Heavy chain mutant resulting in increased immunoglobulin production

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

 

The application describes methods and nucleic acid to produce antibodies in mammalian cells.

Background of invention

Expression systems for production of recombinant polypeptides are known in this field and published. To obtain polypeptides used pharmaceutically acceptable mammalian cells, for example cells, Cho cells, cap cells, NSO cells, Sp2/0 cells, COS cells, SOME cells, PER.C6®, and others. Nucleic acid encoding a polypeptide that is introduced into cells, for example, using plasmids, for example, expressing plasmids. Important elements expressing plasmids are prokaryotic unit propagation of plasmids, such as plasmids Escherichia coli, including the beginning of replication (Replicator) and the selective marker, eukaryotic selective marker and expressing one or more cassettes for the expression of a target nucleic acid (acid), each of which includes the promoter, structural gene and the terminator of transcription, including the polyadenylation signal. For temporal expression in mammalian cells can be included Replicator mammals, for example, SV40 Ori or OriP. As a promoter can be selected constitutive or inducible promoter. To optimize transcription Kozak sequence may be included in the 5'-netransliruemuyu. For mRNA processing also may include a polyadenylation signal.

Proteins, especially immunoglobulins, occupy an important place in the Arsenal of modern medicine. To assign people to each pharmaceutical substance must meet certain criteria. To ensure the safety of biopharmaceutical agents for people, substances that can cause serious damage, should be carefully removed.

Splicing of mRNA is regulated by the availability of donor site splicing in combination with the site of acceptor splicing, which are located at the 5'-end and 3'-end of the intron, respectively. In kN.: "Recombinant DNA: A Short course", 1983, Ed. by Watson and others, published by Scientific American Books, book distribution is carried out by the firm W.H.Freeman and Company, new York, new York, USA, describe the consensus sequence ag|gtragt 5'-donor site of splicing (exon|intron) and (y)nNcag|g 3'-site of acceptor splicing (exon|intron) (r=purine; y=pyrimidine base; n=an integer; N=any natural basis).

In 1980 we published our first article, dealing with the Replicator secreted and membrane-bound forms of immunoglobulins. The formation of secreted (sIg) and is associated with the membrane (mIg) isoforms occurs as a result of a different splicing mRNA, precursor of the heavy chain. The isoforms mIg say the splicing donor in exon, encoding a C-terminal domain of the secreted form (i.e. the domain of CH3 or CH4, respectively), and the site of acceptor splicing, located their distance from him down the chain, is used for communication constant region located below the chain exons encoding the transmembrane domain.

The method of producing molecules of synthetic nucleic acids with reduced inappropriate or unintended indicators transcription or expression in individual cells of the host are described in WO 2002/016944. In WO 2006/042158 described molecules, nucleic acids, modified for enhanced expression of recombinant protein and/or reduction or elimination mistakenly playerowner and/or intron-products formed as a result of continuous read.

Thus, there is a need for a method of producing recombinant products in relation to the immunoglobulins with reduced content of by-products.

Brief description of the invention

The first object of the present invention provides nucleic acid encoding the amino acid sequence C-terminal part of CH3-domain antibodies of class IgE, or IgM, in which the dipeptide glycine-lysine included in the primary amino acid sequence C-terminal part of CH3 or CH4-the house is on, encoded nucleic acid ggaaaa, or nucleic acid ggcaaa, or nucleic acid gggaaa, or nucleic acid gggaag, or nucleic acid ggcaag, or nucleic acid ggaaag.

In one embodiment, the implementation of the present invention the nucleic acid encodes the amino acid sequence selected from the amino acid sequence SEQ ID NO:1, 3, 4, 5, 6, 7 or 8. In another embodiment, the present invention is a nucleic acid encoding the dipeptide glycine-lysine, pradorey the nucleotide g or A. In Drugov embodiment of the present invention nucleic acid that encodes a dipeptide glycine-lysine, is a nucleic acid ggaaaa, or nucleic acid ggcaaa, or nucleic acid gggaaa.

The second object of the present invention is a plasmid comprising the nucleic acid of the present invention, the third object of the present invention are cells comprising the nucleic acid of the present invention.

Another object of the present invention is the nucleotide sequence of SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 30 or 31.

The fifth object of the present invention is a method of producing immunoglobulin in mammalian cells, comprising the following stages:

a) transfection of mammalian cells nucleotide sequence that is the new acid, encoding a heavy chain immunoglobulin representing the nucleic acid SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 30 or 31, which encodes the C-terminal part of the heavy chain of the immunoglobulin,

b) culturing the transfected cells of the mammal under conditions suitable for expression of immunoglobulin,

C) selection of an antibody from the culture or cells.

In one of the embodiments of the present invention the cells are mammalian cells are Cho cells, cap cells, NSO cells, Sp2/0 cells, COS cells, SOME or cells, PER.C6®. Preferably mammalian cells are cells of Cho, or cells KSS, or cells, PER.C6®. In another embodiment, the present invention mammalian cells transfected with the two nucleic acid, the first nucleic acid includes expressing cassette encoding a light chain immunoglobulin and a second nucleic acid comprises expressing cassette encoding a heavy chain immunoglobulin comprising the nucleic acid SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 30 or 31 encoding C-terminal portion of the heavy chain of the immunoglobulin.

The final object of the present invention is a method for improving the expression of immunoglobulin in mammalian cells in which the nucleic acid encoding a heavy chain immunoglobulin includes nuclein the first acid ggaaaa, or nucleic acid ggcaaa, or nucleic acid gggaaa, or nucleic acid ggaaag, or nucleic acid ggcaag, or nucleic acid gggaag encoding the dipeptide glycine-lysine contained in CH3 or CH4-domain.

Detailed description of the invention

The present invention includes a nucleic acid encoding the amino acid sequence C-terminal part of CH3-domain of an immunoglobulin of the class IgA or IgG, or the C-terminal part of CH4-domain immunoglobulin class IgE, or IgM, in which the dipeptide glycine-lysine included in the amino acid sequence C-terminal part of CH3 or CH4-domain encoded by nucleic acid ggaaaa, or nucleic acid ggcaaa, or nucleic acid gggaaa, or nucleic acid ggaaag, or nucleic acid ggcaag, or nucleic acid gggaag, and nucleic acid that encodes a dipeptide glycine-lysine, not necessarily precedes or nucleotide g or nucleotide.

The methods and techniques that can be used in the present invention, well-known experts in this field and are described, for example, in the book: "Current Protocols in Molecular Biology", 1997, Ed. by Ausubel F.M., T.I-III, and in the book. Sambrook and others: "Molecular Cloning: A Laboratory Manual", 1989, 2nd ed., ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Specialists in this field it is known that the application of methods of recombination the NC allows you to get numerous derivatives of nucleic acid and/or polypeptide. These derivatives may, for example, be modified in one particular or several positions by substitution, modification, exchange or insertions. Modification or derivatization may, for example, be performed by site-directed mutagenesis. Such modifications can be easily done by experts in this field (see, for example, Sambrook J. and others in kN.: "Molecular Cloning: A laboratory manual", 1999, ed. Cold Spring Harbor Laboratory Press, new York, USA). The use of recombinant techniques helps professionals in this area to transform different cells are the owners of heterologous nucleic acids. Although the mechanisms of transcription and translation, i.e. expression in different cells used the same elements, cells belonging to different species can have addition to other differences between different used codons. Thus, identical polypeptides (amino acid sequence) can be encoded by different nucleic acids. In addition, due to the degeneracy of the genetic code of different nucleic acids may encode the same polypeptide.

In the context of the present invention, the term "nucleic acid" refers to a polymeric molecule composed of individual nucleotides (also called bases) a, C, g and t (or u in RNA), for example, DNA, RNA, or their modifications. Polynucleotide forefront of the La can be a molecule of natural polynucleotide, or molecule synthetic polynucleotide, or a combination of one or more natural molecules polynucleotide with one or more synthetic molecules polynucleotide. This concept also applies to natural polynucleotide molecules in which one or more nucleotides are replaced (for example, using mutagenesis), or delegated added. Nucleic acid can also be isolated or integrated into another nucleic acid, for example, in expressing cassette, plasmid or the chromosome of the host cell. Nucleic acid is also characterized by a sequence of nucleic acid, consisting of individual nucleotides.

Professionals in this field are well known methods for the conversion of amino acid sequence, for example, polypeptide, in the appropriate sequence of nucleic acid encoding the amino acid sequence. Therefore, the nucleic acid is nucleic acid sequence consisting of a single nucleotide and amino acid sequence of the polypeptide encoded by this nucleic acid.

The term "plasmid" refers, for example, vector and expressing plasmids, and plasmid transfection. The term "vector" in the present invention is used as Sinani is the concepts of "plasmid". Usually "plasmid" also includes the beginning of replication (Replicator), for example, the Replicator ColE1 or oriP, and selective marker (e.g., marker ampicillin, kanamycin, tetracycline, or chloramphenicol) for replication and selection, respectively, plasmids in bacteria.

The concept of "expressing cassette" refers to a structure that contains the required regulatory elements such as promoter and a polyadenylation site for the expression of at least caged nucleic acids.

The concept of "selective marker" means a nucleic acid which allows the cells bearing the selective marker, to be specifically selected on this basis the type of "for" or "against" in the presence of an appropriate selective agent. Usually selective marker can confirm the stability of a drug or to compensate for a metabolic or catabolic defect in the cell host. The marker can be positive, negative or bifunctional. Convenient positive selective marker is the gene of resistance to the antibiotic. Such a selective marker allows the cells to the owners, transfected by this token, to be positively selected in the presence of an appropriate selective agent, i.e. antibiotic. Untransformed cells-owners are unable to grow and survive under conditions of selective culture i.e. in the presence of a selective agent in culture. Positive selective markers allow selection of cells carrying the marker, and markers for negative selection can selectively eliminate cells carrying the marker. To selective markers used in eukaryotic cells include, for example, genes aminoglycosidetherapy (aminoglycoside phosphotransferase - ARN), for example, selective markers hygromycin (hyg), neomycin (neo) and G418, dihydrotetrazolo (DHFR), thymidine kinase (tk), glutamylcysteine (GS), asparaginase, cryptotanshinone (selective agent is indole), histidinolovorans (selective agent histidinol D) and nucleic acids, causing resistance to puromycin, bleomycin, bleomycine, chloramphenicol, zeocin and morphophonology acid. Other markers described in WO 92/08796 and WO 94/28143.

In the context of the present invention the term "expression" refers to the processes of transcription and/or translation in the cell. The level of transcription of the analyzed nucleic acid sequence can be set based on the amount of corresponding mRNA that is present in the cell. For example, transcribed from the analyzed sequence mRNA can be quantified by the method of RT-PCR or norsen-hybridization (see above Sambrook and others, 1989). The polypeptides encoded who's the analyzed nucleic acid, can be quantified by different methods, such as ELISA, assessment of biological activity of the polypeptide or analysis, depending on the activity, for example, Western blotting or radioimmunoassay using antibodies that recognize and bind to the polypeptide (see above Sambrook and others).

The term "cell" or "a host cell" in the context of the present invention means a cell in which the nucleic acid, for example, encoding a heterologous polypeptide, can be or has already been introduced/transfected. The term "cell" includes prokaryotic cells used for propagation of plasmids and eukaryotic cells, which are used for expression of the nucleic acid. Preferably, the eukaryotic cells are mammalian cells. Preferably mammalian cells selected from the group of mammalian cells, including cells Cho (e.g., Cho K1, CHO DG44)cells KSS cells, NSO cells, SP2/0 cells, SOME 293 cells, SOME 293 EBNA cells, PER.C6® cells and COS. The concept of expressing the "cells" in the context of the present invention means a cell of the subject and her offspring. Thus, the term "transformant" and "transformed cells" include the primary cell of the subject and cells in cultures derived from it, regardless of the number of transfers. Also obvious is IDNO, that posterity may not be exactly identical in DNA content, due to deliberate or unintended mutations. This version also includes the option of offspring with the same function or the same biological effects that are identified in the originally transformed cell.

The term "polypeptide" means a polymer comprising amino acids connected by peptide bonds, whether, whether they are natural or synthetic. Polypeptides, the length of not more than about 20 amino acid residues, can be labeled peptides, and molecules comprising two or more polypeptides, or comprising a single polypeptide of more than 100 amino acid residues, can be called "proteins". The polypeptide may also include components that are not amino acids, for example, carbohydrate groups, metal ions or esters of carboxylic acids. Components that are not amino acids, can be attached cells, which expressed the polypeptide, and can vary depending on cell type. Polypeptides in the present invention are expressed from the point of view of their amino acid structure, or nucleic acids, their coding. Add, such as carbohydrate groups, usually not precisely determined, but nevertheless, they may be present.

The term "amino acid" in the context of this is part II of the invention means a group of carboxy α-amino acids, which directly or in the form of a precursor can be encoded by nucleic acid. Individual amino acids are encoded by nucleic acids, each of which consists of three nucleotides is called a codon or triplet. Each amino acid is encoded by at least one codon. For example, the amino acid glycine can be encoded by each of the four nucleic acids (codons) gga, ggc, ggg and ggt, and the amino acid lysine is encoded by two nucleic acids (AAA and aag. This phenomenon is called "degeneracy of the genetic code". The group of amino acids include alanine (three letter code: ala, one letter designation: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), Proline (pro, P), series (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

The term "immunoglobulin" in the context of the present invention means a protein consisting of one or more polypeptides, mainly encoded by immunoglobulin genes. This definition includes options, such as mutant forms, i.e. forms with substitutions, divisions and an insertion of one or several amino acids truncated from the N-end form, a hybrid form, him the forms, and humanized forms. Installed immunoglobulin genes include the genes of different constant regions, as well as genes incalculable number of variable regions of immunoglobulin, for example, in primates or rodents. Preferred are monoclonal antibodies. Each of the heavy and light polypeptide chains of the immunoglobulin may include a constant region (usually the carboxyl terminal part).

The term "monoclonal immunoglobulin in the context of the present invention means an immunoglobulin derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally mutations that may be present in minor amounts. Monoclonal immunoglobulins of vysokospetsifichnymi, directed against a specific antigenic sites. In addition, unlike drugs polyclonal immunoglobulin, which include different antibodies directed against different antigenic sites (determinants or epitopes), each monoclonal immunoglobulin directed against a single antigenic site on the antigen. The advantage of monoclonal antibodies, in addition to their specificity, is that they can be synthesized without primase is in the form of other immunoglobulins. The definition of "monoclonal" refers to the immunoglobulin as obtained from the population of immunoglobulins, which are largely homogeneous, and does not mean that you want to retrieve immunoglobulin a certain way.

"Humanized" forms of antibodies, non-human immunoglobulins (e.g., immunoglobulins rodent)are chimeric immunoglobulins that contain incomplete sequence derived from the immunoglobulin non-human immunoglobulin and immunoglobulin. Mostly humanized immunoglobulins derived from human immunoglobulin (recipient of an antibody)in which residues from a hypervariable region replaced by the residues of the hypervariable region of immunoglobulins of other species, not human, which is the donor immunoglobulin, such as a mouse, rat, rabbit or monkey with the desired specificity or affinity (see, for example, Morrison S.L., and others, Proc. Natal. Acad. Sci. USA 81, 1984, SS-6855, US 5202238, US 5204244). In some cases the remains of the skeleton of the plot (framework region (FR) of a human immunoglobulin are replaced by corresponding residues not from man. Furthermore, humanized antibodies may include additional modifications, for example, amino acid residues that neobsahuje the immunoglobulin recipient or the donor immunoglobulin. These modifications lead to variants of such immunoglobulins recipient or donor, which are homologous, but not identical to the corresponding original sequence. Such modifications get for additional cleaning. In General humanitarianly immunoglobulin can include substantially all of at least one, typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to the loops of the donor immunoglobulin, which is not of man, and all or substantially all of the FR plots are plots FR recipient of a human immunoglobulin. Humanitarianly immunoglobulin optionally may also include at least a portion of constant region of immunoglobulin, usually immunoglobulin. Methods humanitarian immunoglobulin non-human immunoglobulin, known in this area. Preferably humanitarianly immunoglobulin contains one or more amino acid residues introduced into it from a source which is not man. Such amino acid residues that are not associated with a person, often denoted "import" residues, which are typically taken from an "import" variable domain. Humanitarian can be essentially performed according to the method of Winter and co-authors by the Deputy is possible sequences of hypervariable region for the corresponding sequences of the immunoglobulin, non-human immunoglobulin. Therefore, such "humanized" antibodies are chimeric immunoglobulins, and significantly less as compared with intact variable domain of human substituted by the corresponding sequence from a representative of the species, not the person. In practice, humanized antibodies are typically human immunoglobulins in which some remnants of the hypervariable region and perhaps some remnants of the frame section substituted by residues from analogous sites of immunoglobulins rodents or monkeys.

Depending on the amino acid sequence of the constant region of heavy chains, immunoglobulins are divided into classes: IgA, IgD, IgE, IgG and IgM. Some of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. In the constant regions of the heavy chains of different classes of heavy chains of immunoglobulins are designated α-, δ-, ε-, γ -, and µ-chain, respectively. The constant region of the heavy chain of human immunoglobulin full length of the class IgA, IgD and IgG is composed of a constant domain 1 (referred to in the present invention, "CH1"), a hinge region, a constant domain 2 (CH2) and a constant domain 3 (CH3). The human immunoglobulins of class IgE and IgM additionally include the th fourth constant domain (C H4). In addition, the immunoglobulins of the IgM class are polymers that include a variety of immunoglobulins, for example, five or six, covalently linked by disulfide bonds. Amino acid sequence C-terminal constant domain such different classes of immunoglobulin are listed in table 1. The first amino acid sequence SEQ ID NO:01-08 may or may not be present, since this amino acid is encoded by two exons: exon, encoding the C-terminal constant domain, and exon coding for the previous domain.

Table 1.
C-terminal amino acid sequence of immunoglobulins of different classes. Underlined the dipeptide glycine-lysine.
The class of immunoglobulin (C-terminal constant domain)Amino acid sequence C-terminal part (in the direction from N-Terminus to the C-end)SEQ ID NO:
IgA1 (CH3), IgA2 (CH3)GNTFRPEVHL LPPPSEELAL NELVTLTCLA RGFSPKDVLV RWLQGSQELP REKYL TWASR QEPSQGTTTF AVTSILRVAA EDWKKGDTFS CMVGHEALPL AFTQK TIDRL AGKPTHVNVS VVMAEVDGTC Y01
IgD (CH3) AAQAPVKLSLN LLASSDPPEA ASWLLCEVSG FSPPNILLMW LEDQREVNTS GFAPARPPPQ PGSTTFWAWS VLRVPAPPSP QPATYTCVVS HEDSRTLLNA SRSLEVSY02
IgE (CH4)GPRAAPEVYA FATPEWPGSR DKRTLACLIQ NFMPEDISVQ WLHNEVQLPD ARHSTTQPRK TKGSGFFVFS RLEVTRAEWE QKDEFICRAV HEAASPSQTV QRAVSVNPGK03
IgM (CH4)GVALHRPDVY LLPPAREQLN LRESATITCL VTGFSPADVF VQWMQRGQPL SPEKYVTSAP MPEPQAPGRY FAHSILTVSE EEWNTGETYT CVVAHEALPN RVT ERTVDK STGKPTLYNV SLVMSDTAGT CY04
IgGI (CH3)GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK05
IgG2 (CH3)GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK06
IgG3 (CH3)GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE ALHNRFTQKS LSLSPGK07
IgG4 (CH3)GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK08

The term "C-terminal part of CH3-domain of an immunoglobulin of the class IgA or IgG, or the C-terminal part of CH4-domain immunoglobulin class IgE or IgM" means amino acid posledovatelno and the heavy chain of the immunoglobulin, which localized with C-end of the full length or nature of the heavy chain of the immunoglobulin, and With the end of the specified part is identical With the end of the primary amino acid sequence of the heavy chain of the immunoglobulin. The term "primary amino acid sequence" means an amino acid sequence of the heavy chain of immunoglobulin after the broadcast of the corresponding mRNA. This primary amino acid sequence may also be modified in expressing cells after the broadcast mRNA, e.g., peptidases, chip off one or more C-terminal amino acids from the primary amino acid sequence. Hence, the primary amino acid sequence and secreted amino acid sequence can be identical, but may differ some amino acids on the C-end. In one of the embodiments of the present invention C-terminal part includes at least 100 C-terminal amino acids of the primary amino acid sequence of the heavy chain of the immunoglobulin or preferably at least 50 C-terminal amino acids of the primary amino acid sequence of the heavy chain of the immunoglobulin, or preferably at least 20 C-terminal amino acids of the primary amino acid sequence of tarlouze immunoglobulin. In one of the embodiments of the present invention the nucleic acid of the present invention encodes the amino acid sequence C-terminal part of CH3-domain immunoglobulin class IgG, or the C-terminal part of CH4-domain immunoglobulin of the IgE class. In another embodiment of the present invention the nucleic acid of the present invention encodes the amino acid sequence C-terminal part of domain CH3 immunoglobulin of the IgG class.

Amino acid sequence C-terminal constant domain of different human immunoglobulins encoded by the corresponding DNA sequences. In the genome such DNA sequences contain coding (exon) and noncoding (intron) sequences. After transcription of DNA into mRNA, precursor of the indicated mRNA, the precursor also contains intron and exon sequences. Before the broadcast of non-coding intron sequences are removed during processing of mRNA by splicing of the primary transcript mRNA to obtain the Mature mRNA. Splicing of the primary mRNA is controlled by the site splanirovanie donor in combination with properly located at the distance of the site splanirovanie acceptor. The site splanirovanie donor localized at the 5'end, and the site is planirovanie acceptor localized at the 3'end of the intron sequence, and both are partially removed during the process of splicing mRNA predecessor.

The concept of "properly placed" in the context of the present invention means that a website splicing donor and the site of the splicing acceptor in nucleic acid assembled in such a way that all the required elements for splicing are available and are in a position that allows you to be the process of splicing.

The present invention includes a nucleic acid encoding the amino acid sequence C-terminal part of CH3-domain of an immunoglobulin of the class IgA or IgG, or the C-terminal part of CH4-domain immunoglobulin class IgE, or IgM, in which the dipeptide glycine-lysine contained in the amino acid sequence C-terminal part of CH3 or CH4-domain encoded by nucleic acid ggaaaa, or nucleic acid ggcaaa, or nucleic acid gggaaa.

Unexpectedly, it was found that with the nucleic acid of the present invention the formation of undesirable by-products as a result of episodes of undesirable splicing may be reduced.

The term "dipeptide glycine-lysine" in the context of the present invention means a peptide comprising from N-Terminus to the C-end of two amino acids, glycine and lysine, related peptide bond. P is the adoption of "the dipeptide glycine-lysine" in the context of the present invention means the fraction of the dipeptide of a larger polypeptide or protein, which can be found at the beginning, inside or end of a larger polypeptide. The amino acid lysine can be encoded by nucleic acid AAA or aag. Thus, in another embodiment, the present invention dipeptide glycine-lysine included in the amino acid sequence C-terminal part of CH3 or CH4-domain heavy chain immunoglobulin, is encoded by nucleic acid ggaaag, or nucleic acid ggcaag, or nucleic acid gggaag. In another embodiment, the present invention nucleic acid that encodes a dipeptide glycine-lysine is a precursor of the nucleotides a or g.

Nucleic acid sequences encoding the C-terminal constant domain of different classes and subclasses of human immunoglobulin, are listed in table 2. For domain CH3 IgG1 and IgG2 man known two variant forms. In one of the embodiments of the present invention the nucleic acid encodes part of the C-terminal constant domain of the heavy chain of immunoglobulin.

Table 2.
Nucleic acid sequence encoding C-terminal portion of the heavy chain of human immunoglobulins of different classes.
The class of immunoglobulin (C-terminal constant domain)The sequence of a nucleic acid encoding a C-terminal part of the amino acid sequence of the heavy chain immunoglobulinSEQ ID NO:
IgA1 (CH3), IgA2 (CH3)ggcttcagcc ccaaggacgt gctggttcgc tggctgcagg ggtcacagga gctgccccgc gagaagtacc tgacttgggc atcccggcag gagcccagcc agggcaccac caccttcgct gtgaccagca tactgcgcgt ggcagccgag gactggaaga agggggacac cttctcctgc atggtgggcc acgaggccct gccgctggcc ttcacacaga agaccatcga ccgcttggcg ggtaaaccca cccatgtcaa tgtgtctgtt gtcatggcgg aggtggacgg cacctgctac09
IgD (CH3)taccacccaa cgtccgtgac tgtcacctgg tacatgggga cacagagcca gccccagaga accttccctg agatacaaag acgggacagc tactacatga caagcagcca gctctccacc cccctccagc agtggcgcca aggcgagtac aaatgcgtgg tccagcacac cgccagcaag agtaagaagg agatcttccg ctggccaggt aggtcgcacc ggagatcacc cagaagggcc ccccaggacc cccagcacct tccactcagg gcctgaccac aaagacagaa gcaagggctg10
IgE (CH4)tttgcgacgc cggagtggcc ggggagccgg gacaagcgca ccctcgcctg cctgatccag aacttcatgc ctgaggacat ctcggtgcag tggctgcaca acgaggtgca gctcccggac gcccggcaca gcacgacgca gccccgcaag accaagggct ccggcttctt cgtcttcagc cgcctggagg tgaccagggc cgaatgggag cagaaagatg agttcatctg ccgtgcagtc catgaggcag cgagcccctc acagaccgtc cagcgagcgg tgtctgtaaa tcccggtaaa11
IgM(CH4)acgggcttct ctcccgcgga cgtcttcgtg cagtggatgc aggggggca gcccttgtcc ccggagaagt atgtgaccag cgccccaatg cctgagcccc aggccccagg ccggtacttc gcccacagca tcctgaccgt gtccgaagag gaatggaaca cgggggagac ctacacctgc gtggtggccc atgaggccct gcccaacagg gtcaccgaga ggaccgtgga caagtccacc ggtaaaccca ccctgtacaa cgtgtccctg gtcatgtccg acacagctgg cacctgctac 12
The class of immunoglobulin (C-terminal constant domain)The sequence of a nucleic acid encoding a C-terminal part of the amino acid sequence of the heavy chain immunoglobulinSEQ ID NO:
IgG1 (CH3) option 1gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa13
IgG1 (CH3) option 2gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc ctctccctgt ctccgggtaa a28
IgG2 (CH3) option 1gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac acctcccatg ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa14
IgG2 (CH3) option 2gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat ctccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca cacctcccat gctggactcc gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a29
IgG3 (CH3)gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcag cgggcagccg gagaacaact acaacaccac gcctcccatg ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acatcttctc atgctccgtg atgcatgagg ctctgcacaa ccgcttcacg cagaagagcc tctccctgtc tccgggtaaa15
IgG4 (CH3)gtgtacaccc tgcccccatc ccaggaggag atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac agcaggctaa ccgtggacaa gagcaggtgg caggagggga atgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tctgggtaaa16

In one of the embodiments of the present invention the nucleic acid of the present invention encodes the amino acid sequence selected from the amino acid sequence SEQ ID NO:1,3, 4, 5, 6, 7 or 8.

In one of the embodiments of the present invention the nucleic acid encodes part of the C-terminal constant domain of the heavy chain immunoglobulin CL the SSA IgA, IgE, IgM or IgG and selected from the nucleic acid SEQ ID NO:17, 18, 19, 20, 21, 22, or 23, or 30, or 31. In another embodiment of the present invention the nucleic acid encodes part of the C-terminal constant domain of the heavy chain immunoglobulin of the class IgA, IgE, IgM or IgG and selected from the nucleic acid SEQ ID NO:17, 18, 19, 22, 23, 30 or 31. In another embodiment of the present invention the nucleic acid encodes part of the C-terminal constant domain of the heavy chain immunoglobulin of the class IgA, IgE, IgM or IgG and selected from the nucleic acid SEQ ID NO:17, 18. 19, 22 or 23.

Nucleic acids with nucleotide sequence SEQ ID NO:17-23 and 30-31 are also the object of the present invention. In one of the embodiments of the present invention the nucleotide sequence of SEQ ID NO:17, 18, 19, 22, 23, 30 or 31 are the object of the present invention. In another embodiment of the present invention the nucleotide sequence of SEQ ID NO:17, 18, 19, 22 or 23 are the object of the present invention.

In one of the embodiments of the present invention the nucleic acid encodes a C-terminal constant domain of an immunoglobulin of the class IgA, IgE, IgM or IgG and includes nucleic acid selected from the nucleic acid SEQ ID NO:17, 18, 19, 20, 21, 22 or 23, or 30, or 31, which encodes part of the C-terminal domain of the heavy chain of immunoglobulin the and. In another embodiment of the present invention the nucleic acid encodes a C-terminal constant domain of an immunoglobulin of the class IgA, IgE, IgM or IgG and includes nucleic acid selected from the nucleic acid SEQ ID N0: 17, 18, 19, 22, 23, 30 or 31, which encodes part of the C-terminal domain of the heavy chain of the immunoglobulin. In another embodiment of the present invention the nucleic acid encodes a C-terminal constant domain of immunoglobulins IgA, IgE, IgM or IgG and includes nucleic acid selected from the nucleic acid SEQ ID NO:17, 18, 19, 22 or 23, which encodes part of the C-terminal domain of the heavy chain of the immunoglobulin.

Table 3
The nucleic acid sequences of the present invention, encoding part of the amino acid sequence C-terminal constant domain of the heavy chain of immunoglobulins of different classes
The class of immunoglobulinThe sequence of the nucleic acid that encodes a C-terminal amino acid sequenceSEQ ID NO:
IgAggcaaaccca cccatgtcaa tgtgtctgtt gtcatggcgg aggtggacgg cacctgctac 17
IgEcatgaggcag cgagcccctc acagaccgtc cagcgagcgg tgtctgtaaa tcccggcaaa18
IgMggcaaaccca ccctgtacaa cgtgtccctg gtcatgtccg acacagctgg cacctgctac19
IgG1 option 1atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggcaaa20
IgG1 option 2atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tccgggcaaa30
IgG2 variant 1atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggcaaa21
IgG2 variant 2atctccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacacctccc atgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggc aaa31
IgG3atgcatgagg ctctgcacaa ccgcttcacg cagaagagcc tctccctgtc tccgggcaaa22
IgG4atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tctgggcaaa23

Nucleic acid of the present invention encodes at least part of the C-terminal constant domain of an immunoglobulin of the class IgA, gE, IgM or IgG. The term "C-terminal constant domain" means either CH3 domain of the heavy chain immunoglobulin of the class IgA or IgG, or CH4-domain heavy chain immunoglobulin of class IgE or IgM. The expression of "parts" means the C-terminal fraction of C-terminal constant domain of the heavy chain immunoglobulin of the class IgA, IgE, IgM or IgG, at least 20 consecutive amino acids, or at least 50 consecutive amino acids, at least 100 consecutive amino acids of the primary amino acid sequence counted from the end in the direction of the N-end of the heavy chain of the immunoglobulin.

Recombinant obtaining immunoglobulins known in this field and are described, for example, in reviews Makrides, S.C., Protein Expr. Purif. 17, 1999, SS-202, Geisse, S., and others, Protein Expr. Purif. 8, 1996, SS-282, Kaufman R.J., Mol. Biotechnol. 16, 2000, s-160, R.G. Werner, Arzneimittelforschung - Drug Research, 48, 1998, SS-880.

To obtain immunoglobulin comprising amino acid sequence encoded by a nucleic acid of the present invention also provides a method of producing immunoglobulin in mammalian cells, comprising the following stages:

a) transfection of these cells to the mammal a nucleic acid that encodes a heavy chain of an antibody, and a nucleic acid selected from posledovatel the values of the nucleic acid SEQ ID NO:17, 18, 19, 20, 21, 22, or 23, or 30, or 31 encodes the domain C-terminal domain of the heavy chain of the immunoglobulin,

b) culturing the transfected cells of the mammal under conditions suitable for expression of immunoglobulin,

C) selection of an antibody from the culture or cells. The term "conditions permitting expression of immunoglobulin in the context of the present invention means the terms and conditions for the cultivation of mammalian cells expressing immunoglobulin, which are known and can be easily identified by experts in the field. Specialists in this field it is also known that these conditions may vary depending on the type of cultured mammalian cells and the type of expressed immunoglobulin. In General, mammalian cells are cultured at a temperature of, for example, from 20°C to 40°C, for a time sufficient for the effective formation of the protein, namely immunoglobulin, for example, for 4-28 days.

In one of the embodiments of the present invention mammalian cells transferout nucleic acid that encodes a heavy chain of an antibody, and a nucleic acid selected from the nucleic acid SEQ ID NO:17, 18, 19, 22, 23, 30 or 31, which encode part of the C-terminal domain of the heavy chain of the immunoglobulin. In another one of the embodiments in the present invention, mammalian cells transferout nucleic acid, encoding a heavy chain of an antibody, and a nucleic acid selected from the nucleic acid SEQ ID NO:17, 18, 19, 22 or 23, encodes part of the C-terminal domain of the heavy chain of the immunoglobulin. In one of the embodiments of the present invention mammalian cells transferout one nucleic acid comprising expressing cassette that encodes a heavy chain immunoglobulin, and expressing cassette that encodes the light chain of the immunoglobulin. In another embodiment, the present invention provides mammalian cells, transfected with the two nucleic acids, one of which includes expressing cassette encoding a light chain immunoglobulin, and the other includes expressing cassette encoding a heavy chain immunoglobulin.

The immunoglobulin produced by the method of the present invention, is preferably heterologous immunoglobulin. The term "heterologous immunoglobulin" means an immunoglobulin, which normally produces the specified cells of the mammal. The immunoglobulin produced by the method of the present invention, receive recombination methods. Such methods are widely known in the field and include protein expression in prokaryotes and eukaryotes, followed by separation and purification of heterologous IMM is noglobulin, and usually clear up pharmaceutically acceptable purity. To obtain, i.e. expression of the nucleic acid that encodes a light chain of an antibody, and a nucleic acid that encodes a heavy chain that comprises the nucleic acid of the present invention, these nucleic acid insertyour in expressing cassette standard methods. Nucleic acid encoding the immunoglobulin, easily distinguished and sequeiros according to standard procedures. Hybridoma cells can be a source of such nucleic acids. Expressing cassettes can be insertion in expressing plasmids (plasmid), which then transferout in cell owners who otherwise cannot produce immunoglobulins. Expression is performed in appropriate prokaryotic or eukaryotic cells-the owners and immunoglobulin isolated from the cells after lysis or from the supernatant of the culture.

Well-known and widely-used methods used for separation of protein and purification, for example, affinity chromatography with microbial proteins (for example, affinity chromatography using protein a or protein G), ion-exchange chromatography (e.g. cation-exchange (carboxymethyl resins), anion exchange (iminoethylene resins) and mixed-type exchange chromatography), theofilou adsorption (n is an example, with beta-mercaptoethanol and other SH ligands), hydrophobic interaction chromatography or aromatic adsorption (for example, phenyl-separate, Aza-uranopilite resins, or m-aminophenylarsonic acid), affinity chromatography with chelation of metal (for example, Ni(II)- and Cu(II)-affinity material), exclusion chromatography and electrophoresis methods (e.g., methods, gel electrophoresis, capillary electrophoresis) (M.A. Vijayalakshmi, Appl. Biochem. Biotech. 75, 1998, SS-102).

The present invention also includes nucleic acid encoding a heavy chain immunoglobulin representing nucleic acid of the present invention. In addition, the present invention provides a plasmid comprising a nucleic acid of the present invention, and cells containing this plasmid.

Another object of the present invention is a method for improving the expression of immunoglobulin in mammalian cells in which the nucleic acid encoding a heavy chain immunoglobulin includes a nucleic acid ggaaaa, or nucleic acid ggcaaa, or nucleic acid gggaaa, or nucleic acid ggaaag, or nucleic acid ggcaag, or nucleic acid gggaag that encode the dipeptide glycine-lysine contained in CH3 or CH4-domain. With such a nucleic acid is undesirable splicing signal is to be reduced or suppressed.

In one of the embodiments of the present invention a heavy chain immunoglobulin is either a heavy chain of an antibody of human subclass IgG4, or a heavy chain immunoglobulin of an antibody of human subclass IgG1, IgG2 or IgG3. In one of the embodiments of the present invention a heavy chain immunoglobulin a heavy chain of human immunoglobulin and preferably any of the subclass IgG4 human or mutant heavy chain immunoglobulin of the IgG1 subclass of person. In another embodiment of the present invention a heavy chain immunoglobulin is derived from IgG1 subclass humans with mutations L234A and L235A. In another one of the embodiments of the present invention a heavy chain immunoglobulin a heavy chain immunoglobulin IgG4 person with mutation S228P. In one of the embodiments of the present invention a heavy chain immunoglobulin IgG4 subclass or subclass IgG1 or IgG2, mutation L234, L235 and/or D265, and/or contains a mutation PVA236. In other embodiments, implementation of the present invention mutations are S228P, L234A, L235A, L235E and/or PVA236 (PVA236 means that the amino acid sequence ELLG (single-letter code of amino acids) of the amino acid at position 233 through 236 in IgG1 or EFLG immunoglobulin IgG4 replaced by PVA). Preferred mutations S228P in IgG4, and L234 and L235A in IgG1 (numbering according to EU index in Kabat).

The concept of "regulatory elements" in the context of the present invention means a nucleotide sequence in the cis position, necessary for transcription and/or translation of nucleic acid sequence that encodes the analyzed polypeptide. Normal elements of the regulation of transcription include a promoter that is located above expressed chain nucleotide sequence that encodes a target polypeptide. Normal elements of the regulation of transcription include a promoter that is located above the expressed nucleic acid sequence, the sites of initiation and termination of transcription and signal sequence for polyadenylation. The concept of "site of transcription initiation" refers to the base of a nucleic acid comprising a nucleic acid corresponding to the first nucleic acid included in the primary transcript, i.e. mRNA, the precursor; the site of transcription initiation may overlap with the promoter sequence. The concept of "site of transcription termination" refers to a nucleotide sequence, normally located on the 3'-end of a target gene under the transcription, which leads to termination of transcription by RNA-polymerase. The signal sequence for polyadenylation or additional signal poly-And represents a signal for the cleavage of the specific site from the 3'-end of eukaryotic mRNA and post-transcriptional addition to the core sequence of about 100-200 Denisovich nucleotides (tail poly-(A) to split the 3'-end. Signal polyadenylation sequence may include a consensus sequence AATAAA located approximately 10-30 nucleotides above the chain from the site of cleavage.

To obtain a secreted polypeptide target nucleic acid includes a DNA segment that encodes a signal sequence/leader peptide. The signal sequence directs the newly synthesized polypeptide to the membrane of the endoplasmic reticulum and through it to the place to which the polypeptide can be directed for secretion. The signal sequence otscheplaut signal peptidase during the crossing of the protein membrane of endoplasmic reticulum. For the operation of the signal sequence is important recognition mechanism of secretion of the host cell. Therefore, used the signal sequence should be recognized proteins and enzymes mechanism of secretion of the host cell.

Elements of regulation of translation include codon of translation initiation (AUG) and stop codon (TAA, TAG or TGA). The inner side of the entrance ribosomes (internal ribosome entry site (IRES may be included in some designs.

The term "promoter" in the context of the present invention means a polynucleotide sequence that controls transcription of the gene/structural gene or a follower of the awn nucleic acid, with which it is functionally connected. The promoter includes signals for the binding of RNA polymerase and initiation of transcription. Used promoters can be functional in cells of the owners of this type in which the expression of the selected sequence. A large number of promoters, including constitutive, inducible and repressed promoters from different sources, it is known in this area (they are identified, for example, in GenBank and are available by themselves or as part of the cloned polynucleotides (e.g., deposited in the collection of ATS, as well as their other commercial or individual sources). The term "promoter" includes a nucleotide sequence that directs transcription of a structural gene. Typically, the promoter is located at the 5'end non-coding or noncoding region of a gene, proximal in relation to the site of the start of transcription of the structural gene. The elements of the sequences in the composition of the promoters, the function of which is to initiate transcription, often differ consensus nucleotide sequences. These promoter elements are binding sites for RNA polymerase, TATA sequence, sequence SAAT specific in differentiating cells (differentiation-specific elements - DSE; McGehee R.E., and others, Mol. Endocrinol. 7, 993, SC-560), response elements of camp (cyclic AMP response elements - CRE), the elements of serum response (serum response elements - SRE; Treisman R. Seminars in Cancer Biol. 1, 1990, SS-58), the glucocorticoid response (glucocorticoid response elements - GRE), and binding sites for other transcription factors, e.g., CRE/ATF (Treisman R., Seminars in Cancer Biol. 1, 1990, SS-58; O'reilly M.A. and others, J. Biol. Chem. 267, 1992, s), AR (Ye J., and others, J. Biol. Chem. 269, 1994, s), SP1, proteins that bind response elements of the cyclic amp (cAMP response element binding protein - CREB; loeken on M.R., Gene Expr. 3, 1993, s) and octamine factors (see General kN.: "Molecular Biology of the Gene", 1987, Ed. by Watson and others, 4th ed., ed. The Benjamin/Cummings Publishing Company, Inc., and Lemaigre F.P., G.G. Rousseau, Biochem. J. 303, 1994, SS-14). If the promoter is an inducible promoter, the rate of transcription increases in response to signaling agent. On the contrary, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter. Also known repressive promoters. For example, the promoter of c-fos specifically activated upon binding of growth hormone to its receptor on the cell surface. Expression regulated by tetracycline (tet), can be achieved artificial hybrid promoters, which include, for example, from a CMV promoter followed two sites Tet-operator. Tet-repressor binds to two sites Tet-operator and blocks transcription. Adding inductor tetracycli is and Tet-repressor is released from sites Tet-operator and transcription occurs (Gossen M. and Bujard H. PNAS 89, 1992, SS-5551). On the other induced by the promoters, including metallothionein and promoters of heat shock, see, for example, Sambrook and others (above) and Gossen M., Curr. Opin. Biotech. 5, 1994, SS-520. Among the eukaryotic promoters, identified as a strong promoter for high level expression are early SV40 promoter, the major late promoter of adenovirus, the promoter metallothionein-1 mouse, long terminal repeat sarcoma virus of Rausch, elongation factor 1 alpha Chinese hamster (Chinese hamster elongation factor - CHEF-1; see, for example, US 5888809), EF-1 alpha human ubiquitin and very early promoter of human cytomegalovirus (CMV IE).

The term "promoter" in the context of the present invention means a constitutive or inducible promoter. Enhancer (i.e., the element cis-acting DNA, which acts on a promoter to increase transcription) may be required for operation in conjunction with a promoter to increase expression level obtained only with the promoter, and may be included as part of the regulation of transcription. Often polynucleotide segment containing the promoter, may also include an enhancer sequence (e.g., CMV or SV40).

The term "enhancer" in the context of the present invention means a polynucleotide sequence that promotes transcription of a gene or codereuse the sequence, with which it is functionally connected. Unlike promoters, enhancers do not depend on the orientation and the position and were installed with the 5'-end or 3'-end (Lusky, M. and others, Mol. Cell Bio., 3, 1983, SS-1122) to the transcription unit within an intron (Banerji J. and others, Cell, 33, 1983, SS-740), as well as in the coding sequence (T.F. Osborne and others, Mol. Cell Bio., 4, 1984, SS-1305). Thus, enhancers can be placed above or below the chain from the site of transcription initiation or at significant distances from the promoter, although in practice enhancers may overlap physically and functionally with the promoters. A large number of enhancers from different sources in this area (and identified in the databases, for example, in GenBank) and is available as a cloned polynucleotide sequences or included them in sequence (e.g., deposited in the collection of ATS, as well as in other commercial or individual sources). A number of polynucleotides representing promoter sequences (e.g., commonly used CMV), also include an enhancer sequence. For example, all strong promoters listed above, can also include a strong enhancers (see, for example, kN.: Bendig M.M. "Genetic Engineering", 1988, ed. Academic Press, Vol.7, SS-127).

The concept of "internal side of the entrance ribosomes (internalribosome entry site - IRES)" means a sequence that is functionally induces the initiation of translation regardless gene 5'-end sequence of the IRES and allows two cistrons (open reading frames) transmitted from a single transcript in a cell of the animal. IRES provides an independent entrance ribosomes for translation of an open reading frame directly down the chain (in the present invention the term "down the chain" is used interchangeably with 3') from it. Unlike bacterial mRNA that can be polycistronic, i.e. can encode different polypeptides, which consistently translated from mRNA molecules, most mRNA molecules in animal cells monocistronic and encode the synthesis of only one protein. If monocistronic transcript in eukaryotic cells, translation can be initiated from the 5'-end of the site of initiation of translation, terminates on the first stop-codon, and a transcript can be released from the ribosome, leading to broadcast only the first of the encoded polypeptide in mRNA. In eukaryotic cells a polycistronic transcript, in which the IRES is functionally connected with the second or subsequent open reading frame in the transcript, allows a consistent translation of the transcript, below the chain open reading frame for the Holocene two or more polypeptides, encoded by the same transcript. The use of IRES elements in the construction of the vector was described previously, see, for example, Pelletier J., and others, Nature 334, 1988, SS-325, S.K. Jang, etc., J. Virol. 63, 1989, s-1660, Davies, M.V., etc., J. Virol. 66, 1992, SS-1932, Adam, M.A., and others, J. Virol. 65, 1991, SS-4990, Morgan, R.A., and others, Nucl. Acids Res. 20, 1992, SS-1299; Y. Sugimoto and others, Biotechnology 12, 1994, s-698, Ramesh N., and others, Nucl. Acids Res. 24, 1996, s-2700, D.D. Mosser, etc., BioTechniques 22, 1997, SS-161).

The term "functionally linked" means the immediate proximity of two or more components, and these components interact, allowing them to function in the intended way. For example, the promoter and/or enhancer functionally linked to the coding sequence if it acts in cis-position to control or modulate the transcription of a linked sequence. Usually, but not necessarily, a DNA sequence, which is "functionally related", located close by and if you combine the two areas that encode proteins, for example, a secretory leader and polypeptide adjacent and in frame (reading). However, although functionally linked promoter is usually located in front of the coding sequence, it does not necessarily located next to it. Enhancer functionally linked to the coding sequence if the enhancer increases the transcription of the coding consistent is Telenesti. Functionally related enhancers can be located above the chain, inside or below the coding sequence and at a considerable distance from the promoter. The polyadenylation site is functionally linked to the coding sequence if it is located below the chain to the end of the coding sequence such that transcription proceeds through the coding sequence for polyadenylation sequence. Stop-codon broadcast functionally associated with a sequence of exon nucleic acid, if it is located below the chain end (3'-end) of the coding sequence in such a way that translation occurs with the coding sequence to the stop codon and ends. Linking perform recombination methods known in this field, for example, by PCR and/or legirovaniem on the appropriate restriction sites. If appropriate restriction enzymes cut sites don't, then use synthetic oligonucleotide adaptors or linkers in accordance with the usual practice.

The concept of "heterologous DNA" or "heterologous polypeptide" refers to a DNA molecule or polypeptide, or to a population of DNA molecules, or a population of polypeptides, which in natural conditions in this cell the owner is absent. Molecules NAM Prov., heterologous for certain host cells may contain DNA derived from the types of host cells (i.e. endogenous DNA), the length of which is such that the host DNA combined with DNA not belonging to the owner (i.e. exogenous DNA). For example, a DNA molecule containing a segment of DNA from the owner that encodes a polypeptide that is functionally associated with a segment of DNA of the host, including the promoter, evaluate as molecules are heterologous DNA. Conversely, the molecule is heterologous DNA may include endogenous structural gene functionally linked to an exogenous promoter.

The peptide or polypeptide encoded by the DNA molecule, which is not the host DNA, is "heterologous peptide or polypeptide.

The concept of "expressing plasmid in the context of the present invention means a nucleic acid molecule encoding a protein expressed in the cell host. Usually expressing plasmid includes prokaryotic plasmid unit of reproduction, for example, for E. coli, comprising the replicon, and a selective marker, eukaryotic selective marker and expressing one or more cassettes for the expression of the investigated structural gene (genes), each of which includes the promoter, structural gene and the terminator of transcription, including the polyadenylation signal. Gene expression is usually on escaut under the control of a promoter, and such a structural gene called "functionally associated" with the promoter. Similarly a regulatory element and a core promoter functionally linked, if a regulatory element modulates the effect of the crustal promoter.

The term "isolated polypeptide" means a polypeptide that is substantially free from contaminating cellular components, such as carbohydrates, lipids, protein, and other related polypeptides in nature. Usually the drug is selected polypeptide includes polypeptide in high purity form, i.e. at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% purity or greater than 99% purity. One way to show that the drug is a protein contains a selected polypeptide, is the formation of a single lane after sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the protein preparation and staining of the gel dye Kumasi blue. However, the term "isolated" does not exclude the same polypeptide in other physical forms, such as dimers or otherwise glycosylated or derivatives.

The term "transcription terminator" in the context of the present invention means a DNA sequence from 50-750 base pairs in length, which takes the RNA-polymerase signal termination of the synthesis of Il is K. Highly effective (potent) terminator 3'-end expressing cassettes can be used to prevent reading of RNA polymerase, especially when using strong promoters. Inefficient transcription terminators can lead to the formation of operon-like mRNA, which can cause undesirable, for example, encoded by plasmid, gene expression.

The examples given below, the sequence listing and figures are provided to better explain the present invention, the true scope of which is given below in the attached claims. It is obvious that modifications can be obtained in the following without deviation from the spirit of the present invention.

Description sequences

SEQ ID NO:01. C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin of the class IgA person.

SEQ ID NO:02. C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin IgD class person.

SEQ ID NO:03. C-terminal part of the amino acid sequence of the constant domain (CH4) the heavy chain immunoglobulin of the IgE class person.

SEQ ID NO:04. C-terminal part of the amino acid sequence of the constant domain (CH3) heavy zeekanaal IgM person.

SEQ ID NO:05. C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin class IgG1 human.

SEQ ID NO:06. C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin IgG2 class person.

SEQ ID NO:07. C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin class IgG3 human.

SEQ ID NO:08. C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin of the class IgG4 human.

SEQ ID NO:09. Nucleic acid encoding a C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin of the class IgA person.

SEQ ID NO:10. Nucleic acid encoding a C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin IgD class person.

SEQ ID NO:11. Nucleic acid encoding a C-terminal part of the amino acid sequence of the constant domain (CH4) the heavy chain immunoglobulin of the IgE class person.

SEQ ID NO:12. Nucleic acid encoding a C-terminal part of the amino acid sequence of the constant domain (CH4) the heavy chain immunoglobulin of the IgM class person.

SEQ ID NO:13, 28. Well Leonova acid, encoding C-terminal portion of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin class IgG1 human (options 1 and 2).

SEQ ID NO:14, 29. Nucleic acid encoding a C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin IgG2 class person (options 1 and 2).

SEQ ID NO:15. Nucleic acid encoding a C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin class IgG3 human.

SEQ ID NO:16. Nucleic acid encoding a C-terminal part of the amino acid sequence of the constant domain (CH3) the heavy chain immunoglobulin of the class IgG4 human.

SEQ ID NO:17. Nucleic acid of the present invention, the coding part of the amino acid sequences of C-terminal constant domain (CH3) immunoglobulin of the class IgA person.

SEQ ID NO:18. The sequence of the nucleic acid of the present invention, the coding part of the amino acid sequences of C-terminal constant domain (CH4) immunoglobulin of the IgE class.

SEQ ID NO:19. The sequence of the nucleic acid of the present invention, the coding part of the amino acid sequences of C-terminal constant domain (CH4) immunoglobulin class is IgM.

SEQ ID NO:20, 30. The sequence of the nucleic acid of the present invention, the coding part of the amino acid sequences of C-terminal constant domain (CH3) of immunoglobulin class IgG1 (options 1 and 2).

SEQ ID NO:21,31. The sequence of the nucleic acid of the present invention, the coding part of the amino acid sequences of C-terminal constant domain (CH3) immunoglobulin IgG2 class (options 1 and 2).

SEQ ID NO:22. The sequence of the nucleic acid of the present invention, the coding part of the amino acid sequences of C-terminal constant domain (CH3) immunoglobulin class IgG3.

SEQ ID NO:23. The sequence of the nucleic acid of the present invention, the coding part of the amino acid sequences of C-terminal constant domain (CH3) immunoglobulin IgG4 class.

SEQ ID NO:24-27. Primers nucleic acids used in the examples.

Description of figures

Figure 1. Annotated map of plasmid R.

Figure 2. SDS-PAGE and Western blot analysis of antibody secreted by clone No. 23; a) staining dye Kumasi blue, b) analysis by the method of Western blotting using peroxidase connected with the antibody against the immunoglobulin gamma chain of human rights, analysis by the method of Western blotting using peroxidase, linked to the antibody p is otiv light chain immunoglobulin Kappa man. Lane 1: anti-IGF-1R control human antibody; lane 2: culture supernatant of cells CHO-DG44 clone No. 23, including antibody clone No. 23.

Figure 3. Annotated map of plasmid R.

Figure 4. Analysis by the method of Western blotting of immunoglobulin secreted by cells CHO-DXB11, transfected R or 4855. Lane 1: cells SNO, transfected R, lane 2: cells SNO, transfected R.

Figure 5. Annotated map of plasmid R.

6. SDS-PAGE analysis of antibody secreted by the cells of SOME 293-EBNA after transfection with plasmid R or R; band 1+2: R, band 3+4: R, lanes 5-8: antibody against IGF-1R acts as a control antibody; 5: 0,2 μg, 6: 0,7 µg, 7: 2 mg, 8: 6 mcg.

7. Cells CHO-DG44, transfected with plasmid R and selected for stable integration of the plasmid with methotrexate. Antibodies from six clones was purified and analyzed by the method of SDS-PAGE and staining Kumasi.

Materials and methods

Methods of recombinant DNA

To work with DNA using standard methods described by Sambrook and others, 1989 (see above). All reagents from molecular biology commercially available (unless otherwise noted) and used according to manufacturer's instructions.

Determination of nucleic acid sequence

DNA sequences define double-stranded sequencing, done the subject MediGenomix GmbH (Martinsried, Germany).

Analysis of DNA sequences and proteins and work with the results obtained

Software GCG's (Genetics Computer Group, Madison, Wisconsin, USA), version 10.2 and Infbmax''s Vector NTI Advance suite version 8.0 is used to create the sequence, mapping, annotation and illustration.

Methods cell cultures

Cells CHO-DXB11 grown in medium MEM alpha (firm Invitrogen Corp., Gibco®, catalog number: 22571) with 10% FTS (fetal calf serum, obtained from the company Hyclone, Thermo Fisher Scientific Inc., catalog number: SH3007.03).

Cells HEK-293-EBNA (collection of ADS, No. CRL-10852) were cultured in DMEM (modified by way of Dulbecco environment Needle) supplemented with 2 mm glutamine, Gibco®, catalog number: 25030 casual), 1% vol. MEM (minimal supportive environment) amino acids, non-essential (product of Gibco®, catalog number: 11140), 10% vol. FTS with ultra-low IgG (product of Gibco®, catalog number: 16250) and 250 μg/ml G418 (firm Roche Applied Sciences, Roche Diagnostics GmbH, Germany, catalog number: 1464981).

Environment for cell culture CHO-DG44 is Wednesday MEM alpha (product of Gibco®, Cat. No.: 22561) supplemented with 10% vol. detalizirovannoi FST (product of Gibco®, catalog number: 26400) and 2 vol.% HT (product of Gibco®, catalog number: 41065). For selection of stably transfected cell lines Cho DG44 additive NT do not contribute, and add from 20 to 500 nm methotrexate is or separately, or in combination with 400 μg/ml of hygromycin In (company Roche Diagnostics GmbH, Roche Applied Sciences, Germany, catalog number: 10843555001).

All cell lines support in a humid incubator at 37°C in an atmosphere of 5% CO2. Transfection of cells is carried out either by nucleofection (firm Amaxa GmbH, Germany), or by lipofection using FuGENE 6 (firm Roche Diagnostics GmbH, Roche Applied Sciences, Germany, catalog number: 1815075).

In addition, standard methods of cell culture are used as described, for example, in the book: "Current Protocols in Cell Biology", 2000, Ed. by J.S. Bonifacino and others, ed. John Wiley and Sons, Inc.

The deposition of protein A, SDS-PAGE and Western blotting

Immunoglobulins from supernatants cell cultures precipitated on the protein granules And-sepharose and then analyzed by the method of SDS/polyacrylamide gel electrophoresis (sodium dodecyl sulphate, SDS-PAGE) and Western blot testing.

For the deposition of immunoglobulin supernatant cultures of cells containing up to 7 µg immunoglobulin, diluted with TBS buffer (50 mm TRIS/HCl, pH 7.5, 150 enriched mm Nad), 1% vol. Nonidet-P40 (firm Roche Diagnostics GmbH, Roche Applied Sciences, catalog number 1754599) to a final volume of 1 ml and then incubated for 1 h with 15 ál of wet volume of protein a-granules sepharose. Granules recover by centrifugation and washed with TBS with 1% vol. Nonidet P-40, then double the concentration of the FSB (phosphate saline buffer) and finally to 10 mm superaccurate sodium, pH 5. After the last wash, the supernatant is completely removed from the granules. Associated immunoglobulins elute 20 μl of the sample buffer with double concentration of lithium dodecyl sulfate (firm Invitrogen Corp.), containing 50 mm dithiothreitol. After 5 min incubation at 95°C, the suspension is centrifuged and the supernatant collected for further analysis.

SDS-PAGE: SDS-PAGE carried out using the gel NuPAGE® (firm Invitrogen Corp.) according to the manufacturer's recommendations. Samples are loaded into the gels 10% NuPAGE® Novex Bis/TRIS (firm Invitrogen Corp., catalog number: NP0301) and separated proteins in running buffer with decreasing NuPAGE® MES SDS (4-morpholinepropanesulfonic acid/sodium dodecyl sulphate). Usually load 2-3 µg immunoglobulin on the strip for painting Kumasi with the preparation of the dye Simply Blue Safe Stain® (firm Invitrogen Corp.) 0.4-0.6 ág for Western blotting.

Western blotting: for elektroprenos proteins from SDS/polyacrylamide gels using standard polyvinylidene defteritnye (polyvinylidene difluoride - PVDF) or nitrocellulose membranes. After elektroprenos membrane was washed in TBS (saline, buffered by tricom, 50 mm TRIS/HCl, pH 7.5, 150 mm NaCl). The nonspecific binding sites blocked by incubation in TBS with 1 wt.% reagent for Western blotting (firm Roche Diagnostics GmbH, Roche Applied Sciences, catalog number: 11921673001). Chain heavy chain and light chain chain immun the gamma globulin is detected using polyclonal antibodies for detection, United with peroxidase (see next paragraph), diluted in TBS with 0.5 wt.% reagent for Western blotting. After three stages of washing in TBS with 0.05 to about.% Tween® 20 and one stage of washing in TBS associated with peroxidase antibodies to detect detect using chemoluminescence using the substrate solution LumiLightPlus (firm Roche Diagnostics GmbH, Roche Applied Sciences, catalog number: 12015196001) and the analyzer LUMI-Imager F1 (firm Roche Diagnostics GmbH, Roche Applied Sciences).

The heavy chain (H) immunoglobulin gamma man and a light chain (L) immunoglobulin Kappa person discover at the same time or separately. For simultaneous detection using peroxidase connected with the antibody goat against IgG (H+L) human (firm Jackson ImmunoResearch Laboratories Inc., catalog number: 109-035-088) at a dilution of 1:2500 (by volume). For the subsequent detection of the first membrane probe peroxidase, linked to the antibody rabbit against human Ig (firm DAKO GmbH, Germany, catalog number: R), at a dilution of 1:1000 (by volume), or peroxidase, linked to the fragment F(ab')2goats against Fc gamma people (firm Jackson ImmunoResearch Laboratories, catalog number: 109-036-008), at a dilution of 1:7500 (by volume). After the discovery of the heavy chain of immunoglobulin gamma membrane cleaned for 30 min in 62.5 mm TRIS/HCl, pH 6,7, with the addition of 2 wt.% SDS and 100 mm β-mercaptoethanol at 50°C. For the second detection is of membrane re-probe peroxidase connected with the antibody rabbit against Ig Kappa person (firm DAKO GmbH, catalog number: R), at a dilution of 1:1000 (vol./vol.).

Example 1. Obtaining expressing plasmids for immunoglobulin class IgG1

Plasmid 4831 (hereinafter "R") is expressing a plasmid for the expression of anti-IGF-1R antibodies (genome organized expressing cassette stored by the organization of the exon-intron) in eukaryotic cells (sequence see, for example, in US 2005/0008642 or EP 1 646 720). It includes the following functional elements:

- replicon derived from the vector pUC18 (pUC replicon),

gene β(beta)-lactamase, which determines resistance to ampicillin in E. coli (Amp),

- expressing cassette for the expression of the gamma 1 heavy chain comprising the following elements:

the main very early promoter and enhancer of the human cytomegalovirus (hCMV promoter IE1),

- synthetic 5'UTR, including a Kozak sequence,

signal sequence of the heavy chain of mouse immunoglobulin, including the intron, signal sequence (L1__L2),

- cDNA for the variable regions of the heavy chain (VH)connected to the site splanirovanie donor 3'-end,

the enhancer of the immunoglobulin µ mouse

gene heavy chain gamma 1 immunoglobulin (human immunoglobulin heavy chain gamma 1-gene - IGHG1), including E. the areas CH1, the hinge region, CH2 and CH3, inverted introns and 3'UTR bearing signal polyadenylation sequence, and optionally containing a mutation,

- expressing cassette for the expression of Kappa-light chain comprising the following elements:

the main very early promoter and enhancer from the human cytomegalovirus (hCMV promoter IE1), including a Kozak sequence,

signal sequence of the heavy chain of mouse immunoglobulin, including intron (L1__L2),

- cDNA for the variable region of the light chain (VL)connected to the site splanirovanie donor 3'-end,

- enhancer region of intron Ig-Kappa mouse

gene immunoglobulin Kappa man (human immunoglobulin kappa gene - IGK), including exon IGKC and IGK 3'UTR bearing signal polyadenylation sequence,

- unit transcription hygromycin In-phosphotransferase applicable for selection in eukaryotic cells, including

- early promoter and the Replicator virus SV40,

sequence encoding hygromycin In phosphotransferase (HygB),

- early polyadenylation signal SV40,

- expressing cassette digidrofolatreduktazy (dihydrofolate reductase - DHFR), applicable for the selection of auxotrophs in eukaryotic cells, including

- a shortened version of the early promoter and the port Replicator SV40,

- sequentially encoding the th mouse DHFR,

- early polyadenylation signal SV40.

Annotated map of plasmid R presented in figure 1.

R transferout cells CHO-DG44 and produce stable cell lines after selection on the background of hygromycin In and methotrexate. Antibody secreted by clone No. 23, precipitated using protein a-separatrix pellets and analyzed by the method of SDS-PAGE with staining Kumasi (figa)). In addition to the expected heavy chain immunoglobulin gamma-1 mass of 50 kDa and the light chain of the immunoglobulin Kappa mass of 25 kDa find a significant quantity of by-product protein mass of 80 kDa. This protein recognizes using antibodies against the immunoglobulin gamma chain man (figb)), and antibodies against chain immunoglobulin Kappa person (pigv)).

Example 2. Obtaining expressing plasmids for immunoglobulin class IgG1 with modified CH3 domain

To prevent the formation of by-products resulting from incorrectly splanirovano gamma 1 mRNA precursor, internal site splanirovanie CH3 exon exon plasmids R destroy by mutation engine T in position 4573 S. At the same time T replace at With removal of the restriction site BsmA I.

R design as follows. Plasmid R, the original plasmid to plasmid R with the same transcription unit heavy chain gamma 1, consists of a followed by the elements:

the Replicator from the vector pUC18 which allows replication of this plasmid in E. coli (pUC Ori)

gene beta-lactamase, which causes resistance to ampicillin in E. coli (Amp)

- expressing cassette for expression of the heavy chain gamma, which includes the following elements:

the main very early promoter and enhancer of the human cytomegalovirus (hCMV promoter IE1),

- synthetic 5'UTR, including a Kozak sequence,

signal sequence of the heavy chain of mouse immunoglobulin, including the intron, signal sequence (L1__L2),

- cDNA for the variable regions of the heavy chain (VH)connected to the site splanirovanie donor 3'-end,

the enhancer of the immunoglobulin µ mouse

gene heavy chain gamma 1 immunoglobulin (human immunoglobulin heavy chain gamma 1-gene - IGHG1), including exons SN, hinge region, CH2 and CH3, introducing introns and 3'UTR bearing signal polyadenylation sequence, and optionally containing mutations.

Map of plasmid R shown in figure 3.

Plasmid R change site-directed mutagenesis using the kit for site-directed mutagenesis QuickChange Site-Directed Mutagenesis Kit (firm Stratagene, catalog number: 200518) and oligonucleotides specific sequence 1 agcctctccc tgtccccggg caaatgagtg cgacggccg (SEQ ID NO:24) and 2 cggccgtcgc actcatttgc ccggggacag ggagaggct (SEQ ID NO:25).

Frag the UNT SfiI/SgrAI size 839 BP mutant plasmids R cut and are ligated with a fragment of SgrAI/SfiI size 13133 BP plasmid R for forming plasmids R.

Example 3. The expression of the nucleic acid according to examples 1 and 2, the allocation of the produced immunoglobulin and analysis of the produced immunoglobulin

Plasmids R and R temporarily transferout cells CHO-DXB11. Cells cultured in non-selective conditions. After three days of culturing supernatant cultures of cells is collected and secreted immunoglobulins purified protein a-sivaratnam granules. Analysis by the method of Western blotting of immunoglobulin antibodies against human IgG (H+L) shows that a byproduct mass of 80 kDa is expressed by cells transfected with plasmid R, but not cells transfected with plasmid R (figure 4).

Example 4. Obtaining expressing plasmids for immunoglobulin IgG4 class

Plasmid 5031 create for transient and stable expression of the antibody against P-selectin of human rights in the tissue culture of eukaryotic cells. Examples of antibodies against P-selectin see, for example, in EP 1737891 or US 2005/0226876. Plasmid R consists of the following elements:

the Replicator from the vector pUC18 which allows replication of this plasmid in E. coli (Replicator pUC),

gene B-lactamase, which determines resistance to ampicillin in E. coli (Amp),

p> - expressing cassettes for the expression of gamma 4 heavy chain of a human, comprising the following elements:

the main very early promoter and enhancer from the human cytomegalovirus (hCMV promoter IE1),

- synthetic 5'UTR, including a Kozak sequence,

signal sequence of the heavy chain of mouse immunoglobulin, including the intron, signal sequence (L1__L2),

- cDNA for the variable regions of the heavy chain (VH)connected to the website splicing donor 3'-end,

- area enhancer Ig µ mouse

gene heavy chain gamma 4 immunoglobulin (human immunoglobulin heavy chain gamma 4-gene - IGHG4), including exons SN, hinge region, CH2 and CH3, intermediate introns and 3'UTR bearing signal polyadenylation sequence, and optionally containing a mutation,

- expressing cassettes for the expression of light chain Kappa person, including the following elements:

the main very early promoter and enhancer of the human cytomegalovirus (hCMV promoter IE1),

- synthetic 5'UTR, including a Kozak sequence,

signal sequence of the heavy chain of mouse immunoglobulin, including the intron, signal sequence (L1__L2),

- cDNA for the variable region of the light chain (VL)connected to the site splanirovanie donor 3'-end,

- enhancer region is ü intron Ig-Kappa mouse

gene immunoglobulin Kappa man (human immunoglobulin kappa gene - IGK), including exon IGKC and IGK 3'UTR bearing signal polyadenylation sequence,

unit transcription hygromycin In-phosphotransferase applicable for selection in eukaryotic cells, including

- early promoter and the Replicator SV40,

sequence encoding hygromycin In phosphotransferase (HygB),

- early polyadenylation signal SV40,

- expressing cassettes for expression digidrofolatreduktazy (dihydrofolate reductase - DHFR) mouse, applicable for auxotrophic selection in eukaryotic cells, including

- a shortened version of the early promoter and the port Replicator SV40,

- the coding sequence of the DHFR rodent,

- early polyadenylation signal SV40.

Plasmid map of plasmid R presented in figure 5.

If cells HEK-293-EBNA transfected with plasmid R, they produce immunoglobulins, including byproduct mass of 80 kDa (6, lane 1 and 2). According to analysis by the method of Western blotting the protein binds an antibody against Ig gamma man, as well as antibody against Ig Kappa man.

Example 5. Obtaining expressing plasmids for immunoglobulin class with modified IgG4 CH3 domain

The modification of the implement according to example 3. Briefly, T mutate on With together with the second nucleotide, T, which is also substituted With removal of BsmAI restriction site. Oligonucleotide 3 gcctctccct gtccctgggc aaatgagtgc cagg (SEQ ID NO:26) and oligonucleotide 4 cctggcactc atttgcccag ggacagggag aggc (SEQ ID NO:27) is used for site-directed mutagenesis. The obtained plasmid denote R.

Example 6. The expression of the nucleic acid according to example 5, the selection received immunoglobulin and analysis of immunoglobulin

Plasmids R and R temporarily transferout in the cells of SOME 293-EBNA. Cells grown in non-selective conditions. After three days of cultivation supernatant cell cultures are harvested and formed immunoglobulins purified using protein a-separatrix granules. Analysis of antibodies by the method of Western blotting using antibodies against human IgG (H+L) shows that cells transfected with plasmid R, Express a byproduct mass of 80 kDa (6, lanes 1+2), but by-products does not Express the cells transfected with plasmid R (6, lanes 3+4).

In cells HEK-293-EBNA, transfected with plasmid R, protein mass of 80 kDa is not expressed (6, lanes 3 and 4). This result clearly points to the fact that a hybrid protein mass of 80 kDa occurs due to impaired splicing of the mRNA precursor, therefore, development of such unwanted protein during temporal expression can be effectivedate mutation internal customers splicing snz gene heavy chain immunoglobulin gamma 4 (immunoglobulin heavy chain gamma 4 gene - IGHG4).

Mutation of the internal site splicing CH3 in IGHG4 prevents the expression of the connection weight of 80 kDa in stable cell lines. Cells CHO-DG44 were transfected with plasmid R and selected for stable integration of the plasmid with methotrexate. Antibodies 6 clones was purified and analyzed by SDS-PAGE and staining dye Kumasi (Fig.7). None of the antibodies does not contain a subunit mass of 80 kDa.

1. Nucleic acid encoding the amino acid sequence of the heavy chain immunoglobulin of the IgG1 subclass, characterized in that the glycine-lysine dipeptide, located in the region C-terminal part of CH3-domain encoded by nucleic acid ggaaaa, or nucleic acid ggcaaa, or nucleic acid gggaaa.

2. Nucleic acid according to claim 1, characterized in that the C-terminal part includes at least 20 C-terminal amino acids of the primary amino acid sequence of the heavy chain of the immunoglobulin.

3. Nucleic acid according to claim 1, characterized in that the specified part of the C-terminal constant domain of the heavy chain selected from the nucleic acid SEQ ID NO: 20 or 30.

4. Nucleic acid according to claim 3, characterized in that it contains the nucleotide sequence of SEQ ID NO: 20.

5. Nucleic acid according to claim 3, characterized in that it contains a nucleotide sequence EQ ID NO: 30.

6. A plasmid encoding a heavy chain immunoglobulin of the IgG1 subclass and comprising a nucleic acid according to claim 1.

7. The cells providing the expression of the heavy chain immunoglobulin of the subclass IgGl and comprising a nucleic acid according to claim 1.

8. The cells providing the expression of the immunoglobulin subclass IgG1 and comprising a nucleic acid according to claim 1.

9. Cells according to claim 7 or claim 8, characterized in that the cells are mammalian cells.

10. Cells according to claim 9, characterized in that the cells are mammalian cells selected from the cell SNO, SOME cells or cells KSS.

11. Nucleic acid encoding the amino acid sequence of the heavy chain immunoglobulin of the IgG1 subclass, characterized in that it contains the nucleotide sequence of SEQ ID NO:20, or 30, where the glycine-lysine dipeptide, located in the region C-terminal part of CH3-domain encoded by nucleic acid ggcaaa or nucleic acid gggaaa.

12. Nucleic acid according to claim 11, containing the nucleotide sequence of SEQ ID NO:20.

13. Nucleic acid according to claim 11, containing the nucleotide sequence of SEQ ID NO:30.

14. The method of producing immunoglobulin in mammalian cells, comprising the following stages:
a) transfection of these cells mammalian two nucleic acids, in which the first nucleic acid encodes a light chain of the immunoglobulin, and the second nucleic acid encodes a heavy chain immunoglobulin and is a nucleic acid according to claim 1 or claim 11,
b) culturing the transfected cells of the mammal under conditions suitable for expression of immunoglobulin,
C) selection of an antibody from the culture or cells.

15. The method according to 14, characterized in that the mammalian cells transfected with a single design, including expressing cassette encoding a heavy chain immunoglobulin, and expressing cassette encoding a light chain immunoglobulin.

16. The method according to 14, characterized in that the mammalian cells transfected with the two designs, the first includes expressing cassette encoding a light chain immunoglobulin, and the second involves expressing cassette encoding a heavy chain immunoglobulin.

17. The method according to one of p-16, characterized in that the said transfected mammalian cells contain a nucleic acid encoding a heavy chain of an antibody, and nucleic acid SEQ ID NO: 20 or 30 encodes the C-terminal part of the heavy chain of the immunoglobulin.

18. Method for improving the expression of immunoglobulin in mammalian cells, in which a nucleic acid encoding a heavy chain of immunoglobulin, which nucleic acid is th according to claim 1 or claim 11.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: application of yeast strain Komagataella pastoris RNCIM Y-727 as the recipient to construction of producers of target protein is characterised, optionally comprising introduction of mutations into it, providing the use of auxotrophic selective markers.

EFFECT: solution can be applied in preparation of recombinant proteins without the use of methanol as inductor of gene expression.

8 dwg, 4 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: nucleotide sequences are formed, encoding the hybrid proteins EPO-TR 1.6, EPO-TR 4 and EPO-TR 6. Protein EPO-TR 1.6 is recombinant human erythropoietin fused with a fragment TR 1.6 of the human protein MUC1. Protein EPO-TR 4 is recombinant human erythropoietin fused with a fragment TR 4 of the human protein MUC1. The hybrid protein EPO-TR 6 is recombinant human erythropoietin fused with a fragment TR 6 of the human protein MUC1. Hybrid proteins are produced by the roller cultivation in the suitable conditions the modified mammalian cell line CHO containing a nucleotide sequence encoding the protein with subsequent isolation of the hybrid protein from the culture fluid.

EFFECT: invention enables to produce the hybrid recombinant human erythropoietin having the prolonged action.

4 cl, 4 dwg, 7 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to muteins of human tear lipocalin, and can be used in medicine. Mutein of human tear lipocalin (hTLc) has identifiable affinity of binding with human receptor Met (c-Met) receptor tyrosine kinase, or its domain, or fragment of human c-Met. Mutein contains from 6 to 18 amino acid substitutions relative to amino acid sequence of mature lipocalin of human tear liquid (SWISSPROT DATABANK ENTRY P31025; SEQ ID NO:36), selected from group, consisting of Arg 26→Thr, Val, Pro, Ser, Gly; Glu 27→Gln, Gly, Val, Ser; Phe 28→Met, Asp; Pro 29→Leu, Ile, Ala, Trp; Glu 30→Leu, Gly, Arg, Phe; Met 31→Ser; Asn 32→Leu, Arg, Val, Gln; Leu 33→Tyr, Val, Ile, Thr, Phe; Glu 34→Val, Arg, Ala; Leu 56→Asn; Ile 57→Gln; Ser 58→Ile, Val; Asp 80→Tyr; Lys 83→Ala; Glu 104→Asp; Leu 105→Thr; His 106→Trp and Lys 108→Gly. Mutein can also additionally contain the following substitutions: Cys 61→Ser; Cys 101→Ser; Cys 153→Ser; Arg 111→Pro; Lys 114→Trp; Thr 37→Ser; Met 39→Ile, Leu; Asn 48→Ser; Lys 52→Thr, Met; Met 55→Leu; Lys 65→Arg, Leu; Ala 79→Leu, Ser; Ala 86→Thr; Ile 89→Ser, Gln, Thr, His; Thr 40→Cys; Glu 73→Cys; Arg 90→Cys; Asp 95→Cys; Lys 121→Cys; Asn 123→Cys and Glu 131→Cys.

EFFECT: invention makes it possible to efficiently treat pathological disorders, which involve pathway HGF/c-Met, as well as to perform identification of human c-Met in sample.

40 cl, 16 dwg, 9 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of microbiology and deals with method of obtaining RTX-toxin ApxI. Claimed method is realised by cultivation of bacteria Actinobacillus pleuropneumoniae in culture medium, which provides growth of bacteria, and said culture medium contains borogluconate in concentration lower than 60 mmol/l in order to form in medium calcium-bologluconate complex.

EFFECT: invention makes it possible to increase output of RTX-toxin ApxI, which can be applied in production of vaccines.

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SUBSTANCE: invention refers to a method for obtaining an antibody, the pharmacokinetic properties of which have been changed at maintaining antigen-binding activity of a variable area, which provides for the following stages: (a) obtaining antibodies in which there has been modified a charge of amino-acid residues chosen from amino-acid residues in positions 31, 61, 62, 64 and 65 of the variable area of a heavy chain and in positions 24, 27, 53, 54 and 55 of the variable area of a light chain in compliance with numbering as per Kabat system, where modification of the charge of amino-acid residues leads to the change of 1.0 or more at a theoretical isoelectric point of the variable area of the antibody, and (b) extracting an antibody with stored antigen-binding activity from antibodies obtained at stage (a).

EFFECT: invention allows effective change in pharmacokinetic properties of an antibody, thus maintaining its antigen-binding activity.

FIELD: biotechnologies.

SUBSTANCE: invention relates to method for obtaining of RTX-toxins Apxl or ApxIII by culturing of Actinobacillus pleuropneumoniae bacteria in liquid culture media. Characterised method consists in the following: during exponential growth phase of bacteria and production of RTX-toxins air passes through the medium, carbon dioxide content in air is above normal atmospheric level and is up to 10 % vol.

EFFECT: invention allows increasing Apxl or ApxIII toxins output, this may be used during vaccines production.

7 cl, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

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FIELD: biotechnologies.

SUBSTANCE: method involves cultivation in the appropriate conditions of yeast Saccharomyces cerevisiae and release of target protein; besides, release is directed with leader polypeptide, which has amino acid sequence SEQ ID NO1 and representing a variant of a pro-area of leader polypeptide of protein PpPIRl Pichia pastoris.

EFFECT: invention enlarges the range of methods for obtaining target protein in yeast Saccharomyces cerevisiae and increases possibilities for effective synthesis of such proteins.

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FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant human blood coagulability factor VIII with deletion of B-domain (hFVIII-BDD). Recombinant plasmid DNA pAP227 coding polypeptide with sequence hFVIII-BDD also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 2H5 producing recombinant hFVIII-BDD with highly stable yield at the level of about 20 IU/ml/24 h. Cultivation of cells-producers is performed in medium DME/F12 containing 2-4% of Fetal Bovine Serum, 1% of dimethylsulphoxide and 50 IU/l of insulin.

EFFECT: improvement of the method.

4 cl, 5 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.

EFFECT: improvement of the method.

5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly a polypeptide bearing a human BNP(1-32) epitope, for producing ligands that are directed against the human BNP(1-32) or human proBNP(1-108), where said polypeptide has the formula α1-R1-X1-FGRKMDR-X2-R22. Disclosed is use of said polypeptide to produce ligands directed against the human BNP(1-32) or human proBNP(1-108) and for producing a hybridoma which secretes a monoclonal antibody directed against the human BNP(1-32) or human proBNP(1-108). Disclosed is a method of producing a hybridoma which secretes a monoclonal antibody directed against the human BNP(1-32) or human proBNP(1-108), as well as the obtained hybridoma. Disclosed is a ligand which is specific to an epitope with the sequence FGRKMDR, as well as use thereof to detect human BNP(1-32) or human proBNP(1-108) in a biological sample. Disclosed are methods of detecting human BNP(1-32) or a human proBNP(1-108) derivative in a biological sample, a method for in vitro diagnosis, prediction, risk stratification or subsequent observation of long-term results of cardiac and/or vascular pathology in an individual, as well as a method for in vitro diagnosis of stroke in an individual using said ligand. Disclosed is a multi-epitope calibrator designed to obtain calibration curves for analysis of BNP(1-32), proBNP(1-108), as well as a kit for detecting human BNP(1-32) or human proBNP(1-108).

EFFECT: invention enables to efficiently detect cardiac and/or vascular pathologies in an individual.

15 cl, 17 dwg, 12 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a lymphotoxin-alpha (LTα) antibody, including chimeric and humanised versions thereof. Where are disclosed compositions containing it, using it for preparing a medication for autoimmune disorders, a method of inhibiting ex vivo lymphotoxin-alpha activated cell proliferation with using the antibody under the invention, as well as a nucleic acid, an expression vector, a host cell and a method of producing the antibody that proceeds from using them.

EFFECT: present invention can find further application in a therapy of the autoimmune diseases.

51 cl, 21 ex, 27 dwg, 7 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 2B8 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-20.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 5G6 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-19.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 2 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: osteons and intermediate lamellas are delineated and framed as members of analysis; osteocyte lacunae are numbered in each formed image; total number is presented, as well as a number of lacunae containing minerals in an amorphous phase. A percentage of the latter is described as a coefficient of osteocyte involvement in bone matrix mineralisation. If the minerals in the amorphous phase are found in less than 30% of lacunae, mineralisation activity of osteocyte is considered to be low. The presence of the amorphous minerals in 30-80% of lacunae shows moderate activity, and if more than 80% of lacunae contain the minerals in the amorphous phase, high activity is stated.

EFFECT: improved assessment accuracy.

1 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: what is presented is a hybrid cell strain of Mus musculus 10G4 deposited in the collection of microorganisms Federal State Research Institution State Research Centre for Applied Microbiology and Biotechnology, No. N-13.

EFFECT: strain may be used for producing monoclonal antibodies to capsular F1 antigen Yersinia pestis and is applicable for constructing the based plague agent test systems.

7 dwg, 4 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention is an isolated nucleic acid comprising a canine RNA polymerase I regulatory sequence and containing (i) at least 250 or at least 350 or at least 450 adjoining nucleotides or an entire nucleotide sequence, which is in form of SEQ ID NO:26, (ii) a nucleotide which is at least 80% identical to said nucleotide sequence (i) or includes a complementary or reverse complementary (i) or (ii) sequence. The nucleotide sequence (i) or (ii) is operably linked to cDNA which encodes influenza virus vRNA, and when produced in MDCK cells, is capable of directing expression of said influenza virus vRNA. The present invention also describes expression vectors and cells containing such nucleic acids, as well as methods of using such nucleic acids to obtain influenza viruses, including infectious influenza viruses.

EFFECT: canine plasmid rescue system pol I enables to obtain recombinant influenza viruses in a canine cell culture with high titre.

25 cl, 16 dwg, 7 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention describes Mus museums hybrid cultivated cell strains of Russian National Collection of Industrial Microorganisms H-117, Russian National Collection of Industrial Microorganisms H-116, Russian National Collection of Industrial Microorganisms H-115, Russian National Collection of Industrial Microorganisms H-113 and Russian National Collection of Industrial Microorganisms H-114 - producers of the monoclonal antibodies showing various specificity to human recombinant erythropoietin (EPO) and enabling distinguishing between a glycolised form of EPO and accompanying hypo- and deglycolised impurities. Estimating the strain efficacy and the produced antibody specificity is ensured by immune-enzyme assay with using a number of specificity markers: EPO recombinant protein; hypoglycolised EPO (o-glycolised) produced by natural antigen periodate oxidation; deglycolised EPO (de-EPO) produced by enzymatic treatment of recombinant EPO (PNGasa).

EFFECT: hybridoma produced monoclonal antibodies with said specificity are required for recovery of glycolised forms of EPO, and the quantitative and qualitative analysis of EPO mixtures.

5 cl, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: there is constructed a recombinant plasmid DNA pAP271 containing a rhFVII protein gene, a MAR matrix attachment region of an avian lysozyme gene, CMV virus transcription amplifier, a promoter of translational factor of human EF-1α elongation, an internal site of translational initiation of encephalomyocarditis IRES virus, a mouse DHFR gene, a SV40 virus polyadenylation signal, a cartridge comprising all elements required for aminoglycoside-3'-phosphotransferase (APH) gene expression, a cartridge for expression in bacterial cells of β-lactamase gene, as well as unique recognition sites of the following restriction endonucleases: Agel (850 base pairs), BbvCI (1657 base pairs), BmgBI (4202 base pairs), BssHII (6672 base pairs), Eco47III (11269 base pairs), EcoRI (10929 base pairs), EcoRV (11863 base pairs), Fsel (1455 base pairs), NotI (4812 base pairs), RsrII (6790 base pairs), Sbfl (2330 base pairs), Sfil (6027 base pairs), Tthllll (6390 base pairs), Xcml (2404 base pairs).

EFFECT: presented invention provides producing stably high-yield recombinant human blood coagulability factor VII.

2 cl, 4 dwg, 2 ex

Fused partner cell // 2431667

FIELD: medicine.

SUBSTANCE: invention describes fused partner cells making it possible to produce heterohybridomas of cells of biological species different from a mouse. Heterohybridomas are produced by fused myeloma cells produced from an animal of a first biological species with leukemia cells produced from an animal of a second biological species which have an additional S-phase in a cell cycle and exhibit a diploidisation property. The fusion partner cell SPYMEG is deposited, No. FERM BP-10761 and can be used for hybridoma production. What is described is hybridoma producing antibodies produced by fusion of fusion partner cell and a third cell with antibody-producing ability. The invention describes methods for producing a fusion partner cell, a hybridoma and an antibody-producing cell, and also methods for producing antibodies. Use of the invention provides stable and simple production of the substances with the use of hybridomas in a wide range of species of animals.

EFFECT: hybridomas stably keep a phenotype throughout the whole process of cloning.

20 cl, 10 dwg, 2 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: inventions relate to the field of immunology. Claimed are a single-chain antibody, specific to a carcinoembryonic antigen, a chimeric mononuclear T-cell receptor, a vector, a host cell and a method of diagnostics or treatment of diseases, characterised by the presence of antigens, capable of binding with the chimeric receptor. Described is a genetic construction, coding chimeric monomolecular T-cell receptors, in which an effector fragment of the T-cell receptor is combined with an antigen-recognising part, which represents variable fragments of two different antibodies to the carcinoembryonic antigen (CEA).

EFFECT: claimed inventions can be used in T-cell cancer therapy.

7 cl, 4 dwg, 3 ex, 1 tbl

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