ointment foundation of cryoprotective ointment could contain water-free lanolin, Vaseline and Vaseline oil, at the following ratio of components: 2.5;3.5:1 - 6.5:0.5:1. The innovation provides maximal safety of recombinant interferon-α2 activity in cryoprotective ointment at multiple alteration of positive and negative environmental temperature and at keeping cryoprotective ointment under these conditions.

EFFECT: higher efficiency of application.

8 cl, 8 ex

FIELD: medicine, pharmaceutics, pharmacology.

SUBSTANCE: one should apply mammalian anti-HBP-antibodies. The ways are being suggested to identify monoclonal antibody bound, at least, with one epitope upon native HBP (heparin-binding protein) and methods to detect whether a mammal produces HBR being bound with a monoclonal antibody and, also, the kits for the above-mentioned purpose. The present innovation provides the opportunity to apply the mentioned antibodies in preventing and treating disorders associated with bradykinin releasing.

EFFECT: higher efficiency of application.

25 cl, 11 dwg, 3 ex, 1 tbl

FIELD: veterinary science.

SUBSTANCE: animals should be introduced with antihistamine serum (AHS) subcutaneously at the dosage of 4.0-5.0 ml in combination with myxoferon at the quantity of 60-75 dosages and vitamin C at the dosage of 1.0-1.5 ml/animal, once daily, thrice at interval of 5-7 d. Application of AHS in combination with myxoferon and ascorbic acid provides active stimulation of immunological reactivity, increases total body resistance I animals and causes no toxic effects and allergic reactions.

EFFECT: higher efficiency of correction.

3 tbl

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