Improved expression vectors of mammals and their use

FIELD: biotechnologies.

SUBSTANCE: expression vector includes: (a) replication origin OriP obtained from Epstein-Barr virus (EBV), where replication origin contains: 1) symmetry element of the second order (DS); and 2) duplication section (FR) that contains fixation point EBNA; (b) replication origin SV40; (c) insertion section for inserting a gene of concern; (d) promoter EF-1b functionally bound to the insertion section; (e) poly-A signal; (f) bacterial replication origin; (g) selected marker; and unnecessarily containing (h) sequence of nucleic acid, which codes constant area of heavy or light chain of antibody, which is functionally bound to the insertion section. With that, replication origin OriP is bound to an initiation factor of replication EBNA 1, which acts from outside and is not coded with an expression vector.

EFFECT: use of an expression vector in an extracted host cell, a set and a method for obtaining recombinant protein provides production of abundant protein expression.

26 cl, 25 dwg, 3 tbl, 4 ex

 

Related applications

This application claims the priority of provisional application U.S. No. 61/021282, filed January 15, 2008, and provisional application U.S. No. 61/104546, filed October 10, 2008, the contents of which are incorporated in this description by reference in its entirety.

The prior art inventions

Stable production of proteins, including the production of biological products, can be achieved by transfection of host cells with vectors containing DNA encoding the protein. Save the vector in the cell line may provide a number of ways, including extrachromosomal replication using episomal of original replication. Episomal vectors contain an origin of replication, which initiates replication of the vector after associating a sequence with a factor of replication initiation. Episomal vectors have several advantages compared with vectors, which require the introduction into the host genome. For example, the use of episomal vectors can reduce the phenotypic changes resulting from integration of the vector into the host genome. In addition, episomal vectors can be extracted from transfected cells using standard methods of DNA extraction.

With the development of the significance of therapeutic proteins, i.e. biologics, it is necessary to undertake efforts op is imitatie production of proteins while increasing the overall efficiency of the production process. Thus, the efficiency must be weighed against the performance vector for the production of one protein. There is a need for improved expression systems, which provide conditions for efficient cloning and high levels of the target protein product. It is preferable to reduce the number of stages of the cloning process of production of biological products, in particular antibodies, since it allows to reduce the amount of time and minimize cost. It is also preferable to use vectors that provide satisfactory products of protein in small-scale and large-scale cell cultures. The present invention is directed to overcoming the shortcomings associated with conventional vectors, by using additional advantages that will become apparent from the following detailed description.

The invention

Recombinant proteins can be obtained by transient transfection of mammalian cells, in particular, in the process of developing pharmaceuticals. For protein expression, you can use a number of host cells, including mammalian cells such as COS cells and embryonic stem cells human kidney (HEK). Activity episomal vectors due to the presence of the origin of replication and acting the th external factor of replication initiation, that is associated with the origin of replication. Factors of replication initiation, such as nuclear antigen of Epstein-Barr (EBNA), which binds to OriP of Epstein-Barr, can be cloned into the episomal vector or, alternatively, it is possible to Express in the cell-master, transtitional this vector. Thus, episomal vectors can be specific for certain cell lines, which Express the current external factor required for activation of replication origin of replication.

The present invention eliminates the need for different skeletons episomal vectors used for expression of recombinant proteins. The present invention provides episomal vectors containing at least two different episomal origin of replication that allow you to use the same vector for protein expression in different cell types. Different origin replication allow you to use the vector in different types of mammalian cells, which provide the necessary operating outside factors replication and allow for replication of the vector. By eliminating the need to re-clone the genes of interest in obtaining protein present invention improves efficiency and reduces cost by reducing the number of vectors, keeping p and this same level of production of the protein. An unexpected aspect of the present invention is that the addition of nucleotides to the vector, i.e. the second origin of replication, there is a negative impact on the ability of the vector to produce the protein at the desired level.

In a preferred embodiment, the vectors of this invention contain a constant areas of heavy or light chain. Therefore, variable plot heavy or light chain can be cloned into the vector above constant plot heavy or light chain, respectively, further improving the efficiency of the expression system. Episomal vectors provide efficient production of the protein in mammalian cells expressing the SV40 T Ag or nuclear antigen of Epstein-Barr (e.g., COS7 cells or HEK293-6E).

The present invention provides a combination of elements, optimum for high protein yield, high production efficiency and low cost, and these elements play an important role in the production of protein, especially in the pharmaceutical industry and in obtaining biological proteins such as antibodies. Other characteristics and advantages of the present invention described in the detailed description and the claims below.

In one aspect the invention provides an expression vector, provided the: (a) origin of replication, OriP, derived from Epstein-Barr (EBV); (b) the origin of replication of SV40; (c) plot of insertion to insert a gene of interest; and (d) a nucleotide sequence encoding a constant plot heavy or light chain antibodies, functionally associated with the site of insertion. In one embodiment of interest, the gene encodes variable plot heavy or light chain of the antibody, for example, variable plot heavy or light chain of mouse, gumanitarnogo, chimeric or human antibodies. In a specific embodiment, the flexible section of the heavy chain antibody is a variable plot heavy chain antibodies selected from the group consisting of adalimumab ABT-325 and ABT-874. In another specific embodiment, the variable area light chain antibody is a variable area light chain antibodies selected from the group consisting of adalimumab, ABT-325 and ABT-874. Constant plot heavy chains can be obtained, for example, from mouse, gumanitarnogo, chimeric or human antibodies, and can be a constant site of the heavy chain antibody is selected from the group consisting of gamma 1, z, a; gamma 1, z, non-a; gamma 2, n+; gamma 2, n-; and gamma 4. Constant plot heavy chain antibodies gamma 1, z, non-a may optionally with erati alanine mutation at position 234 const plot heavy chain. In another embodiment, const plot heavy chain antibodies gamma 1, z, non-a may further comprise alanine mutation at position 235 or 237 const plot heavy chain antibodies.

In one embodiment, the constant-area light chain of the antibody belongs to the Kappa-isotype human chain or lambda isotype human chain. In one embodiment, const plot heavy chain antibody refers to gamma 1 isotype mouse chain or gamma 2a the isotype mouse chain. In another embodiment, the constant-area light chain of the antibody belongs to the Kappa-isotype mouse chain. In one embodiment, const plot heavy chain antibody is an Fc domain. In one embodiment, variable plot heavy or light chain of the antibody is located in the 5'direction relative to the site of insertion.

In one embodiment, the expression vector further comprises a promoter functionally linked with the site of insertion, where the promoter is either a promoter EF-1α, or the promoter of cytomegalovirus (CMV).

In one embodiment, the expression vector further comprises a breeding marker such as a gene for resistance to ampicillin.

In one embodiment, the CMV promoter contains nucleate the percent sequence, which, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide 1-608 sequence SEQ ID NO:1. In a specific embodiment, the CMV promoter contains nucleotides 1-608 sequence SEQ ID NO:1.

In one embodiment, the promoter EF-1α is a human. In one embodiment, the promoter EF-1α contains a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide 76-1267 sequence SEQ ID NO:2. In a specific embodiment, the promoter EF-1α contains nucleotides 76-1267 sequence SEQ ID NO:2.

In one embodiment, the origin of replication OriP contains a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide 1795-3545 sequence SEQ ID NO:1.

In one embodiment, the origin of replication of SV40 contains a nucleotide sequence which is less than the least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide 5834-6140 sequence SEQ ID NO:1. In a specific embodiment, the origin of replication of SV40 contains nucleotides 5834-6140 sequence SEQ ID NO:1.

A typical expression vector of the present invention contains a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence selected from the group consisting of sequences SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32. In specific embodiments, the implementation of the expression vector contains the nucleotide sequence selected from the group consisting of sequences SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32.

The expression vectors of the present invention is also shown in figure 1, 2 and 14-25. Other vectors of the present invention depicted in Fig-13.

In another aspect, the invention provides cell-host mammal containing the vector of the present invention. As a host cell of a mammal, you can use the tapped COS, such as COS cell 7, or embryonic cell of the human kidney (HEK), such as cell HEK-293.

In another aspect the invention provides a set containing the vector of the present invention.

In another aspect the invention provides a method of obtaining a recombinant protein, comprising introducing the expression vector of the present invention in cells of the host mammal, culturing the host cell of the mammal in suitable conditions ensuring the expression of the protein and isolating the protein.

In another aspect the invention provides an expression vector containing the nucleotide sequence encoding a signal peptide. In one embodiment, gene of interest functionally linked to a nucleic acid that encodes a signal peptide.

Brief description of figures

The above and other objectives, features and advantages of the present invention, and the invention are explained more fully in the following detailed description of preferred embodiments in conjunction with the accompanying figures, in which:

Figure 1 shows a map of the empty vector pHyb-C. elements of the vectors include eukaryotic origin of replication of SV40, eukaryotic expression the promoter of cytomegalovirus (pCMV), a three-membered leader sequence (TPL), donor site splicing (D), major late adenovirus enhancer (enh MLP), acceptor site of splicing (SA), plot open reading frame (ORF)containing the gene of interest with the subsequent signal poly-A (pA), the element of symmetry of the second order (DS), eukaryotic origin of replication of Epstein-Barr (OriP), a site duplications (FR), a marker of resistance to ampicillin (AmpR) and a bacterial origin of replication (pMB1ori).

Figure 2 shows a map of the empty vector pHyb-e vector Elements include eukaryotic origin of replication of SV40, the eukaryotic promoter EF-1a, plot open reading frame (ORF)containing the gene of interest with the subsequent signal poly A (pA), the element of symmetry of the second order (DS), eukaryotic origin of replication of Epstein-Barr (OriP), a site duplications (FR), a marker of resistance to ampicillin (AmpR) and a bacterial origin of replication (pMB1ori).

Figure 3 shows the titers of recombinant Fc hybrid protein produced by cells of COS, transfitsirovannykh pBOS vectors, pTT3, pHybC and pHybE using the electroporation method.

Figure 4 shows the titers of recombinant Fc hybrid protein produced by cells HEK-293-6E, transfitsirovannykh pBOS vectors, pTT3, pHybC and pHybE using the PEI method.

Figure 5 shows the titers of antibodies produced by HEK-293-6E, transfitsirovannykh using the method of PEI is cerami pBOS, pTT3, pHybC and pHybE ensuring the expression of IgG antibodies.

Figure 6 shows the titers of antibodies produced by cells of COS, transfitsirovannykh using the method of electroporation pBOS vectors, pTT3, pHybC and pHybE ensuring the expression of IgG antibodies.

7 shows the titers of antibodies produced by cells of COS, transfitsirovannykh using the method of electroporation vectors pHyb-E-SwaI (v1) or pHyb-E (v2), which provides the expression of IgG antibodies.

On pig show a map of vector pHybC-mBR3-mCg2a (also called "pHybC-mBR3-Fc").

Figure 9 shows a map of pHybE vector-mBR3-mCg2a (also called "pHybE-mBR3-Fc").

Figure 10 shows a map of vector pHybC-E7-hCk (also called "pHybC-E7").

Figure 11 shows a map of vector pHybC-D2-hCg1,z,a (also called "pHybC-D2").

On Fig displayed a map of pHybE vector-D2-hCg1,z,a (also called "pHybE-D2").

On Fig displayed a map of pHybE vector-E7-hCk (also called "pHybE-E7").

On pig show a map of pHybE-hCg1,z,a, V2 (also called "pJP182").

On pig show a map of pHybE-hCgl,z,non-a V2 (also called "pJP183").

On pig show a map of pHybE-hCg1,z,non-a,mut(234,235) V2 (also called "pJP184").

On pig show a map of pHybE-hCg1,z,non-a,mut (234,237) V2 (also called "pJP185").

On pig show a map of pHybE-hCg2,n+ V2 (also called "pJP186").

On pig show a map of pHybE-hCg2,n - V2 (also called the th "pJP187").

On pig show a map of pHybE-hCg4 V2 (also called "pJP188").

On pig show a map of pHybE-mCg1 V2 (also called "pJP189").

On pig show a map of pHybE-mCg2a V2 (also called "pJP190").

On pig show a map of pHybE-hCk V2 (also called "pJP191").

On pig show a map of pHybE-hCl V2 (also called "pJP192").

On pig show a map of pHybE-mCk V2 (also called "pJP193").

Detailed description of the invention

I. Definitions

To facilitate understanding of the present invention, in the present description first provides definitions for certain terms.

The term "nucleic acid" or "nucleic acid molecule", as used herein, refers to DNA, RNA, mRNA, cDNA, genomic DNA and their analogues. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is a double-stranded DNA. The nucleic acid may be isolated or integrated into another nucleic acid molecule, such as an expression vector or a eukaryotic chromosome of the host cell.

"Isolated" nucleic acid molecule is a molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, in relation to genomic DNA, the term "isolated" includes molecules NUS is einwich acids, separated from the chromosome with which the genomic DNA is associated in nature. Preferably, an "isolated" nucleic acid does not contain sequences that are in the nature flank the nucleic acid (i.e. adjacent to the 5'- and 3'-ends of the nucleic acid) in the genomic DNA of the organism from which the obtained nucleic acid. Moreover, an "isolated" nucleic acid molecule such as a cDNA molecule, essentially does not contain other cellular substances or culture medium in the case of obtaining recombinant methods, essentially does not contain chemical precursors or other chemicals in the case of obtaining chemical synthesis.

The terms "recombinant vector" or "vector" in this description are used interchangeably and refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. One type of vector is a "plasmid", which is a circular double-stranded loop of DNA, which can be legirovanyh other segments of DNA. Alternatively, the vector may be linear. Another type of vector is a viral vector, allowing ligitamate other segments of DNA in the viral genome. Some vectors can autonomously replicate in the cell host into which they are introduced (e.g., bacterial, vecto is s, contains a bacterial origin of replication and episomal vectors mammals). Other vectors (e.g., episomal vectors mammals) can be integrated into the genome of the host cell and, therefore, be replicated together with the genome of the host. In a preferred embodiment, the vectors of this invention are episomal vectors mammals. The term "design" in this description also applies to the vector.

Certain vectors are capable of driving the expression of genes with which they are functionally linked. "Expression vector" or "recombinant expression vector" is a nucleic acid molecule that encodes a gene expressed in a cell-master, and, in addition, contains the necessary elements to control gene expression. Typically, the expression vector contains a promoter transcription, gene of interest and a transcription terminator. Gene expression is usually under the control of the promoter, and in this case we say that the gene is functionally linked to the promoter. Similarly, a regulatory element and the main promoter called functionally related if a regulatory element modulates the activity of the core promoter. In one embodiment, the expression vector of the present invention contains multiple origin is in replication that allows not to limit the application of the vector by one cell type.

Used in this description, the term "episomal can replicate vector" or "episomal vector" refers to a vector, which is typically and preferably does not integrate into the genome of the host cell, and exist in parallel. Episomal can replicate the vector, in accordance with the present description, is replicated during the cell cycle, and copies of the vector resulting from this replication, distributed in new cells statistically, depending on the number of copies present before and after cell division. Preferably, episomal can replicate the vector is in the kernel of the host cell, and preferably is replicated during the S phase of the cell cycle. In addition, episomal can replicate the vector is replicated at least once, i.e. one or more times, in the nucleus of the host cell during the S-phase of the cell cycle. In a more preferred embodiment, episomal can replicate the vector is replicated once in the nucleus of the host cell during the S-phase of the cell cycle.

In this description, the terms "sequence, origin of replication" or "origin of replication" is used interchangeably to denote sequences, which, if present in the vector, the trigger is aplikazio. The origin of replication may be recognized factor in the initiation of replication or, alternatively, a DNA helicase.

Used in this description, the term "recombination" refers to the process by which the substance is nucleic acid, such as DNA, is distributed between the two nucleic acid molecules, for example, in the microorganism. Used in this description, the term "homologous recombination" refers to the process by which the substance is nucleic acid is distributed between two molecules of nucleic acids through the sections or segments of homology sequences or, preferably, identical sequences (e.g., having a high degree of sequence identity). In illustrative embodiments, the implementation of the substance nucleic acid is located on the chromosome or the formation of the episome of the microorganism. In other illustrative embodiments, the implementation of the substance nucleic acid is outside the chromosome, for example, on the plasmid. Recombination can occur between a linear and/or circular DNA molecules.

Used in this description, the term "gene of interest" refers to an exogenous DNA sequence, which is added to the vector of the present invention. Interest gene, for example, may contain the serial encoding gnosti, which may be either separated by introns, or which may be a cDNA encoding the open reading frame. The term "gene of interest", as used herein, refers to a DNA sequence, which is added to the vector of the present invention for the ultimate expression of the protein. Plot vector, in which the cloned gene of interest, in this description referred to as "the site of insertion". Preferably, the gene of interest contains a sequence encoding a fragment of the antibody or hybrid protein, which Express using the vector of the present invention. For example, a sequence encoding a variable plot heavy chain antibody adalimumab, i.e. interest gene clone in the vector of the present invention, which contains a constant site of the heavy chain.

In one embodiment of the present invention, the vector contains the sequence encoding the constant-area light or heavy chain antibodies, which is located in the 3'direction with respect to the site of insertion of gene of interest functionally linked with it. Thus, in one embodiment of interest, the gene encodes a variable area light or heavy chain antibodies and functionally associated with the sequence, the code is highlighted constant area light or heavy chain antibodies, which is part of the vector of the present invention.

The nucleotide sequence is "operatively linked" with another nucleotide sequence if it is a functional dependency from it. For example, DNA encoding a signal peptide, the functionally linked to DNA that encodes a protein or polypeptide, if the expression of the sequence encoding the signal peptide in the same reading frame with the protein or polypeptide. Similarly, the promoter or enhancer functionally linked to a nucleotide sequence that encodes a protein or polypeptide, if the expression of the protein or polypeptide is regulated by a specified promoter or enhancer. In one embodiment, functionally linked to a nucleotide sequence adjacent to each other (for example, in the case of signal sequences). Alternatively, functionally related nucleotide sequences may not be adjacent to each other (for example, in the case enhancers). In one embodiment, the nucleotide sequence encoding the constant-area light or heavy chain antibodies, functionally connected with the gene of interest, for example, coded variable plot heavy or light chain.

The term "promoter" includes any nucleotide follower of the spine, sufficient to control transcription in eukaryotic cells, including inducible promoters, repressed promoters and constitutive promoters. Typically, the promoter includes elements sufficient to ensure control over the expression driven by the promoter of a gene in a cell-specific, Dane-specific or time-dependent manner, or for induction under the influence of external signals or factors. Such elements may be located on the 5'- or 3'-ends, or in the regions of the sequence of the intron-specific gene. Usually use the constitutive expression of a gene, although if necessary, in the present invention can be used in regulated promoters. Gene expression can also be controlled by the regulation of transcription under the action of heat, light or metals, for example, using the genes of metallothionine or genes heat shock protein.

The terms "above in the course of reading and following along reading" refer to the relative location of the two elements present in the nucleotide sequence or vector. The element that is above in the course of reading"than the other, is located closer to the 5'-end sequence (i.e. closer to the end of the molecule, which contains a phosphate group attached to the 5'-carbon atom ribosome or deoxyribose skeleton, if forefront of the La is linear), than another element. They say that the item is below during reading", if it is located closer to the 3'-end sequence (i.e. closer to the end of the molecule, which contains a hydroxyl group attached to the 3'-carbon atom ribosome or deoxyribose skeleton linear molecules)than the other element.

In this description, the term "odd sequence" refers to nucleotide sequences, preferably contained in the vector, which planiruetsja areas enzymatic restriction on the 5'- and 3'-ends. Extra sequence is the vector plot of the insertion of a nucleic acid that encodes a gene of interest. In the cloning process extra sequence cut from the vector using the appropriate restriction enzymes, then the vector in place of the extra sequences are ligated or inserted by homologous recombination nucleic acid encoding a gene of interest. Preferably, once the sequence is large enough to provide sufficient distance between the 5'- and 3'sites of enzymatic restriction, allowing the enzymes to effectively cut the vector. In addition, the extra length sequence preferably differs from the length of nucleic acid that encodes a p is establishe the gene of interest, for example, extra sequence, the length of which is approximately 300 base pairs or less, or about 400 base pairs or more, can be used to insert a nucleic acid that encodes a gene of interest, the length of which is approximately 350 base pairs. In another embodiment, the size of the extra sequence is about 1 so

The term "recombinant a host cell" (or simply "a host cell") in this description is used to refer to cells in which the introduced recombinant expression vector. It should be understood that such terms are intended to denote not only the concrete investigated cells, but also to the progeny of such cells. Because in subsequent generations may occur some changes due to mutations or environmental influences, such progeny may in fact be identical to the parent cell, but it is still included in the scope of the term "a host cell", as used in this description.

The term "antibody" in accordance with this description includes whole antibodies and any antigen binding (i.e., "antigen-binding fragment") or single-stranded fragments. "Antibody" is a glycoprotein containing at least two heavy (H) chains and two light (L) chain, the United disulfi the governmental relations, or antigen-binding fragment. Each heavy chain consists of a variable segment of the heavy chain (referred to in this description VHand constant plot heavy chain. Constant plot heavy chain consists of three domains, CH1, CH2 and CH3. Each light chain consists of a variable area light chain (referred to in this description VLand constant area light chain. Constant area light chain consists of one domain, CL. Plots of VHand VLcan be further subdivided into areas of hypervariability, called plots, complementarity determining (CDR), interspersed more conservative areas, which are called frame sections (FR). Each VHand VLconsists of three CDRs and four FR located from amino end to the carboxy-end, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Variable areas of heavy and light chains contain a binding domain that interacts with the antigen. The combination of the six CDR VHand VLforms the antigen-binding site. If the antibody consists of two H chains and two L chains, it may contain two identical antigen-binding site, two different antigen-binding site, which bind the same antigen, or two antigen-binding site that svyazyvaetsya antigens. The constant parts of the antibodies may mediate the binding of immunoglobulin with tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"in this description refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., IL-1α, IL-1β). The antigen-binding function of an antibody can be performed by fragments of a full-sized antibodies. Examples of binding fragments included in the scope of the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of domains VLVH, CL and CH1; (ii) the fragment F(ab')2, a bivalent fragment comprising two Fab fragment linked by a disulfide bridge at the hinge area; (iii) a Fd fragment consisting of domains VHand CH1; (iv) a Fv fragment consisting of domains VLand VHone shoulder antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of domain VHor VL; and (vi) selected hypervariable segment (CDR). In addition, although the two domains of the Fv fragment, VLand VHthat are encoded by different genes, they can be combined recombinant methods importantmessage linker, which allows to obtain a single-chain protein, which plots the VLand VHconnected with the formation of monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also included in the scope of the term "antigen-binding fragment" of an antibody. These antibody fragments obtained by traditional methods known to experts in this field, is subjected to the screening efficiency in the same manner as are intact antibodies. In one embodiment of this invention, the antibody fragment selected from the group consisting of Fab, Fd, Fd', single-chain Fv (scFv), scFvaand a domain antibody (dAb).

In addition, the antibody or antigen-binding fragment may be part of larger molecules immunoadhesin formed by covalent or non-covalent Association of the antibody or antibody fragment with one or more other proteins or peptides. Examples of such molecules immunoadhesin include tetramer molecules scFv obtained using streptavidine Central plot (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101)and bivalent and biotinylated scFv molecules obtained using a cysteine residue, a marker peptide and a C-terminal polyhistidine the marker (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody fragments, such as fragments of the Fc, Fab and F(ab')2you can get from whole antibodies by conventional methods, such as cleavage by papain or pepsin, respectively. Moreover, antibodies, antibody fragments and molecules immunoadhesin can be obtained using standard methods of recombinant DNA.

The term "domain" refers to a stacked structure of a protein, which retains the tertiary structure independently from the rest of the protein. Typically, domains are responsible for specific functional properties of proteins and can often be added, deleted or moved to other proteins without loss function the rest of the protein and/or domain. Under single variable domain antibodies implied laid polypeptide domain comprising sequences typical for the variable domains of antibodies. Therefore, it includes the variable domains of whole antibodies and modified variable domains, for example, in which one or more loops are replaced by sequences which are not typical for the variable domains of antibodies, or the variable domains of the antibodies that have been shortened, or which contain N - or C-terminal additions, and laid the fragments of the variable domains, which remain, at least partly, the binding activity and specificity of the full-domain.

The variable domains of the present invention can be combined with obtaining the set of domains; for example, can be combined in complementary domains, such as domains VLand domains VH. Can also be combined not complementary domains, for example, the domain of VHand a second domain VH. Domains can be combined with a number of ways including connection domains covalent or non-covalent bonds.

The term "dAb" or "domain antibody" refers to a polypeptide representing a single variable domain antibodies (VHor VL), which specifically binds the antigen. In one embodiment, the vector of the present invention is used for the expression of dAb.

The phrase "recombinant antibody" refers to antibodies that receive, Express, create or produce, using recombinant methods, such as antibodies expressed using a recombinant expression vector, transfitsirovannykh in the cell host, antibodies isolated from a recombinant, combinatorial libraries of antibodies, antibodies isolated from an animal (e.g. a mouse)that is transgenic, because it carries the genes of the human immunoglobulin (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies that receive, Express, create or select a using l is any other ways including a connection sequence of the gene-specific immunoglobulin (for example, the gene sequence of a human immunoglobulin) with other DNA sequences. Examples of recombinant antibodies include chimeric, CDR-grafted, and humanized antibodies.

The term "human antibody" refers to antibodies that contain variable and constant regions with the sequences of human embryonic immunoglobulin, as described, for example, Kabat et al. (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), or derived from them. However, the human antibodies of the present invention may contain amino acid residues that are not encoded by the sequences of human embryonic immunoglobulin (e.g., mutant residues, introduced by nonspecific or specific mutagenesis in vitro or by somatic mutation in vivo), for example, CDR, in particular, in CDR3.

Recombinant human antibodies of the present invention contain variable parts and possibly constant plots obtained from sequences of human embryonic immunoglobulin (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, in some embodiments, the implementation is such recombinant human antibodies are subjected to in vitro mutagenesis (or, if you use an animal transgenic for human sequences Ig somatic mutagenesis in vivo) obtaining amino acid sequence of sections VHand VLrecombinant antibodies, which, though derived from human embryonic sequences VHand VLand are related to them, may not exist in the natural set of human embryonic antibodies in vivo. However, in some embodiments, the implementation of such recombinant antibodies are formed as a result of selective mutagenesis or reverse mutation, or both.

The term "reverse mutation" refers to a process in which some or all of the amino acids introduced into the human antibody in the result of somatic mutations, are replaced with the corresponding residues from homologous sequences embryonic antibodies. Sequences of the heavy and light chains of a human antibody of the present invention is compared separately with embryonic sequences from the VBASE database and identify the sequence with the highest homology. Differences between human antibody of the present invention and embryonic sequence treated by mutations of nucleotides at certain positions that encode amino acids that cause such various spare parts of the shafts. Then each amino acid is identified so as a candidate for reverse mutations, are examined to determine whether it is directly or indirectly involved in the binding to the antigen, and amino acids, after which mutations affect any of the desirable characteristics of human antibodies that are not included in the final human antibodies. To minimize the number of amino acids that are subject to the reverse mutation, you can leave the amino acids that differ from the amino acids in the relevant provisions of the nearest embryonic sequence but identical to the corresponding amino acids of the second embryonic sequence, provided that the second embryonic sequence identical to and collinear sequence of the human antibody of the present invention, at least 10, preferably 12 amino acids on both sides of the studied amino acids. Reverse mutation can be performed at any stage of the optimization antibodies.

The term "chimeric antibody" refers to antibodies that contain sequences of variable regions of heavy and light chains of antibodies derived from one species and the constant sequence of plots from other species, such as antibodies, containing mouse variable parts are heavy and agcih circuits, United with human constant regions.

The term "CDR-grafted antibody" refers to antibodies that contain sequences of variable regions of heavy and light chain from one species but in which the sequences of one or more areas CDR VHand/or VLreplaced by a CDR sequence from other species, such as antibodies containing a variable plots murine heavy and light chains, in which one or more murine CDRs (e.g., CDR3) replaced by human CDR sequence.

The term "humanitariannet antibody" refers to antibodies that contain sequences of variable regions of heavy and light chains of the non-human species (e.g., a mouse), but in which at least part of the sequence VHand/or VLmodified to be more similar to the human sequence, i.e. more similar to human embryonic variable sequence. One of the types gumanitarnogo antibody is a CDR-grafted antibody in which human CDR sequence introduced into nonhuman sequence VHand VLinstead of the corresponding nonhuman CDR sequences.

In this description, the terms "connected", "heriditary" or "hybrid" is used it is to be interchangeable. These terms refer to the combination of two or more elements or components using any methods, including chemical conjugation or recombinant methods. The terms "hybridization in the frame read" or "functionally linked" refers to the combination of two or more open reading frames (ORFS) with a more solid long ORF to maintain the correct reading frame of the original ORF. Thus, the obtained recombinant hybrid protein is a single protein that contains two or more segments corresponding to the polypeptides encoded by the original ORF (these segments in nature are usually not connected). Although the thus obtained reading frame is continuous throughout hybridized segments, these segments can be divided physically or spatially, for example, is contained in the frame reading of the linker sequence.

In this description, the term "site of Fc" includes an amino acid sequence derived from the constant plot heavy chain antibodies. In some embodiments, the implementation of the section Fc includes a polypeptide containing a constant site of an antibody excluding the first constant domain of the site of the immunoglobulin.

Section Fc may represent functionally equival ntny similar plot Fc. Functionally equivalent analogue section Fc may be a variant of the plot of Fc containing one or more amino acid modifications compared with a plot of Fc wild-type or natural area Fc. In some embodiments option plot Fc, at least 50% homologous to the natural area Fc, preferably, 80%-99%, including at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%. Functionally equivalent analogues plot Fc can contain one or more amino acid residues added or deleted at the N - or C-end of the protein, preferably not more than 30, most preferably, not more than 10. Functionally equivalent analogues plot Fc include lots Fc, functionally associated with the partner hybridization.

Used in this description, the terms "hybrid Fc" or "Fc hybrid protein" includes a protein in which one or more proteins, polypeptides or small molecules functionally connected with a plot of Fc or its derivatives. The term "hybrid Fc" in this description is used as a synonym for terms such as "hybrid Ig", "Ig Chimera", and "receptor globulin" (sometimes with dashes)used in the prior art technology is key (Chamow et al, 1996, Trends Biotechnol. 14:52-60; Ashkenazi et al, 1997, Curr Opin. Immunol. 9:195-200). Hybrid Fc contains one or more plots Fc immunoglobulin or their variants, and partner hybridization, which generally represents any protein, polypeptide, peptide or small molecule. In some embodiments, the implementation is different from the Fc part of the hybrid Fc, i.e. partner hybridization, can be used for mediating binding of the target, and therefore, it may be functionally similar to the variable portions of an antibody.

In the present invention for covalent connection of Fc polypeptides with a partner hybridization or conjugation, or to obtain hybrid Fc, you can use a number of linkers. In this description, the terms "linker", "linker sequence", "spacer", "connecting sequence" or their equivalents refer to a molecule or group of molecules (such as a monomer or polymer)that connect two molecules and can serve to create a preferred configuration of the two molecules. For covalent molecular compounds can be used several ways. Such methods include, without limitation, education polypeptide linkages between N - and C-ends of proteins or protein domains, the connection via a disulfide bond and connection through chemical perekrestnolistaja reagents.

II. The vectors of this and is gaining

The invention provides episomal vectors for protein expression in the cells of the host mammal. The vectors of this invention is based on the use of two episomal of original replication, which allow you to use the vector in any cell line containing the factors of replication initiation, acting from outside on any origin of replication. Although the vector can also contain a factor of replication initiation, which binds the origin of replication, in the preferred embodiment, operating outside the replication factor is the host-cell. In addition, in one embodiment, the vectors of this invention are effective in obtaining antibodies and Fc hybrid proteins as vectors contain a constant areas of heavy or light chains that are functionally related gene of interest. Examples of the vectors of the present invention is shown in figures 1, 2 and 8-25. In addition, the typical sequence of vectors represented in SEQ ID NO:1-32. Figures 1 and 2 (respectively SEQ ID NO:1 and 2) shows the "open" vector, i.e. the vector of the present invention, which does not contain a constant areas of heavy or light chains and gene of interest. On Fig-25 shows a map of vectors of this invention which also contain different mouse or human constant plots and castke cloning gene of interest.

The vector of the present invention contains at least two different origin of replication, for example, the origin of replication, OriP, derived from Epstein-Barr (EBV), and the origin of replication of SV40. The origin of replication may be derived from a DNA virus, more preferably, a DNA virus capable of episomal replication, and includes originy replication, obtained, for example, Epstein-Barr, herpes simplex virus, herpes virus Saimiri, murine Gammaherpesvirus 68, human cytomegalovirus, mouse cytomegalovirus, virus pseudoleskeella, monkey virus 40, virus polyoma, human BK virus, human papilloma virus of cattle and adeno-associated virus.

In one embodiment, use of the origin of replication of Epstein-Barr, for example, oriP, or its functional fragments (examples of functional fragments of the origin of replication of Epstein-Barr described in Aiyar et al. (1998) EMBO Journal, 17:6394). The origin of replication of Epstein-Barr (OriP) consists of 2 main elements, several CIS-acting elements that provide DNA synthesis in the cell, and the element of the maintenance of the virus. The first of two main elements contains the family of repeats (FR), which contains plots of the binding of EBNA (shown in figure 1 and 2). EBNA is a factor of replication initiation, which initiates R is plicatio vector through OriP (access number sequence EBNA in Genbank V01555 (gi:94734074)). The second element that is part of OriP contains the so-called symmetry element of the second order (DS), which functions as an element of recognition of the origin of replication. Typically, the elements DS and FR are separated in the space of a few base pairs, typically 1000 BP Relative orientation OriP, in particular, DS and FR can be changed without affecting the function of OriP. The orientation of OriP, in particular, DS and FR, relative to other elements on the expression vectors of this invention can be changed without affecting the function of OriP. In a preferred embodiment of the present invention, where the origin of replication is an origin of replication of Epstein-Barr (OriP), and where OriP contains the family of repeats (FR) and a symmetry element of the second order (DS), DS is located between the gene of interest and an element of FR. In one embodiment, the vector of the present invention contains the origin of replication OriP (Epstein-Barr), comprising nucleotides 1795-3545 sequence SEQ ID NO:1, or a sequence that is identical to her 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

In another embodiment, the vector contains the origin of replication of SV40. To initiate replication of the vector through the origin of replication of SV40 (monkey virus 40) (indicated, for example, figures 1 and 2 as SV40 Ori") requires the presence of one viruses the protein, large T-antigen. The origin of replication of SV40 can be used in episomal vectors for the replication and maintenance of these vectors (see Calos (1996) Trends in Genetics 12:462; Harrison et al (1994) J Virol. 68:1913; Cooper et al (1997) PNAS 94:6450; and Ascenziono et al. (1997) Cancer Lett 118:135). In one embodiment, the vector of the present invention contains the origin of replication of SV40, including nucleotides 5834-6140 sequence SEQ ID NO:1, or a sequence that is identical to her 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

Functional variants described in this document the origin of replication is also included in the scope of determining the origin of replication in accordance with the present application.

In addition episomal origin of replication, the vector of this invention may also contain an origin of replication that provides replication of the vector in bacteria. An example of such origin, shown in figures 1 and 2, includes, without limitation, pMB1 ori, which functions in E. coli.

The vector of this invention can also contain breeding marker. Marker selection can facilitate cloning and amplification of the sequence of the vector in prokaryotic and eukaryotic organisms. In some embodiments, the implementation of marker selection confers resistance to the compound or class of compounds, such as antibiotics. An example of marker selection, which can be used in combination with the mole is Olami nucleic acids and systems of the present invention, is the token that gives resistance to puromycin. Alternatively, you can use the markers selection, which give resistance to hygromycin, gpt, neomycin, zeocin, ouabaine, blasticidin, kanamycin, geneticin, gentamicin, ampicillin, tetracycline, streptomycin, spectinomycin, nalidixic acid, rifampicin, chloramphenicol, zeocin or bleomycin, or markers like DHRF, hisD, trpB or glutamylcysteine.

The composition of the vector of the present invention also comprises the regulatory elements necessary for transcription of the gene of interest (as well as breeding marker) and broadcast them into proteins. Transcriptional regulatory elements typically include a promoter that is joined to the 5'-end sequence of the gene to be expressed, the sites of initiation and termination of transcription and sequence of the polyadenylation signal. The term "site of transcription initiation" refers to a nucleic acid corresponding in construction to the first nucleic acid included in the primary transcript, i.e. precursor mRNA; the site of transcription initiation may overlap with promoter sequences. The term "site of transcription termination" refers to a nucleotide sequence, usually present on the 3'-end of the gene of interest or outline the s sequence subject to transcription, which causes RNA polymerase to terminate transcription. Signal polyadenylation sequence, or signal adding poly-A, provides cleavage at a specific site on the 3'-end of eukaryotic mRNA posttranscriptional addition to the core sequence, containing about 100-200 adenine residues (tail poly-A), to split the 3'-end. The sequence signal includes polyadenylation sequence AATAAA located approximately 10-30 nucleotides above the site of cleavage, and the underlying sequence.

Regulatory element that can be included in the vector of the present invention is the promoter. The promoter may be constitutive or inducible. Enhancer (i.e. CIS-acting element DNA, which acts on the promoter, increasing transcription), which operates in combination with the promoter, may be needed to increase the level of expression compared to the level provided only by the promoter, and may be included in the vector as a regulatory element. In many cases, the polynucleotide segment containing the promoter, also include enhancer sequences (e.g., CMV IE P/E; SV40 P/E; MPSV P/E). If you want to receive playerowner transcripts, the vector can be included whitefish is Aly splicing. To get the secretory polypeptide in the composition of breeding sequence is usually injected signal sequence encoding a leader peptide that directs the synthesized polypeptide to the ER membrane and through it, after which the polypeptide may secretariats. Leader peptide is often, but not always, located at the amino-end of the secreted protein and is cleaved under the action of signal peptidase after crossing a protein of the ER membrane. Breeding sequence, usually, but not always, contains its own signal sequence. If the native signal sequence is absent, to the breeding sequence you can attach a heterologous signal sequence. In this area there are many signal sequences that can be found in databases for sequences, such as GenBank and EMBL. Translational regulatory elements include the site of translation initiation (AUG), the stop codon and the signal poly-A used for each individual expressed polypeptide. In some designs is a part of the internal landing ribosomes (IRES).

The promoters used in the present invention include viral promoters, the promoters of mammalian and yeast promoters such as the promoter of the mouse beta-globin, a promotion is R of ubiquitin, the promoter polyoma, the promoter of cytomegalovirus mammals (CMV)promoter of the yeast alcoholiday, the promoter phosphoglyceromutase induced by lactose promoters, promoter galactosidase, the promoter of adeno-associated virus, a poxvirus promoter, a promoter of retrovirus, a promoter of rous sarcoma virus, the promoters of adenovirus, SV40 promoter, the promoter hydroxymethylglutaryl-coenzyme A, the promoter timedancing, poxvirus promoters H5R, the late promoter of adenovirus type 2MPC, the promoter of the alpha-antitrypsin deficiency, the promoter Fox IX, immunoglobulin promoter, the promoter surfactants CFTR, the promoter of albumin and transferrin promoter. The promoter selected for use in combination with nucleic acids and expression vectors of this invention should provide (1) high levels of expression, for example, when controlling the expression of the gene of interest, or (2) decreased levels of expression (after a decline in efficiency modifications), for example, when the control expression of a gene of breeding marker. Preferably, the promoter that controls expression of the gene of interest, is a strong promoter such as the promoter of ubiquitin, CMV, EF-1α and SR alpha, providing increased expression and initiation of correct splicing of the target product.

what one embodiment, the vector of the present invention contains the CMV promoter to control expression of the gene of interest. The use of the CMV promoter is described in U.S. patent No. 5385839 and 5849522 included in this description by reference. In one embodiment, the CMV promoter used in the vector of the present invention, functionally associated with the gene of interest and the nucleotide 1-608 sequence SEQ ID NO:1. In the scope of the present invention also includes the sequence of the CMV promoter, which 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide 1-608 sequence SEQ ID NO:1.

Other promoters suitable for use in the vector of the present invention is the promoter of the elongation factor 1α (EF-1α), for example, human EF-1α. The sequence of the promoter of the human EF-1α can be found in GenBank under access number NM_001402 (gi:83367078). In one embodiment, the vector of the present invention contains a nucleotide 76-1267 sequence SEQ ID NO:2. In the scope of the present invention also includes the sequence of the promoter EF-1α, which 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide 1-608 sequence SEQ ID NO:1.

In one embodiment, for carrying out the cloning part of the vector is administered section of the SwaI restriction.

Typically, genes (e.g., breeding markers and GOI) is placed between the promoter and the polyadenylation site. You can use a sequence of poly-A, derived from interest g is on (i.e. native sequence poly-A), or heterologous sequence poly-A (i.e. of a gene, differing from GOI), for example, poly-A BGH and poly-A SV40. mRNA is transcribed with the participation of promoters and stabilized under the action of polyadenylation signals located from the 3'ends of the coding stations. Signals poly-A well-known in this field can be selected based on suitability for use in combination with vectors and cells of the host used in the present invention. Examples of suitable signals poly-include A poly-A human BGH, poly-A SV40, poly-A human beta-actin, poly-A, rabbit beta-globin and poly-A Kappa-chain immunoglobulin.

The vector of the present invention contains a gene of interest, through which the vector is expressed in cell culture. Of interest, the gene can encode a functional nucleic acid molecule (e.g., RNA, such as a molecule, antisense RNA) or, more preferably, it encodes a peptide, polypeptide or protein that it is desirable to produce at a higher level. The vectors of this invention may contain a gene of interest inserted into the site of insertion so that the interest gene was functionally associated with a regulatory nucleotide sequence, Kotor, which provides the expression of the gene of interest. In one embodiment, the vectors of this invention can be used for the expression of virtually any gene of interest, especially genes encoding recombinant proteins with therapeutically useful activity or with other commercially significant application.

Non-limiting examples of genes of interest include genes, hormones, chemokines, cytokines, lymphokines, antibodies, receptors, adhesion molecules and enzymes. A partial list of target products include, for example, human growth hormone, bovine growth hormone, parathyroid hormone, thyrostimulin hormone, follicle-stimulating hormone, luteinizing hormone; hormone releasing factor; lipoproteins; alpha-1-antitripsin; A-chain, insulin B-chain of insulin; proinsulin; calcitonin; glucagon; such molecules as renin; coagulation factors such as factor VIIIC, factor IX, tissue factor, and the factor a background of Villebranda; antikoaguliruyuschee factors such as protein C, trially natriuretic factor, lung surfactant; plasminogen activator, such as urokinase or human plasminogen activator urine or tissue plasminogen activator (t-PA); bombezin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (chemokine granted aemy T-cell activation); human macrophage protein inflammation (MIP-I alpha); a serum albumin such as human serum albumin; müller inhibiting substance; A - or B-chain of relaxin; prolactin; mouse gonadotropin-associated peptide; Tnkase; inhibin; activin; receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), growth factors, including growth factor vascular endothelial (VEGF), nerve growth factor such as NGF-beta; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF, bFGF, FGF-4, FGF-5, FGF-6; epidermal growth factor (EGF); transforming growth factor (TGF)such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and-II (IGF-I and IGF-II); des(1-3)-IGF-I (IGF-I brain), proteins that bind insulin-like growth factor; CD proteins such as CD-3, CD-4, CD-8 and CD-19; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta and-gamma; colony stimulating factors (CSFs)such as M-CSF, GM-CSF and G-CSF; interleukins (IL)such as IL-1)-(IL-33); peroxydisulfate; receptors of T-cells; surface membrane proteins, for example, HER2; complementability stimulator hemolysis; irony antigen, such as, for example, a fragment of the shell of the AIDS virus; transport proteins; homing receptors; Addressin; receptors of growth factors, cytokines, chemokines and lymphokines; regulatory proteins; antibodies; chimeric proteins, for example, contains immunoadhesin and fragments of any of the above polypeptides. Examples of bacterial polypeptides or proteins include, for example, alkaline phosphatase and beta-lactamase.

In one aspect of the present invention, the vector contains a plot heavy or light chain of the antibody, which is functionally associated with the site of insertion. Examples of vectors containing two episomal origin of replication and a constant area light or heavy chain antibodies can be found in SEQ ID nos:3-32.

One of the embodiments of the present invention includes vectors that can be used for the expression of whole antibodies, i.e. the variable segment associated with a constant area for heavy or light chain. Therefore, the gene of interest can encode variable plot heavy or light chain of the antibody, which may be an antibody of any type, for example, murine, chimeric, humanitariannet and human. Of interest, the gene encoding the variable plot heavy chain or light chain may include a sequence encoding a full-size in Realny plot, or, alternatively, only a fragment of the heavy chain or light chain, for example, the site of the antigen-binding fragment. In one embodiment of interest, the gene encodes variable plot murine or human antibody. In this case, a constant area may belong to the same species as the variable plot (SEQ ID NO:3-8, 27 and 28 encode murine constant plots, and SEQ ID NO:9-26 and 29-32 encode human constant segments).

In one embodiment, the vector of the present invention contains a nucleotide sequence encoding a constant plot heavy chain antibodies with certain izotopicheskii and/or alltimecase characteristics. Constant plot heavy chain, for example, may relate to gamma isotype (IgG), such as gamma 1, gamma 2, gamma 3 and gamma 4. In one embodiment, const plot heavy chain gamma 1 refers to a specific allotype, including, without limitation, allotype z,a and z,non-a. Allotype z, a, also known as allotype G1m17 and G1m1, in accordance with IGHG1 contains a Lys at position 214 (CH1), Asp at position 356 (CH3) and Leu at position 358 (CH3) (numbering according to the EU system). Allotype z,non-a, also known as allotype G1m17 and nG1m1 in accordance with IGHG1 contains a Lys at position 214 (CH1), Glu at position 356 (CH3) and the Met at position 358 (CH3) (numeration system U).

In another embodiment, const plot heavy chain gamma 2 (hcG2) refers to a specific allotype, including, without limitation, n - or n+. Allotype hcG2 n+, also known as the G2m (n) or G2m (23), in accordance with IGHG2 contains Thr at position 189 CH1 and the Met at position 282 (numbering according to the EU system). Allotype hcG2 n-, also known as the G2m (n), in accordance with IGHG2 contains Pro at position 189 CH1 and Val at position 282 (numbering according to the EU system). Other features of allotypes n+ and n - are described in Hougs et al. (2001) Immunogenetics 52:242 and Brusco et al. (1995) Immunogenetics 42:414.

In other embodiments, implementation of the constant-area heavy chain may refer to isotype IgM, IgA (IgA1 or IgA2), IgD or IgE.

In one embodiment, const plot heavy chain may have the following human izotopicheskie and alltimecase characteristics: gamma 1,z,a; gamma 1,z,non-a; gamma 2,n+; gamma 2,n-; or gamma 4. In one embodiment, the isotype/allotype gamma 1,z,non-a can contain a mutation at position 234 const plot heavy chain. In another embodiment, the isotype/allotype gamma 1,z,non-a may contain mutations at positions 234 and 235 or 234 and 237 const plot heavy chain. Examples of such vectors is shown in Fig-25.

In another example, the constant-area light chain encoded in the vector of the present invention, can refer to the Kappa-isotype or lambdas is-isotype.

Constant sites encoded by the vector of the present invention are not limited to human, but may include constant sections of mice or other species. In one embodiment, the expression vector of the present invention contains a nucleic acid encoding a constant site of the heavy chain, which refers to either mouse isotype gamma 1, or mouse isotype gamma 2a, or constant area light chain, which refers to mouse Kappa-isotype.

Two vectors of the present invention, pHybC and pHybE represent the empty vectors because they do not contain constant plots, and can be used for cloning genes of interest. Description pHybC and pHybE below, and map the data vectors can be found in figure 1 and 2.

pHybC

Vector pHybC (empty) contains two viral origin of replication which allows replication of the vector in different cell lines. pHybC contains the following elements: origin of replication of SV40 ("SV40 Ori"), which provides replication vector plasmids in cells expressing a protein large T-antigen of SV40 (e.g., COS7 cells); the CMV promoter ("pCMV"), functionally associated with the site of insertion of gene of interest; trinomial leader sequence (TPL); donor site splicing (SD); major late adenovirus enhancer (enh LP); acceptor site of splicing (SA); section open reading frame (ORF)containing the gene of interest with the subsequent signal poly-A (pA); the element of symmetry of the second order (DS); eukaryotic origin of replication of Epstein-Barr (OriP), which provides replication vector plasmids in cells expressing a viral protein EBNA-I (for example, in cells HEK-293-6E); the site duplications (FR); token resistance to ampicillin (AmpR); and bacterial origin of replication (pMB1ori). In the vector pHybC used the pCMV promoter, one of the most effective available promotor elements. Map vector pHybC (blank) is shown in figure 1. The nucleotide sequence of the vector pHybC described in SEQ ID NO:1.

pHybE

The pHybE vector (empty) contains two origin of replication which allows replication of the vector in different cell lines. pHybE contains the following elements: origin of replication of SV40 ("SV40 Ori"), which provides replication vector plasmids in cells expressing a protein large T-antigen of SV40 (e.g., COS7 cells); eukaryotic promoter, EF-1a, functionally associated with the site of insertion of gene of interest; section open reading frame (ORF)containing the gene of interest with the subsequent signal poly-A (pA); the element of symmetry of the second order (DS); eukaryotic origin of replications Epstein-Barr (OriP); the site duplications (FR); token resistance to ampicillin (AmpR); and bacterial origin of replication (pMB1ori). Map of pHybE vector (empty) is shown in figure 2. pHybE differs from pHybC that pHybE contains the promoter EF-1A, functionally associated with the site of insertion of gene of interest, and pHybC contains the CMV promoter. The nucleotide sequence of pHybE vector described in SEQ ID NO:2.

The following vectors are obtained based on the pHybE or pHybC additionally contain a constant areas of heavy or light chains of immunoglobulin. As in the case of pHybE and pHybC, the following vectors contain the areas of cloning, which can be used to insert a gene of interest, such as a sequence encoding a variable segment of immunoglobulin, or antigen-binding fragment. In all cases, the site of the cloned gene of interest is adjacent to a sequence that encodes a constant area, which is part of the vector. Thus, the following vectors can be used for the expression of light and heavy chains of the antibody containing a specific constant area and a specific variable plot. As in the case of pHybC and pHybE, each of the following vectors of the present invention contains several original replication, allowing to Express the light or heavy CE is b antibodies in different cell lines using the same vector. Descriptions of the other vectors of the present invention are given below (see also maps the vectors depicted in Fig-25). It should be noted that the vectors pHyb, designated as version 1 (V1), contain additional plot SwaI above section SrfI restriction, whereas the vectors pHyb, designated as version 2 (V2), do not contain additional SwaI site.

The vectors of this invention containing the murine constant plots

pHybC-mCg2a

Vector pHybC-mCg2a is obtained on the basis of the vector pHybC (hence, it contains all the above elements of the vector pHybC). This vector also contains the sequence encoding the constant plot heavy chain gamma 2a murine immunoglobulin. Thus, in one embodiment, the vector pHybC-mCg2a can be used for the expression of heavy chain antibodies containing a variable plot the heavy chain of the immunoglobulin or its fragment) and a murine constant plot heavy chain gamma 2. Alternatively, pHybC-mCg2 can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 2, for example, a hybrid protein of the Fc. On Fig map shows pHybC-mBR3-mCg2a, which contains the sequence encoding the extracellular domain (ECD) of murine BR3 protein as a gene of interest. The nucleotide sequence pHybC-mBR3-mCg2a described in SEQ D NO:27.

pHybE-mCk

The pHybE vector-mCk receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). pHybE-mCk also contains the sequence encoding the constant-area light chain Kappa mouse immunoglobulin. Thus, in one embodiment, the pHybE vector-mCk can be used for expression of the light chain of the antibody containing the variable area light chain immunoglobulin and a murine constant area light chain Kappa. Alternatively, pHybE-mCk can be used for expression of interest gene hybridizing with murine constant area light chain Kappa. Map of pHybE vector-mCk V2 shown in Fig. The nucleotide sequence of pHybE-mCk V1 described in SEQ ID NO:3 and the nucleotide sequence of pHybE-mCk V2 described in SEQ ID NO:4.

pHybE-mCg1

pHybE-mCg1 receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 1 mouse immunoglobulin. Thus, in one embodiment, the pHybE vector-mCg1 can be used for the expression of heavy chain antibodies containing a variable plot heavy chain immunoglobulin and a murine constant plot heavy chain gamma 1. Alternatively, pHybE-mCg1 can be used for e is cpressey gene of interest, hybridizing with murine constant plot heavy chain gamma 1, for example, a hybrid protein of the Fc. Map of pHybE vector-mCg1 V2 shown in Fig. The nucleotide sequence of pHybE-mCg1 V1 described in SEQ ID NO:5, and the nucleotide sequence of pHybE-mCg1 V2 described in SEQ ID NO:6.

pHybE-mCg2a

pHybE-mCg2a receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 2a murine immunoglobulin. Thus, in one embodiment, the pHybE vector-mCg2a can be used for the expression of heavy chain antibodies containing a variable plot heavy chain immunoglobulin and a murine constant plot heavy chain gamma 2. Alternatively, pHybE-mCg2a can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 2, for example, a hybrid protein of the Fc. Map of pHybE vector-mCg2a V2 shown in Fig. The nucleotide sequence of pHybE-mCg2a V1 described in SEQ ID NO:7, and the nucleotide sequence of pHybE-mCg2a V2 described in SEQ ID NO:8. Figure 9 shows a map of pHybE-mBR3-mCg2a, which in one embodiment is an example of the application of pHybE-mCg2a. The vector described in figure 9, contains the sequence encoding the extracellular domain (ECD) of the mouse protein BR3. The nucleus is Edna sequence of pHybE-mBR3-mCg2a described in SEQ ID NO:28.

The vectors of this invention containing human constant plots

pHybC-E7-hCk

pHybC-E7-hCk is obtained on the basis of the vector pHybC (hence, it contains all the above elements of the vector pHybC). This vector also contains the sequence encoding the constant-area light chain Kappa human immunoglobulin. In addition, pHybC-E7-hCk contains a sequence encoding a variable area light chain of adalimumab (also referred to as "E7"). Map vector pHybC-E7-hCk is shown in figure 10, and the nucleotide sequence pHybC-E7-hCk described in SEQ ID NO:29.

pHybC-D2-hCg1,z,a

pHybC-D2-hCg1,z,a is obtained on the basis of the vector pHybC (hence, it contains all the above elements of the vector pHybC). This vector also contains the sequence encoding the constant plot heavy chain gamma 1,z,a. In addition, pHybC-D2-hCg1,z,a contains a sequence encoding a variable plot heavy chain of adalimumab (also referred to as "D2"). Map vector pHybC-D2-hCg1,z,a, see figure 11. The nucleotide sequence pHybC-D2-hCg1,z,a are described in SEQ ID NO:30.

pHybE-hCk

pHybE-hCk receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant-area light chain Kappa human immunoglobulin. Thus, the example vector pHybE-hCk can be used for expression of the light chain of the antibody containing the variable area light chain immunoglobulin and a human constant area light chain Kappa. Alternatively, pHybE-hCk can be used for expression of interest gene hybridizing with constant area light chain Kappa. Map vector pHybE-hCk V2 shown in Fig. The nucleotide sequence of pHybE-hCk V1 described in SEQ ID NO:9, a nucleotide sequence of pHybE-hCk V2 described in SEQ ID NO:10. Map of pHybE vector-E7-hCk is also depicted on Fig. In addition to all the above described elements of the vector pHybE-hCk, pHybE-E7-hCk contains a sequence encoding a variable area light chain of adalimumab (also referred to as "E7"). The nucleotide sequence of pHybE-E7-hCk described in SEQ ID NO:32.

pHybE-hCl

pHybE-hCl receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant-area light chain lambda human immunoglobulin. Thus, in one embodiment, the pHybE vector-hCl can be used for expression of the light chain of the antibody containing the variable area light chain immunoglobulin and a human constant area light chain lambda. Alternatively, pHybE-hCl can be used to Express the AI gene of interest, hybridizing with constant area light chain lambda. Map of pHybE vector-hCl V2 shown in Fig. The nucleotide sequence of pHybE-hCl V1 described in SEQ ID NO:11, and the nucleotide sequence of pHybE-hCl V2 described in SEQ ID NO:12.

pHybE-hCg1,z,a

pHybE-hCg1,z,a gain based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 1,z,a human immunoglobulin. Thus, in one embodiment, the vector of pHybE-hCg1,z,a, can be used for the expression of heavy chain antibodies containing a variable plot heavy chain immunoglobulin and a human constant plot heavy chain gamma 1,z,a. Alternatively, pHybE-hCgl,z,a, can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 1,z,a, for example, a hybrid protein of the Fc. Map vector pHybE-hCg1,z,a, see Fig. The nucleotide sequence of pHybE-hCg1,z,a, V1 described in SEQ ID NO:13, and the nucleotide sequence of pHybE-hCg1,z,a, V2 described in SEQ ID NO:14. Map of pHybE vector-D2-hCg1,z,a, see Fig. In addition to the elements described above for pHybE-hCg1,z,a, pHybE-D2-hCg1,z,a contains a sequence encoding a variable plot heavy chain of adalimumab (also referred to as "D2"). Nucleotide posledovatel is of pHybE-D2-hCg1,z,a are described in SEQ ID NO:31.

pHybE-hCg1,z,non-a

pHybE-hCg1,z,non-a gain based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 1,z,non-a human immunoglobulin. Thus, in one embodiment, the pHybE vector-hCgl,z,non-a can be used for expression of the heavy chain of the antibody containing the variable plot heavy chain immunoglobulin and a human constant plot heavy chain gamma 1,z,non-a. Alternatively, pHybE-hCg1,z,non-a can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 1,z,non-a, for example, a hybrid protein of the Fc. Map vector pHybE-hCg1,z,non-a V2 shown in Fig. The nucleotide sequence of pHybE-hCg1,z,non-a V1 described in SEQ ID NO:15, and the nucleotide sequence of pHybE-hCg1,z,non-a V2 described in SEQ ID NO:16.

pHybE-hCg1,z,non-a,mut(234,235)

pHybE-hCg1,z,non-a,mut(234,235) receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 1,z,non-a,mut(234,235) human immunoglobulin. Thus, in one embodiment, the vector of pHybE-hCg1,z,non-a,mut(234,235) can be used for the expression of heavy chain antibodies containing variab the local area heavy chain immunoglobulin and a human constant plot heavy chain gamma 1,z,non-a,mut(234,235). Alternatively, pHybE-hCg1,z,non-a,mut(234,235) can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 1,z,non-a,mut(234,235), for example, a hybrid protein of the Fc. Map vector pHybE-hCg1,z,non-a,mut(234,235) V2 shown in Fig. The nucleotide sequence of pHybE-hCg1,z,non-a,mut(234,235) V1 described in SEQ ID NO:17, and the nucleotide sequence of pHybE-hCg1,z,non-a,mut(234,235) V2 described in SEQ ID NO:18.

pHybE-hCg1,z,non-a,mut(234,237)

pHybE-hCg1,z,non-a,mut(234,237) receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 1,z,non-a,mut(234,237) human immunoglobulin. Thus, in one embodiment, the vector of pHybE-hCg1,z,non-a,mut(234,237) can be used for the expression of heavy chain antibodies containing a variable plot heavy chain immunoglobulin and a human constant plot heavy chain gamma 1,z,non-a,mut(234,237). Alternatively, pHybE-hCg1,z,non-a,mut(234,237) can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 1,z,non-a,mut(234,237), for example, a hybrid protein of the Fc. Map vector pHybE-hCg1,z,non-a,mut(234,237) V2 shown in Fig. The nucleotide sequence of pHybE-hCg1,z,non-a,mut(234,237) V1 described in SEQ ID NO:19, and the nucleotide sequence of pHybE-hC1,z,non-a,mut(234,237) V2 described in SEQ ID NO:20.

pHybE-hCg2,n-

pHybE-hCg2,n - receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 2,n - human immunoglobulin. Thus, in one embodiment, the pHybE vector-hCg2,n - can be used for the expression of heavy chain antibodies containing a variable plot heavy chain immunoglobulin and a human constant plot heavy chain gamma 2,n-. Alternatively, pHybE-hCg2,n - can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 2,n-, for example, a hybrid protein of the Fc. Map of pHybE vector-hCg2,n - V2 shown in Fig. The nucleotide sequence of pHybE-hCg2,n - V1 described in SEQ ID NO:21, and the nucleotide sequence of pHybE-hCg2,n - V2 described in SEQ ID NO:22.

pHybE-hCg2,n+

pHybE-hCg2,n+ receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 2,n+ human immunoglobulin. Thus, in one embodiment, the pHybE vector-hCg2,n+ can be used for the expression of heavy chain antibodies containing a variable plot heavy chain immunoglobulin and human intercept the private plot heavy chain gamma 2,n+. Alternative? pHybE-hCg2,n+ can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 2,n+, for example, a hybrid protein of the Fc. Map of pHybE vector-hCg2,n+ is shown in Fig. The nucleotide sequence of pHybE-hCg2,n+ V1 described in SEQ ID NO:23, and the nucleotide sequence of pHybE-hCg2,n+ V2 described in SEQ ID NO:24.

pHybE-hCg4

pHybE-hCg4 receive based on the pHybE vector (thus contains all of the above elements of the vector pHybE). This vector also contains the sequence encoding the constant plot heavy chain gamma 4 human immunoglobulin. Thus, in one embodiment, the pHybE vector-hCg4 can be used for the expression of heavy chain antibodies containing a variable plot heavy chain immunoglobulin and a human constant plot heavy chain gamma 4. Alternatively, pHybE-hCg4 can be used for expression of interest gene hybridizing with constant plot heavy chain gamma 4, for example, a hybrid protein of the Fc. Map of pHybE vector-hCg4 shown in Fig. The nucleotide sequence of pHybE-hCg4 V1 described in SEQ ID NO:25, and the nucleotide sequence of pHybE-hCg4 V2 described in SEQ ID NO:26.

The sequence of the vectors of the present invention is described in SEQ ID NO:1-32. In one embodiment, the vector of the present invention with the contains the sequence presented in any one of the sequences SEQ ID nos:1-32 or sequences that are identical, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

This invention can be used for human and/or humanized antibodies that immunospecificity recognize specific cellular targets, for example, any of the above proteins, such as human EGF receptor antigen her-2/neu antigen CEA, prostatespecific membrane antigen (PSMA), CD5, CD11a, CD18, NGF, CD20, CD45, CD52, Ep-cam, other cancer cell surface molecules, TNF-alpha, TGF-b1, VEGF and other cytokines, alpha 4 beta 7 integrin, IgE, viral proteins (for example, proteins of cytomegalovirus). Examples of antibodies that can be obtained using the compositions and methods of this invention include, without limitation, the antibody against TNFα, antibody against IL-12 antibody against IL-18 antibody against EPO receptor (EPO-R). In one embodiment, the antibody against TNFα is a fully human antibody against TNFα, e.g., adalimumab/D2E7 (see U.S. patent No. 6090382 included in this description by reference; Humira®; Abbott Laboratories). In one embodiment, the antibody against IL-12 represents the FDS is the first fully human antibody against IL-12, for example, ABT-874 (Abbott Laboratories; see U.S. patent No. 6914128 included in this description by reference). In one embodiment, the antibody against IL-18 is a fully human antibody against IL-18 (for example, ABT-325), as well as antibodies, are described in US20050147610 A1 included in this description by reference. In one embodiment, the antibody against EPO/R (also referred to as ABT-007) is a fully human antibody, such as described in published U.S. patent number US 20060018902 A1 included in this description by reference.

In addition, constant areas, encoded by the vector, such as an Fc domain can be functionally related proteins with the formation of the hybrid protein, for example, a hybrid protein of the Fc. Thus, another example of the type of proteins that can be produced using the methods and compositions of the present invention includes a hybrid proteins. Examples of such hybrid proteins include proteins, expressed in the form of a hybrid fragment of immunoglobulin molecules, proteins, expressed in the form of a hybrid with a fragment-"lightning", and the new multifunctional proteins, such as hybrids of cytokines and growth factors (e.g., GM-CSF and IL-3, MGF and IL-3). In WO 93/08207 and WO 96/40918 retrieves various soluble oligomeric forms of the molecule, called CD40L, including hybrid protein immunoglobulin and hybrid protein"zip", accordingly, described herein, the methods are applicable to other proteins. Another hybrid protein is a recombinant TNFR:Fc, also known as entanercept. Entanercept (or Enbrel®; Amgen/Wyeth) is a dimer of two molecules of the extracellular portion of the receptor TNF-alpha p75, each of which contains obtained from TNFR polypeptide size of 235 amino acids, heriditary with Fc fragment of human IgG1 size of 232 amino acids. In fact, in the form of a hybrid protein can Express any molecule, including, without limitation, the extracellular domain of the molecule cell receptor, an enzyme, a hormone, a cytokine, a fragment molecules of the immunoglobulin domain is"lightning" and the epitope.

Methods of determination of identity of the nucleotide and amino acid sequences known in the field. Typically, such methods include determining the nucleotide sequence of the mRNA of the test gene and/or the definition of the encoding of its amino acid sequence, and comparison of these sequences with the second nucleotide or amino acid sequence. As a rule, the term "identity" refers to exact match the nucleotide-nucleotide or amino acid-amino acid of two polynucleotide go polypeptide sequences, respectively. DV is or more sequences (polynucleotide or amino acid) can be compared by determining the "percent identity." The percent identity of two sequences, either nucleotide or amino acid sequences, represents the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. The approximate alignment of the nucleotide sequences is performed using the algorithm of the local homology, Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences using matrix points developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D. C, USA, and normalized Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). A typical application of this algorithm to determine the percent identity of the sequences is Genetics Computer Group (Madison, Wis.) in the helper application "BestFit". The default parameters for this method are described in the Wisconsin Sequence Analysis Package, Program Manual, Version 8 (1995) (provided by the Genetics Computer Group, Madison, Wis.). In the preferred method of determining percent identity in the context of the present invention use a set of programs MPSRCH protected by copyright University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this set of programs can use the algorithm of Smith-Waterman, where to obtain estimates table is C use standard settings (for example, the penalty for opening a gap - 12, the penalty for continued space unit and a space - six). The resulting number of "coincidences" reflects "identity sequence". Other programs that are appropriate for the calculation of the percent identity or similarity of the sequences is well known in this field.

The terms "selective hybridization" of two nucleic acid fragments described herein. The degree of identity of two sequences of nucleic acid molecules affects the efficiency and strength of hybridization events such molecules. Partially identical to the nucleotide sequence, at least partly inhibits the hybridization of the target molecules with completely identical sequence. The inhibition of hybridization of the completely identical sequence can be estimated from the analysis of hybridization are well known in this field (such as southern blotting, Northern blotting, hybridization in solution or the like, see Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York; or Ausubel et al. (Eds.), Current Protocols In Molecular Biology, John Wiley & Sons, Inc., New York (1997)). Such analyses can be performed with varying degrees of selectivity, for example, varying conditions from low to high stiffness. If you are using conditions of low stringency, the absence of nonspecific binding can be defined by means of the secondary probe, which does not have even a partial sequence identity (e.g., a probe that is identical to the sequence of a target molecule is less than 30%), so that in the absence of events of nonspecific binding of the secondary probe is not hybridized with the target.

When the use of a detection system based on hybridization choose a nucleotide probe complementary to the target nucleotide sequence, then in suitable conditions hold for the "selective hybridization" of the probe and the target sequence, or linking them with each other with obtaining hybrid molecules. The molecule is a nucleic acid capable of selectively gibridizatsiya with the target sequence under conditions of "medium hard", usually hybridizes under conditions that allow the detection of a target nucleotide sequence which contains at least about 10 to 14 nucleotides in length and at least about 70% identical to the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow you to detect the target nucleotide sequence, which contains at least about 10 to 14 nucleotides in length and at least about 90-95% identical to the sequence of the selected nucleic acid probe. Conditions of hybridization of probe and target, with opredelennosti identity sequences, can be determined using known in the field of methods (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With regard to conditions of stringency hybridization in this area is well known that various equivalent conditions can be used to establish certain rigidity by varying such factors as the length and nature of the sequences of probe and target, the composition of the bases of different sequences, concentrations of salts and other components of the solution for hybridization, the presence or absence of a blocking means or detergents in the solution for hybridization (e.g., formamide, dextransucrase, polyethylene glycol and sodium dodecyl sulfate), temperature and time parameters of the reaction, hybridization, and washing conditions. The choice of hybridization conditions is performed using the following standard methods known in the art (see, for example, Sambrook, et al., above, or Ausubel et al., above). Consider that the first polynucleotide is "derivative" of the second polynucleotide, if it has the same or essentially the same sequence of base pairs as the plot of the second polynucleotide, its cDNA, or complementary chain, or sequence identity, as described above. Consider that the first polyp is Ted is "derivative" of the second polypeptide, if he (i) is encoded first polynucleotide derived second polynucleotide, or (ii) has a sequence identity relative to the second polypeptide, as described above.

This invention also provides a kit containing one or more vectors of the present invention in a suitable vessel such as a vial. The expression vectors may contain at least one cloning site for insertion of the selected interest sequence, or they may contain a specific gene of interest that was already present in the vector. The vector can be represented in the form of a dehydrated or lyophilized form, or in aqueous solution. The set can include the dilution buffer digidratirovannogo of polynucleotide. The kit can include other reagents, such as buffers for the reaction, the vectors used for comparison as positive and negative controls. Typically, the kit also contains instructions for use are included in the reagents.

III. The use of the vectors of the present invention

The invention includes methods of ekspressirovali proteins with the use described in this document vectors. Thus, this invention provides a method of obtaining a recombinant protein, comprising introducing the vector of the expression of the present invention in cells of the host mammal, culturing the host cell of the mammal in suitable conditions ensuring the expression of the protein and isolating the protein. The advantage of the vector of the present invention is that it provides products of protein at high levels in mammalian cell cultures.

In the present invention as a host cell can be any cell that can Express the gene by means of nucleic acid or expression vector of the present invention. The term "cell host" refers to cells that have been transformed with vectors constructed using methods of recombinant DNA.

Ordinary experts in this field can choose a specific line of master cells, which is best suited to the expression of the GOI and the gene of breeding token using the vector of the present invention. Cells that can be used in this invention include mammalian cells and derived cell lines and cell culture. Mammalian cells, for example, embryonic stem cells or somatic cells can be obtained from mammals such as mice, rats or other rodents or primates, such as humans or monkeys. It should be understood that for the implementation of the methods of this invention can be used primary cell ku is Tory or immortalized cells.

In specific embodiments, the implementation of the use of cell types related to mammalian cells by origin, which include, without limitation, cells of the Chinese hamster ovary (CHO) (e.g., DG44 and DUXB11; Urlaub et al., Som. Cell Molec. Genet. 12:555, 1986; Haynes et al., Nuc. Acid. Res. 11:687-706, 1983; Lau et al., Mol. Cell. Biol. 4:1469-1475, 1984; Methods in Enzymology, 1991, vol.185, pp.537-566. Academic Press, Inc., San Diego, Calif.), fibroblasts Chinese hamster (for example, R1610), cell cervical carcinoma human (e.g., HELA), the cell line of monkey kidney (e.g., CVI and COS), murine fibroblasts (for example, BALBc/3T3)cells, mouse myeloma (P3.times.63-Ag3.653; NS0; SP2/0), the cell line hamster kidney (for example, HAK), mouse L-cells (for example, L-929), human lymphocytes (e.g., RAJI)cells, human kidney (e.g., 293 and 293T). Cell line-owners usually are commercially available (e.g., from BD Biosciences, Lexington, Ky.; Promega, Madison, Wis.; Life Technologies, Gaithersburg, Md.) or they can be obtained from the American type culture collection (ATCC, Manassas, Va.).

In the preferred embodiment, used in this invention is a host cell provides a current from the outside factor of replication initiation, corresponding to at least one origin of replication, which is part of the vector of the present invention. For example, if the vector contains two origin of replication, corresponding to the origin of replication of SV40 and the origin replica the AI OriP, you can use any line of cells, preferably mammalian cells, which Express large T-antigen, or protein EBNA. In one embodiment, the vector is introduced into the COS cell or embryonic cell of the human kidney (HEK). For example, cells obtained from COS7 cells monkeys CV-I transformed initially defective mutant of SV40 (Sigma-Aldrich). EBNA can be obtained, for example, using cells HEK-293-6E.

Cell lines that contain factors of replication initiation, stably integrated into the genome, have the advantage that a stable long-term expression of factor of replication initiation and long-term provision of replication and maintenance of plasmids containing the origin of replication. Examples of commercially available cell lines expressing EBNA-1, are ATCC: 293HEK-EBNA1 and CV1-EBNA1. Specific cell lines carrying the increased expression of at least one factor in the initiation of replication, preferably protein EBNA1 or large T-antigen of SV40, can be obtained by transfection and selection of stable cell clones.

Nucleic acids and expression vectors can be entered or translated into the appropriate cell hosts with a variety of methods known in the art (see, for example, Ridgway, 1973, Vectors: Mammalian Expression Vectors, Chapter 24.2, pp.470-472, Rodriguez and Denhardt, eds., Buttrworths, Boston, Mass.; Graham et al., 1973, Virology 52:456; Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; and Chu et al., 1981, Gene 13:197). The terms "transformation" and "transfection"and their grammatical variations are used herein interchangeably and refer to the introduction of foreign DNA into the cell by any suitable means. A cell is "transformed" after exogenous nucleic acid gets inside the cell membrane. The uptake of nucleic acids leads to the formation of stable transfectant, regardless of the method of carrying out acquisitions, which may include the content (including, electroporation, fusion of protoplasts, the precipitation of calcium phosphate, merging cells with coated DNA, microinjection, and infection of intact virus. Even transient expression at the level higher than normal, is useful for functional studies or for the acquisition and allocation of interest protein. Transformed cells are grown under conditions suitable for the production of protein of interest (for example, in one embodiment, the heavy or light chain antibodies), and then conduct tests to identify the encoded interest of the polypeptide. Examples of analytical methods used for ID is tificatio and quantification of gene products, include solid-phase enzyme-linked immunosorbent assay (ELISA), radioimmunoassay analysis (RIA) or cell sorting with activation of fluorescence (FACS), immunohistochemistry, etc.

Cells used in the present invention, can be grown using standard methods, for example, they can be fixed on a solid surface or to grow in suspension in a suitable nutrient medium.

The cells of the host mammal containing the vectors described here, are also included in the scope of this invention.

Unless otherwise specified, to implement the present invention using conventional methods of molecular biology and the like, known to specialists in this field. Such methods are described in detail in the literature. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989); Current Protocols in Molecular Biology (F. Ausubel et al., eds., 1987 updated); Essential Molecular Biology (T. Brown ed., IRL Press 1991); Gene Expression Technology (Goeddel ed., Academic Press 1991); Methods for Cloning and Analysis of Eukaryotic Genes (A. Bothwell et al. eds., Bartlett Publ. 1990); Gene Transfer and Expression (M. Kriegler, Stockton Press, 1990); Recombinant DNA Methodology (R. Wu et al. eds., Academic Press 1989); PCR: A Practical Approach (M. McPherson et al., IRL Press at Oxford University Press 1991); Oligonucleotide Synthesis (M. Gait ed., 1984); Cell Culture for Biochemists (R. Adams ed., Elsevier Science Publishers, 1990); Gene Transfer Vectors for Mammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian Cell Biotechnology (M. Butler ed., 1991); Animal Cell Culture (J. Pollard et al. eds., Humana Press, 1990); Culture of Animal Cells, 2.sup.nd Ed. (R. Freshney et al. eds., Alan R. Liss 1987); Flow Ctometry and Sorting (M. Melamed et al. eds., Wiley-Liss 1990); the series Methods in Enzymology (Academic Press, Inc.); and Animal Cell Culture (R. Freshney ed., IRL Press 1987); and Wirth, M. and Hauser, H. (1993) Genetic Engineering of Animal Cells, In: Biotechnology Vol.2 Puhler A (ed.) VCH, Weinhcim 663-744.

Examples

The following examples illustrate an innovative approach to eliminate the need to design separate vectors for the various cells of the host mammal, such as COS7 cells and HEK-293-6E. The following examples also offers vectors, containing a nucleic acid encoding the constant parts of the antibody that can be used for the expression of whole light or heavy chain antibodies, or for the expression of hybrid proteins Fc.

Two new vector skeleton, called pHyb-C and pHyb-E design by combining selective elements of the other vectors, i.e. vectors pBOS and pTT3 (see provisional application U.S. No. 60/878165, international application number PCT/US2007/26482, filed December 28, 2007, entitled "DUAL-SPECIFIC IL-1A/IL-1b ANTIBODIES and USSN 12/006068, each of which is incorporated into this description by reference). Control pBOS vector contains the promoter EF-1a, functionally associated with the site of insertion of gene of interest, and the origin of replication of SV40. Control the pTT3 vector contains the CMV promoter functionally linked with the site of insertion of gene of interest, and the origin of replication EBNA (OriP).

The vectors of this invention are tested by analysis what and protein expression, such as mouse hybrid BR3-Fc and human antibody (adalimumab) in COS7 cells and HEK-293-6E. Successful expression of the protein in COS7 cells and HEK-293-6E shows that the vector system suitable for recombinant expression in both cell types.

Example 1

Construction of vectors pHybC and pHybE

Figures 1 and 2 show maps of new vectors, each of which contains two origin of replication. Figures 1 and 2 show an "empty" version vectors, i.e. versions that do not contain nucleic acids encoding the gene of interest or a constant plots antibodies (these are described in more detail below in example 4). pHybC contains the CMV promoter functionally linked with the site of insertion of gene of interest, whereas pHybE contains the promoter EF-1a.

To get pHybC-mBR3-Fc ("mBR3" means the mouse version of the third receptor, BlyS and in this description refers specifically to a sequence that encodes a fragment of the extracellular domain (ECD) protein mBR3), the site of the beginning of replication of SV40 vector pEF-BOS amplified by PCR using primers that provide the introduction of restriction sites PspX I on the 5'- and 3'-ends amplifierarava DNA fragment. Then this fragment insertions split PspX I. Design pTT3-mBR3-Fc containing the plot enzyme Sal I above the CMV promoter, split Sal I. Then the fragment insertions, rasmala the hydrated PspX I? are ligated at the site of Sal I pTT3 vector-mBR3-Fc with obtaining vector pHybC-mBR3-Fc.

To get the design pHybE-mBR3-Fc, first PCR amplified the 5'-end PspX I-modified DNA fragment containing the site of the beginning of the SV40 replication during extracellular domain mBR3. Then the obtained product is cleaved by the 5'-end under the action of PspX I and 3'-end under the action of Bsp68 I, a specific portion of which is located in the leader sequence of the above sequence of the extracellular domain mBR3. This splintered fragment of the clone in the Sal I and Bsp68 I-split design pTT3-mBR3-Fc with obtaining construction pHybE-mBR3-Fc.

Card pHybC-mBR3-Fc and pHybE-mBR3-Fc, each of which expresses a hybrid protein receptor-Fc mBR3-Fc depicted on Fig and 9.

Vector pHybC-E7 expressing such protein, as the light chain of the antibody D2E7 (adalimumab), design as well as pHybC-mBR3-Fc, i.e. by ligating the same PspX I-split plot SV40 Ori, which is isolated and cleaved in the process of getting pHybC-mBR3-Fc (described above), in a pre-designed vector pTT3-E7, pre-split Sal I.

To construct the vector of pHybE-E7, get the fragment insertion by splitting an existing vector pBOS-E7 the enzymes Hind III and BsiW I. Then the resulting fragment inserts are ligated into the vector pHybC-E7, previously cleaved with the same enzymes, to obtain the pHybE-E7, ensuring the expression of this protein,as D2E7 light chain.

To construct vectors pHybC-D2 and pHybE-D2, receive a fragment of the insert containing the parcels encoding the variable and constant parts of the heavy chain of the antibody D2E7 (adalimumab) (i.e., the sequence encoding a heavy chain D2), by splitting an existing vector pTT3-D2 enzymes Bsp68 I and Not I. the resulting fragment inserts are ligated into vectors pHybC-mBR3-Fc and pHybE-mBR3-Fc, previously cleaved with the same enzymes, to obtain the pHybC-D2 and pHybE-D2, respectively, providing for the expression of this protein, as heavy chain antibodies D2E7 (adalimumab).

Example 2

Comparison of the outputs of protein

To determine whether increasing the size of the vector by adding two original replication protein products under the action of the vectors described above vectors pHyb-E and pHyb-C compared with the control vectors pBOS and pTT3, each of which contains only one origin of replication. To compare expression caused by pBOS vectors, pTT3, pHyb-C and pHyb-E, design, encoding a hybrid protein murine BAFF receptor-human Fc (mBR3-Fc), subcloning four vector skeletons and get along with usage set to obtain purified and specific DNA.

Four vector containing the sequence mBR3-Fc injected by the method of electroporation in COS cells or transferout cells HEK-293-6E (methods is written below). Cells are incubated for five or seven days. Take samples of the environment and measure the protein concentration mBR3-Fc secreted into the environment. Titers determined by analysis of IgG using ELISA method and adjusting for the difference in molecular weight standard protein IgG and protein mBR3-Fc conditioned medium after 5 days in the case of COS7 cells and after 7 days in the case of cells HEK-293-6E. Correction of title is required in order to avoid overestimation of protein titer mBR3-Fc due to the fact that in a method ELISA as standard use much larger human protein IgG.

Transfection of 293

In this experiment, as the procedure transient transfection of 293 using modified methods, published in Durocher et al. (2002); Nucleic Acids Research 30(2):E9 and Pham et al. (2005); Biotechnology Bioengineering 90(3):332-44. For transfection using the following reagents:

cells HEK 293-6E (cell line of embryonic human kidney, stably expressing EBNA1; receive from the National research Council of Canada), cultured in disposable Erlenmeyer flasks in a humid incubator at 130 rpm, 37°C and 5% CO2.

- cultural environment: the environment for the expression of FreeStyle 293 (Invitrogen 12338-018), optionally containing 25 μg/ml of geneticin (G418) (Invitrogen 10131-027) and 0.1% Pluronic F-68 (Invitrogen 24040-032).

environment for transfection: the environment for the expression of FreeStyle 293, optionally containing 1 mm HEPES (Invitrogen 15630-080).

- source solution polyethylenimine (PEI): sterile source solution of 1 mg/ml, pH 7.0, obtained using linear PEI 25 kDa (Polysciences), which has been stored at temperatures below -15°C.

- Treponema nutrient medium: 5% wt./about. sterile source solution tryptone N1 (Organotechnie, 19554) in the medium for the expression of FreeStyle 293.

Obtaining cells for transfection: approximately 2-4 hours prior to transfection cells HEK 293-6E are harvested by centrifugation and resuspended in culture medium to obtain a density of approximately 1 million viable cells per ml For each transfection 40 ml of cell suspension is transferred into a disposable Erlenmeyer flask 250 ml and incubated for 2-4 hours.

Transfection: the environment for transfection and the original PEI solution is heated in advance to room temperature (CT). For each transfection 25 μg of plasmid DNA and 50 μg of polyethylenimine (PEI) are combined in 5 ml of medium for transfection and incubated for 15-20 minutes at RT to ensure the formation of complexes with DNA:PEI. For transfection BR3-Ig using 25 µg of plasmid BR3-Ig. Add 5 ml of a mixture of each complex DNA:PEI to culture volume of 40 ml, obtained in advance, and again incubated in a humid incubator at 130 rpm, 37°C and 5% CO2. Through 20-28 hours each transfection mixture add 5 ml triptolemos nutrient medium and the cultivation of prodoljaut within six days.

Transfection of COS7 cells

Two cups of size 150 mm, containing COS7, transferout design using standard electroporation conditions, as described below. For experiments for transfection COS7 cells COS cultured in DMEM + 10%FBS + 1X glutamine. After reaching confluence, the cells from one flask T-150 is used for electroporation. Cells trypsinized and centrifuged in a medium containing serum to inactivate the serum. Cells are washed with 1XPBS.

For each T-150 sediment resuspended in 0.8 ml of buffer for electroporation. Buffer for electroporation COS contains 20 mm Hepes (or buffer P3), 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4and 6 mm dextrose. Buffer for electroporation adjusted to pH 7.0 and sterilized by filtration. For each electroporation using sixty micrograms DNA (30 μg of each plasmid DNA heavy and light chain or 60 µg DNA in the case of the hybrid protein Fc). In each cuvette add 0.8 ml of a mixture of cell/buffer/DNA (the cell size of 0.4 cm - Biorad). In addition, one cell filled with only buffer and used as control. The cuvette was placed on ice. Electroporation of cells is carried out at 250 V and 950 μf within 15-25 milliseconds. Then the cell will return to the ice. The contents of 2 cells are transferred into a single conical tube with a volume of 50 ml containing 20 ml of hybridoma SFM. Pipette 10 ml is used to split nirupa and transfer to two cups for the cultivation of tissues in the size of 150 mm, each containing 20 ml of medium. Thus, the total volume of medium in each Cup is Then 30 ml cups incubated at 37°C, 5% CO2within three days.

Conditioned medium of COS cells (supernatant) is collected in a conical tube with a volume of 50 ml and centrifuged. After centrifugation the supernatant was filtered using filter 2 micron (μm). Take a sample for analysis by ELISA. Supernatant harvested after 5 days and analyzed by the standard method of ELISA for IgG to determine the appropriate yield of protein.

Version vectors pBOS, pTT3, pHybC and pHybE tested individually in mBR3 and adalimumab (D2E7).

Testing proteins

The concentration of the hybrid protein mBR3-Fc determined in the culture supernatant after 5 days (in the case of COS7 cells) or 7 days after transfection (in the case of cells 293-6E), using ELISA and/or Poros A.

Results

Data showing the levels of expression of the protein after the control and experimental transfection, shown in figure 3 (COS cells) and figure 4 (cells HEK-293). The data shown in figure 3, show that pHybC and pHybE provide efficient production of the protein in COS cells, where both vectors are expressed at a higher level than the control vector pTT3. The data shown in figure 4, show that the levels of expression in HEK cells, transfected with the pHyb-E, exceeds the levels expre the FIC, provide other three vectors, whereas the levels of production of the protein pHyb-C compared with control levels. Thus, as pHyb-C, and pHyb-E is able to Express the hybrid protein mBr3-Fc as a control vectors pTT3 and pBOS, if not better.

Example 3

Comparison of the yield of protein, which requires joint transfection of the two constructions DNA

Human monoclonal antibody IgG1/κ against TNFα (adalimumab)/D2E7 subcloning four vector skeletons and get along with usage set to obtain purified and specific DNA.

The four vectors containing sequences that allow expression of adalimumab, a method of electroporation is introduced into COS cells; cells HEK-293-6E transferout using poly(ethylenimine) (PEI).

Use the same procedure transient transfection of 293, as in example 3, except that the transfection of adalimumab use plasmid containing 10 µg D2E7 heavy chain (denoted by "D2"), and the plasmids containing 15 µg D2E7 light chain (denoted "E7").

Experiments transfection COS7 carried out as described above except that in each transfection Cup add 30 μg of each vector containing the heavy and light chain.

The concentration of antibody adalimumab determined in the culture supernatant 7 days after t is inspectie, using ELISA and/or Poros A. Titers determined by analysis of IgG using ELISA method in the conditioned medium after 5 days in the case of COS7 cells and after 7 days in the case of cells HEK-293-6E.

Data showing the levels of expression of the protein after the control and experimental transfection, shown in figure 5 (cells HEK-293) and 6 (COS cells). The data shown in figure 5, show that both vector skeleton pHybC and pHybE able to provide products of adalimumab at a higher level than the control vector pBOS, and comparable (pHybC) or higher (pHybE) quantities than the control vector pTT3 (Durocher, Y. et al. Nucleic Acids Res. 30:E9 (2002)). Similarly, the data shown in Fig.6 show that both vector skeleton pHybC and pHybE able to provide products of protein at a level exceeding the level provided by the control pTT3 vector, and comparable to the level provided by the control vector pBOS.

Example 4

Design vector pHyb-E, containing a constant plot antibodies

To facilitate the creation of vectors that can be used for the production of antibodies, using the new vector skeleton pHyb-E, get a set of twelve different vectors containing the heavy and light chains (General information presented in tables 2 and 3). Design twelve reference matrices vectors pHybE that provide Express the human and mouse IgG.

To obtain the vectors described in Fig-25, fragment SrfI/NotI size 6123 P.O. allot of pHybE-stuffer-hCgl,z,a (pJP167) and are ligated to the restriction fragments SrfI/NotI from pBOS vectors containing section that encodes the signal peptide, the "extra" fragment of lambda, and the sequence encoding the constant segment. To obtain fragments of the enzyme SrfI/NotI, conduct cleavage by enzymes SrfI/NotI, obtaining fragments insertions containing section that encodes the signal peptide, the "extra" fragment of lambda, and the sequence encoding the constant segment (sequence const plots are shown in table 1). These fragments derived from the template matrices pBOS that construct in plasmid DNA pEF-BOS (see Mizushima, S. and Nagata, S. Nucleic Acids Res. 18:5322 (1990); they are also described in the provisional application U.S. No. 60/878165, international application number PCT/US2007/026482, filed December 28, 2007, titled "DUAL-SPECIFIC IL-1A/IL-1b ANTIBODIES") and USSN 12/006068, which are included in this description by reference). Fragment insertions for the design of pHybE-hCl first modify by PCR with overlapping, to get a piece of the AfeI restriction on the 3'-end of the J-section, to facilitate cloning into this vector. All inserts are ligated into the design of pHyBE with a proven earlier sequence, pre-split SrfI and NotI to obtain the following vecto the s.

Then check the new sequence of vectors that contains the constant parts to confirm the presence of sequences encoding the constant sections of mouse and human antibodies (see SEQ ID nos:3-32).

All vectors are given in tables 2 and 3 contain "extra" sequence (the DNA of phage λ) of size ~1 so that it can be replaced by sequences encoding the variable areas. These new template vectors also contain a new section of the SwaI restriction immediately behind the SrfI above during reading. Specified new SwaI site can be useful to transfer open reading frame antibodies of the pHyb-E into other expression vectors, which contain the SwaI site to ensure cloning, such as expression vectors CHO. In addition to the universality of alternative sites cloning, these vectors are also compatible with existing pBOS vectors, pTT3 and CHO.

As shown in Fig.7, preliminary data on transfection of COS7 cells show that these additional SwaI site (the vectors v1) does not have a significant impact on the levels of expression of adalimumab compared with structures that do not contain additional plot SwaI (vector v2).

Conclusions:

The experiments described above in approx the arts 1-4, show that the vectors pHyb-C and pHyb-E can operate in several cell lines, providing abundant expression of the protein, the level of which often exceed the levels provided by the original pBOS vectors and pTT3. This increased expression is especially pronounced when the vector pHyb-E is used for expression of the hybrid protein mBR3-Fc produced by cells HEK-293-6E with low yield. The results show that the vectors pHyb-C and pHyb-E have a significant advantage when using vector technology in comparison with the previously used vectors.

Vectors pHyb, designated as version 1, contain additional plot SwaI above section SrfI restriction.

Vectors pHyb, designated as version 2, do not contain additional SwaI site.

Equivalents

Professionals in this field are known, or they can be installed using no more than routine experimentation, that there are many equivalents described herein specific embodiments of the present invention. It is assumed that such equivalents are included in the scope the following claims.

The incorporation by reference

The contents of all cited references (including literature references, patents, patent applications, and web sites)that may be mentioned on p is ateenyi of this application, specifically included in this description in its entirety for any purpose, as cited in these references. Unless otherwise specified, to implement the present invention using conventional techniques of immunology, molecular biology, cell biology, as well as the production and delivery of drugs is well known in this field. These methods include, without limitation, the methods described in the following publications.

1. The expression vector containing:
(a) origin of replication, OriP, derived from Epstein-Barr (EBV), where the origin of replication contains: 1) a symmetry element of the second order (DS); and 2) the site duplications (FR), which contains the binding site EBNA;
(b) the origin of replication of SV40;
(c) plot of insertion to insert a gene of interest;
(d) the promoter EF-1α, functionally associated with the site of insertion;
(e) signal poly-A;
(f) a bacterial origin of replication;
(g) breeding marker; and optionally containing
(h) a nucleic acid sequence encoding a constant region of a heavy or light chain antibodies, functionally associated with the site of insertion;
where the origin of replication OriP associated with the applicable external factor of replication initiation EBNA 1, which is not encoded by the expression vector.

2. The expression vector according to claim 1, where interest is Yong is a variable plot heavy or light chain of the antibody.

3. The expression vector according to claim 2, where the variable plot heavy or light chain of an antibody selected from the group consisting of mouse, gumanitarnogo, chimeric and human areas.

4. The expression vector according to claim 2, where the variable plot heavy chain antibody is a variable plot heavy chain antibodies selected from the group consisting of antibodies against TNFα antibodies against IL-18 antibodies against IL-12.

5. The expression vector according to claim 2, where the variable area light chain antibody is a variable area light chain antibodies selected from the group consisting of antibodies against TNFα antibodies against IL-18 antibodies against IL-12.

6. The expression vector according to claim 1, where the constant plot heavy chain antibody is murine or human.

7. The expression vector according to claim 1, where the constant plot heavy chain antibodies selected from the group consisting of gamma 1, z, a; gamma 1, z, non-a; gamma 2, n+; gamma 2, n-; and gamma 4.

8. The expression vector according to claim 7, where the constant plot heavy chain antibodies gamma 1, z, non-a additionally contains alanine mutation at position 234 (numbering system of the EU) const plot heavy chain.

9. The expression vector of claim 8, further containing alanine mutation at position 235 or 237 (numbering system of the EU) const plot heavy chain antibodies.

10. The expression vector according to claim 1, where the constant a plot of the light chain of the antibody is either human Kappa the isotype or human lambda isotype.

11. The expression vector according to claim 1, where the constant plot heavy chain antibody refers to either the mouse gamma 1 isotype, or to murine gamma 2A to isotype.

12. The expression vector according to claim 1, where the constant a plot of the light chain of the antibody belongs to the mouse the Kappa isotype.

13. The expression vector according to claim 1, where the constant plot heavy chain antibody is an Fc domain.

14. The expression vector according to claim 1, where breeding marker is a gene for resistance to ampicillin.

15. The expression vector according to claim 1, where the promoter EF-1α is a human.

16. The expression vector according to claim 1, where the promoter EF-1α contains nucleotides 76-1267 sequence SEQ ID NO: 2 or a nucleotide sequence that is at least 80%, 90% or 95% identical to nucleotides 76-1267 sequence SEQ ID NO: 2.

17. The expression vector according to claim 1, where the origin of replication OriP contains a nucleotide sequence that is at least 80%, 90% or 95% identical to nucleotides 1795-3545 sequence SEQ ID NO: 1.

18. The expression vector according to claim 1, where the origin of replication of SV40 contains nucleotides 5834-6140 sequence SEQ ID NO: 1 or a nucleotide sequence that, at m is re, 80%, 90% or 95% identical to nucleotides 5834-6140 sequence SEQ ID NO: 1.

19. The expression vector according to claim 1, where the expression vector contains the nucleotide sequence selected from the group comprising the sequences SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32, or the nucleotide sequence which is at least 80%, 90% or 95% identical to a sequence selected from the group comprising the sequences SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32.

20. The expression vector according to claim 1, where the expression vector contains the nucleotide sequence of SEQ ID NO: 2.

21. The expression vector according to claim 1, additionally containing a nucleotide sequence encoding a signal peptide.

22. Set for expression of recombinant protein in the culture of mammalian cells containing the vector according to claim 1.

23. Selected a host cell of a mammal for expression of recombinant protein in the culture of mammalian cells containing the vector according to claim 1.

24. Selected a host cell of a mammal according to item 23, which is a COS cell, or embryonic cell of the human kidney (HEK).

25. Selected a host cell of a mammal according to paragraph 24, which represents a COS7 cell or cell, HEK-293-6E.

26. A method of obtaining a recombinant protein, comprising century the decline in the expression vector according to claim 1 in selected cells of the host mammal, culturing the host cell of the mammal in suitable conditions ensuring the expression of the protein and isolating the protein.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: invention proposes a method for obtaining recombinant core protein of hepatitis E virus (rtHEV-ORF2) and recombinant vaccine for prophylaxis of hepatitis E virus. Core protein is obtained by cultivation of recombinant yeast strain Hansenula polymorpha "КБТ"-11/pHEV-001, which contains DNA sequence integrated into genom of yeast cell and coding the fragment of amino-acid sequence from position 86 to 607 of core protein of hepatitis E virus of genotype 3 (rtHEV-ORF2) under control of promoter of MOX gene. The method allows obtaining immunogenic antigene of hepatitis E virus, which has properties of natural protein. Based on the obtained antigene there created is recombinant vaccine for prophylaxis of hepatitis E virus. Vaccine includes effective amount of rtHEV-ORF2 protein, adjuvant and a physically acceptable diluter.

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FIELD: chemistry.

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SUBSTANCE: method is proposed to produce a polypeptide, including cell cultivation, which intensely expresses a bicarbonate carrier and has a transferred DNA, which codes the desired polypeptide.

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12 cl, 15 dwg, 6 ex

FIELD: chemistry.

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FIELD: biotechnology.

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FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. There are presented versions (aCt1 and aCt2) of one-domain antibodies specifically binding the Chlamydia trachomatis antigen. There are described versions of the method of inhibiting an infection caused by Chlamydia wherein the method involves the preparation of elementary bodies C.trachomatis by a therapeutically effective amount of the nanoantibody aCt1 or aCt2 before being attached to infected target cells.

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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.

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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.

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FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically connects segment M1' IgE and that induces apoptosis in IgE-expressing B-cells and its antigen-binding fragment. Besides, compositions and curing methods of IgE-mediated abnormalitiy, an item, a specific elimination method of IgE-producing B-cells, methods for prophylaxis and reduction of IgE products induced with an allergen, as well as isolated nucleic acid, an expression vector, a host cell and a method for obtaining an antibody as per the invention together with their use are considered.

EFFECT: invention can be further used in therapy of diseases associated with IgE.

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FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.

4 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: present inventions relate to protein engineering, plant molecular biology and pest control, as well as a hybrid insecticide protein and use thereof. Described is a hybrid insecticide protein which includes from the N-end to the C-end an N-end portion of Cry3A protein which is fused with the C-end portion of Cry1Ab protein, wherein the position of the crossover of the Cry3A protein and the Cry1Ab protein is located in a conservative block 2, in a conservative block 3 or in a conservative block 4 and has anti-western corn rootworm activity. Also disclosed are nucleic acid molecules which code the novel proteins, methods of producing proteins, methods for use thereof, as well as transgenic plants and seeds thereof which contain such proteins.

EFFECT: inventions enable to obtain cheap means of controlling Diabrotica worms.

39 cl, 8 dwg, 9 tbl, 46 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a humanized human monoclonal CD19 antibody prepared of an HB12B antibody, or a fragment thereof characterised by amino acid sequences of variable domains. Also, there are presented nucleic acids coding polypeptides having the sequences of the variable domains, and a cell expressing the antibody under the invention, and a pharmaceutical composition, and a method for treating a B-cell diseases or disorders in a human.

EFFECT: invention can find further application in treating various CD19-associated diseases, including autoimmune diseases, and preventing or treating the graft-versus-host disease (GVHD), and the humoral rejection and post-transplantation lymphoproliferative disorder in a human graft recipient.

21 cl, 45 dwg, 40 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant nucleic acid expresses one or several polypeptides of interest, a vector of expression and bacteria, which contain this recombinant nucleic acid. The recombinant nucleic acid contains a natural promotor of a gene of HU-like DNA-binding protein (PhilA) of Lactococcus type with the sequence SEQ TD NO:28, or its homological or functional version, which at least by 95% identical to the promotor with sequence SEQ ID NO:28, functionally linked with one or several open reading frames, heterological for the promotor RhIIA, where the promotor RhIIA is located above one or several open reading frames. The expression vector contains the above recombinant nucleic acid, preferably, the specified vector is produced from pTINX. A bacterium contains the above recombinant nucleic acid or the above vector.

EFFECT: proposed invention makes it possible to increase level of expression of polypeptide genes of interest and therefore produce sufficient number of expressed proteins.

19 cl, 26 dwg, 12 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant nucleic acid expresses one or several polypeptides of interest, a vector of expression and bacteria, which contain this recombinant nucleic acid. The recombinant nucleic acid contains a natural promotor of a gene of HU-like DNA-binding protein (PhilA) of Lactococcus type with the sequence SEQ TD NO:28, or its homological or functional version, which at least by 95% identical to the promotor with sequence SEQ ID NO:28, functionally linked with one or several open reading frames, heterological for the promotor RhIIA, where the promotor RhIIA is located above one or several open reading frames. The expression vector contains the above recombinant nucleic acid, preferably, the specified vector is produced from pTINX. A bacterium contains the above recombinant nucleic acid or the above vector.

EFFECT: proposed invention makes it possible to increase level of expression of polypeptide genes of interest and therefore produce sufficient number of expressed proteins.

19 cl, 26 dwg, 12 tbl, 9 ex

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