Nucleic acid synthetic molecule (versions), mammal cell expression vector, mammal host cell and method of expressing antigen protein of human epidermal growth factor-2 (her2/neu) or shortened form thereof

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.

EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.

8 cl, 10 dwg, 14 ex

 

The technical field to which the invention relates

The present invention in General relates to the treatment of cancer. More specifically, the present invention relates to synthetic polynucleotides coding genes associated with human tumor polypeptide antigen epidermal growth factor 2/neu, indicated in the present description hHER2.opt, and polynucleotides are codon-optimized for expression in the cellular environment of the person. The present invention also relates to synthetic polynucleotides encoding a shortened form of the antigen HER2/neu, in the present description designated as hHER2ECDTM.opt, and polynucleotides are codon-optimized for expression in the cellular environment of the person. The present invention also relates to recombinant vectors and hosts containing these synthetic polynucleotide. The present invention also provides adenoviral vector and plasmid constructs carrying hHER2.opt, and their use in vaccines and pharmaceutical compositions for the prevention and treatment of cancer.

The level of technology

Epidermal growth factor-2 is a transmembrane-associated tumor antigen encoded by the HER2/neu by protooncogenes (also called c-erbB-2), which is a member of a family of receptors epidermal what about the growth factor receptors on the cell surface. Gene HER2 was originally isolated from neurogeometry rats (Shih et al., Nature 290:261-264 (1981)and later cloned and described from human cells (Coussens et al., Science 230:1132-39(1985); King et al., Science 229:974-76 (1985)).

In addition, HER2/neu classified as a member of HER family receptor tyrosinekinase, which consists of four receptors involved in the regulation of cell growth and differentiation. The HER receptors help to maintain normal cell growth by binding of ligands growth factor in the form of dimers. In particular, the human HER2 forms heterodimer with other members of the EGFR family (HER1, her3, and HER4) (Klapper et al., Adv Cancer Res 77:25-79 (2000)). After the dimerization hHER2 and autophosphorylate tyrosine generated websites clutch for cytoplasmic signaling molecules and trigger the recruitment of secondary signaling molecules. Thus, initiates the intracellular signaling cascades that result in activation of genes that play an important role in cell growth.

Low levels of transcript expression of HER2/neu and encoded a protein of 185 kDa usually detected in adult epithelial cells of various tissues, including the skin, and mammary gland, and tissue of the gastrointestinal tract, genital tract and urinary tract (Press et al., Oncogene 5:953-962 (1990)). Higher levels of expression of HER2/neu also detect, in compliance is affected embryonic tissues during embryonic development (Press et al., see above).

Some studies allow us to consider the HER2 antigen an attractive target for active specific immunotherapy. First, usually overexpression or gene amplification of HER2/neu in various malignant tumors, such as breast carcinoma, ovarian, bladder, colon and prostate cancer, and adenocarcinoma of the lung (see Disis and Cheever, Adv. Cancer Research 71:343-371 (1997)). Overexpression of HER2/neu correlates with poor prognosis and with a higher frequency of relapse in cancer patients (Slamon et al., Science 244:707-712 (1989)). Amplification of the human HER2 leads to increased activity MAR-kinase and cell proliferation and promotes aggressive behavior of cancer cells (Ben-Levy et al., Embo J 13(14):3302-11 (1994)). High expression level of HER2 is observed in tumour cells, is the complete opposite of low level associated with normal Mature tissues.

In addition, many cancer patients suffering from malignant tumors, associated with overexpression of HER2/neu, had immune responses to the protein HER2. Anti-hHER2 cytotoxic T lymphocytes (CTL) were isolated from patients with breast cancer and ovarian cancer (Ioannides et al., Cell Immunol 151(1):225-34 (1993); Peoples et al., Proc Natl Acad Sci USA 92(14):6547-51 (1995)). Identified some HLA-A2.1-associated peptides hHER2, and peptide-specific is by T cells can be generated in vitro (Fisk et al., Cancer Res 57(1):8-93 (1997); Yoshino et al., Cancer Res 54(13):3387-90 (1994); Lustgarten et al., Hum Immunol 52(2):109-18 (1997)).

The above data demonstrate that cancer patients are activated anti-ErbB-2 immune effector mechanisms, and highlight the potential advantage augment immune responsiveness. An effective vaccine that causes the immune response to HER2/neu, should enhance this resistance level, which is a protective and/or preventive and overcome samotolerantnosti.

Based on the above, HER2/neu has been investigated as a target for the development of immunological therapy of malignant tumors. Anti-HER2 monoclonal antibodies was investigated as a means of therapy for breast cancer, while approaches using each of the antibodies showed different levels of success (see discussion Yarden, Oncology 61 (suppl 2):1-13 (2001)).

In addition, it was reported about vaccines based on peptides and DNA, targeted to HER2/neu. Amici et al. (U.S. patent 6127344) disclose a method of inducing immunity to HER2/neu by introducing the expression vector containing the cDNA of human HER2/neu full length, functionally associated with the promoter of human cytomegalovirus. Morris et al. (WO 2004/041065) disclose a method of vaccination of dendritic cells modified adenoviral vectors expressing the non-signaled the gene HER2/neu. Cheever and isis disclose methods of immunization of people peptides against HER2 HER2/neu-associated cancers (U.S. patent 5846538). In addition, vaccines based on peptides HER2/neu have been studied in rodent models (for example, see Disis and Cheever, Adv. Cancer Res. 71:343-71 (1997)).

Development and commercialization of many vaccines prevented the difficulties associated with obtaining high levels of expression of exogenous genes in successfully transformed organisms owners. Therefore, despite the identification of the nucleotide sequences of the wild-type protein coding hHER2 described above, would be highly desirable to develop an easily reproducible source of human HER2 protein, which uses hHER2-encoding nucleotide sequence is optimized for expression in the intended host cell, and this provides an opportunity for the development of cancer vaccine, which is effective and which does not prevent samotolerantnosti.

The invention

The present invention relates to compositions and methods for the generation or amplification of immune proteins expressed by the genome of the human HER2, which is associated with numerous types of adenocarcinomas, including breast cancer and ovarian cancer. In particular, the present invention provides polynucleotide encoding human HER2 protein or a shortened form of the human HER2 protein, which contains vneck mocny and transmembrane domains of the protein HER2 (hereinafter in the present description hHER2ECDTM), moreover, these polynucleotides are codon-optimized for high level expression in human cells. The present invention also provides adenoviral vector and vector-based plasmids containing synthetic polynucleotide, and discloses the use of these vectors in immunogenic compositions and vaccines for the prevention and/or treatment of HER2-associated cancer. Polynucleotide disclosed in the present description, are more effective than polynucleotide HER2 wild type, in the development of cellular and humoral immune response to human HER2.

The present invention also relates to synthetic nucleic acid molecules (polynucleotides)containing a sequence of nucleotides that encode a human antigen epidermal growth factor-2 (hereinafter in the present description hHER2)as shown in SEQ ID NO:2, and the synthetic molecules of the nucleic acid is codon-optimized for high level expression in a human cell (hereinafter in the present description hHER2.opt). The present invention also relates to synthetic nucleic acid molecules (polynucleotides)containing a sequence of nucleotides that encode human HER2ECDTM as shown in SEQ ID NO:14, and synthetic nucleic acid molecule I is are codon-optimized for high level expression in human cells. The nucleic acid molecule disclosed in the present description, can be transliterowany in the selected host cells, and recombinant host cell provides a source for significant levels of expressed functional protein hHER2 (SEQ ID NO:2) or protein hHER2ECDTM (SEQ ID NO:14).

The present invention also relates to a synthetic nucleic acid molecule, which encodes mRNA which expresses a human HER2 protein. A preferred aspect of this part of the present invention disclosed in figure 1, which shows the DNA (SEQ ID NO:1)encoding a protein hHER2 (SEQ ID NO:2). Preferred nucleic acid molecule of the present invention is codon-optimized for high level expression in human cells. The sequence of this preferred polynucleotide also contains a mutation that inhibits the activity of tyrosine kinase (AAA2257GCC, K753A). Nucleotide sequences that do not contain this mutation also included in the scope of the present invention.

The present invention also relates to a synthetic nucleic acid molecule, which encodes mRNA which expresses a human protein HER2ECDTM. A preferred aspect of this part of the present invention disclosed in figa, which shows the DNA (SEQ ID NO:9), which encodes hHER2ECDTM (SEQ ID NO:14). Preferred nucleic acid molecule of the present invention is codon-optimized for high level expression in human cells.

The present invention also relates to recombinant vectors and recombinant host cells, both prokaryotic and eukaryotic, which contain the nucleic acid molecule disclosed in the present description.

The present invention also relates to a method of expression of a codon-optimized human HER2 protein in the recombinant host cell, involving: (a) introducing a vector containing a synthetic polynucleotide encoding human HER2 protein in a suitable host cell, and synthetic polynucleotide is codon-optimized for optimal expression in a human cell; and (b) culturing the host cell under conditions which allow for expression of the indicated human HER2 protein.

The present invention also relates to a method of expression of a codon-optimized human protein HER2ECDTM in the recombinant host cell, involving: (a) introducing a vector containing a synthetic polynucleotide encoding human protein HER2ECDTM, in a suitable host cell, and synthetic polynucleotide is codon-optimized DL is optimal expression in a human cell; and (b) culturing the host cell under conditions which allow for expression of the indicated human protein HER2ECDTM.

Another aspect of the present invention is a method of prevention or treatment of cancer, involving the administration to a mammal a vaccine vector containing a synthetic molecule of nucleic acid, and synthetic nucleic acid molecule contains a nucleotide sequence which encodes a human protein-antigen (hHER2) epidermal growth factor-2 as shown in SEQ ID NO:2, or human protein HER2ECDTM as shown in SEQ ID NO:14, and a synthetic molecule of nucleic acid is codon-optimized for high level expression in human cells.

The present invention also relates to adenoviral vaccine vector containing the adenoviral genome with a deletion in the region E1 and the insert in the area E1, and the insert contains an expression cassette, comprising: (a) a codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (b) a promoter functionally linked with polynucleotides.

The present invention also relates to a vaccine plasmid, the plasmid containing part and the cassette expression, and part of the expression cassette contains: (a) Sint is political polynucleotide, encoding human HER2 protein or a human protein HER2ECDTM, and synthetic polynucleotide is codon-optimized for optimal expression in a human cell; and (b) a promoter functionally linked with polynucleotides.

Another aspect of the present invention is a method of protecting a mammal from cancer or treatment of a mammal suffering from HER2-associated cancer, providing: (a) introducing to a mammal a first vector comprising: i) a codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (ii) a promoter functionally linked with polynucleotides; (b) waiting for a predetermined period of time; and (C) introduction to the mammal a second vector comprising: i) a codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (ii) the promoter functionally associated with polynucleotides.

As used in this description and the attached claims, the singular number include reference to the plural, unless the context clearly otherwise specified.

As used in this description and the attached claims, used the following definitions and abbreviations:

The term “promoter” refers to the website known the project on DNA strand, to bind RNA polymerase. The promoter generates initiating complex with RNA polymerase to initiate and control the transcriptional activity. The complex can be modified by activating sequences called enhancers, or by inhibiting sequences, called “silent”.

The term “cassette” refers to a nucleotide or gene sequence, which must be expressed from the vector, such as a nucleotide or gene sequence encoding a protein or HER2 protein HER2ECDTM. In General, the cassette contains the coding sequence of a gene integrated in the vector, which in some embodiments, the implementation provides a regulatory sequence for expression of the nucleotide or gene sequence. In other embodiments, implementation of the nucleotide or gene sequence provides a regulatory sequence for the expression. In other embodiments, implementation of the vector provides some regulatory sequences, and the nucleotide or gene sequence provides other regulatory sequences. For example, the vector may provide a promoter for transcription of the nucleotide or gene sequence, and the nucleotide or gene sequence p is dostavljaet sequence termination of transcription. Regulatory sequences that can be provided by the vector include, without limitation, enhancers, sequences, transcription termination, acceptor and donor sequences for splicing, introns, sequences for binding to the ribosome and additional poly(A)sequences. The cassette is in principle similar to a cassette tape; each cassette has its own sequence. Thus, when changing the cassette vector will Express another sequence. Due to the restriction sites on the 5' and 3' ends of the cassette can be easily inserted, removed or replaced by another cartridge.

The term “vector” refers to some means by which DNA fragments can be introduced into the host organism or host tissue. There are different types of vectors, including plasmids, viruses (including adenoviruses), bacteriophages and Comedy.

The term “first generation”, as used in respect of adenoviral vectors, describes these adenoviral vector, which are replication-defective. First generation adenoviral vectors usually has deleteriously or inactivated gene E1 and preferably has deleteriously or inactivated gene E3.

The designation “pV1J-hHER2.opt” refers to a plasmid is the second design, disclosed in the present description, containing early (IE) promoter, CMV person with intron a, a codon-optimized gene human HER2 full length, sequence, polyadenylation and transcription termination, selected from growth hormone in cattle, and a minimum basis pUC (see example 2).

The designation “pV1J-hHER2ECDTM.opt” refers to a plasmid constructs disclosed in the present description, containing early (IE) promoter, CMV person with intron a, a shortened codon-optimized gene human HER2 containing the extracellular and transmembrane domains of HER2 gene, sequence, polyadenylation and transcription termination, selected from growth hormone in cattle, and a minimum basis pUC (see example 2).

The designation “pV1J-hHER2.wt” refers to the construction as described above, except that this design contains a gene of the human HER2 full length wild-type instead of the codon-optimized gene human HER2.

The designation “pV1J-hHER2ECDTM.wt” refers to the construction as described above, except that this design contains a truncated HER2 gene human wild-type, and the specified shortened gene contains a nucleotide sequence that encodes the extracellular and transmembrane domains of the protein HER2, instead of the codon-optimizirovannuyu human HER2 full length.

Indicate “MRKAd5-hHER2.opt”, “MRKAd5-hHER2ECDTM.opt” and “MRKAd5-hHER2.wt” refer to the three structures disclosed in the present description, which contains the Ad5 adenovirus genome with deleteregvalue areas E1 and E3. In design “MRKAd5-hHER2.opt” plot E1 replaced by the codon-optimized gene human HER2 full length in a direction parallel to E1, under the control of the CMV promoter without intron a, followed by the polyadenylation signal of growth hormone in cattle. Design “MRKAd5-hHER2ECDTM.opt”, essentially, is the same as described above except that the area E1 of the Ad5 genome substituted codon-optimized truncated version of the human gene HER2, and truncated HER2 gene contains a nucleotide sequence that encodes the extracellular and transmembrane domains of the HER2 receptor protein. Design “MRKAd5-hHER2.wt”, essentially, is the same as described above except that the area E1 of the Ad5 genome replaced by the sequence of the human HER2 wild-type full length (see example 11).

The term “effective amount” means a sufficient amount of a vaccine composition that is administered to produce adequate levels of the polypeptide to obtain an immune response. Specialists in the art it is known that this level may vary.

The term “treatment” is tositsa as to therapeutic, and to prophylactic treatment or prevention. Those in need of treatment include those who already have the disorder, as well as those who are predisposed to the disorder or those in whom the disorder should be warned.

“Disorder” is any condition which is improved by treatment methods or vaccines and immunogenic compositions disclosed in the present description. This term includes chronic and acute disorders or diseases including such pathological conditions in which mammals are genetically predisposed to the disorder. Methods and vaccines of the present invention are intended to treat disorders or conditions associated with impaired HER2/neu-associated expression or signaling, including, without limitation, breast cancer, cancer of the colon, rectum, stomach cancer, ovarian cancer and lung cancer.

“Conservative amino acid substitution” refers to substitution of one amino acid residue other, chemically similar, amino acid residue. Examples of such conservative substitutions are: the substitution of one hydrophobic residue (isoleucine, leucine, valine or methionine) another; the substitution of one polar residue other polar residue with the same charge (e.g., arginine, lysine; glutamic the acid aspartic acid).

“hHER2.wt” and “hHER2.opt” refers to a human antigen epidermal growth factor-2 and codon-optimized human antigen epidermal growth factor-2, respectively.

“hHER2ECDTM.wt” and “hHER2ECDTM.opt” refers to a shortened human antigen epidermal growth factor-2 and shortened the codon-optimized human antigen epidermal growth factor-2, respectively. Truncated forms of HER2, “hHER2ECDTM.wt” and “hHER2ECDTM.opt”, containing the extracellular and transmembrane domains of the human HER2 protein.

The term “mammal” refers to any mammal species, including humans.

The abbreviation “Ag” refers to the antigen.

The abbreviation “Ab” and “mAb” refers to the antibody and the monoclonal antibody, respectively.

The abbreviation “ORS” refers to the open reading frame of the gene.

Brief description of drawings

Figure 1 shows the nucleotide sequence of the codon-optimized polynucleotide (hHER2.opt, SEQ ID NO:1)which encodes a human protein HER2. Cm. example 1. Panel b shows the installed amino acid sequence of the human HER2 protein (SEQ ID NO:2).

Figure 2 shows the identification of immunodominant T-cell epitopes in the human HER2 protein by analysis of the ELISPOT and intracellular staining (ICS). BALB/c mice, immunition the e Ad5-hHER2, were analyzed for the induction of human HER2-specific cellular immunity. The number of T cells secreting IFN-γ against human HER2 was determined by ELISPOT method in splenocytes from groups of mice (listed in the first column) using pools or single peptides. Displayed data are representative of several independent experiments. Values expressed as number of colonies forming the spot (SFC)/106the total number of splenocytes minus background values determined in the absence of peptides (usually less than 10 SFC/106the total number of splenocytes). Number, more than three times above the background measured in the control experiments without antigenic peptides were considered as positive values and are shown in bold. The frequency of T cells CD4+or CD8+secreting IFN-γ, measured manner ICS. Displayed data are representative of several independent experiments. Values are expressed as 1000×[(IFN-γ CD3+and CD4+or CD8+)/(CD3+and CD4+or CD8+)]. Values larger than 1%, was seen as a positive and bold. The sequence covered by the pool or a single peptide used in the studies are listed on the left. The numbers refer to the position of amino the PCI-e slot remnant of the human HER2 protein.

Figure 3 shows expression in vitro hHER2 after transfection in (A) human embryonic stem cells HEK-293 kidney and (B) murine cultured myoblasts SS. Data are expressed as geometric mean channel fluorescence minus signal generated by the empty plasmid pV1JnsA. For cells SS data normalized to the efficiency of transfection with pEGFP DNA.

Figure 4 shows the immune response to human HER2 in BALB/c mice. Panel (A) shows that the codon-optimized HER2 gave significantly improved values ELISPOT compared to wild-type HER2. Shows the results of immunization of four groups, each of which contains two mice, a plasmid pV1J-hHER2.wt or pV1J-hHER2.opt (50 μg/dose, entered by way of electroinjection in the quadriceps muscle). Two weeks after the last injection was determined by the frequency of IFN-γ-secreting T cells in murine splenocytes by analyzing IFN-γ ELISPOT using peptides hNeu15.3 (AA 63-71, including epitope CD8+), hNeu301 (AA 1202-1214, including epitope CD8+and hNeu42 (AA 165-179, including epitope of CD4+). Shows the results of the 2.5×105and 5×105the splenocytes with two replicas of each tested number. Averages calculated by subtracting the background level, as defined in the absence of peptides (usually less than 10 SFC/106the total number of splenocytes). The results are expressed as the number is and SFC/10 6the total number of splenocytes. Panel (C) shows that pV1J-hHER2.opt produces significantly enhanced IgG1 and IgG2 humoral response in comparison with pV1J-hHER2.wt. Serum samples were collected at week 6 (the day before the first immunization, the preliminary drawing blood) and 14 week (two weeks after last injection) from the groups consisting of 4 mice immunized with pV1J-hHER2.wt or pV1J-hHER2.opt plasmid DNA. The titers of anti-hHER2 antibodies in pooled sera from each group of mice was measured using ELISA using dimeric extracellular domain hHER2 (HER2-ECD) as the target antigen. For the detection of related mouse antibodies used AP-conjugated goat anti-mouse IgG1 or IgG2.

Figure 5 shows a comparison R-specific T-cell response generated in mice by immunization with pV1J-HER2 and Ad5-HER2. Mouse BALB/c wild-type and transgenic mice BALB/c, sverkhekspressiya rat HER2 (labeled NeuT, see Lucchini et al., Cancer Lett 64(3):203-9 (1992))were immunized at the age of 6 and 9 weeks or pV1J-hHER2.wt DNA (50 μg/dose, injected in the quadriceps muscle) with subsequent stimulation, or Ad5-hHER2.wt. At the age of 12 weeks were determined by the number of IFN-γ-secreting anti - human cells using ELISPOT analysis of pools of mice, using these peptides. Displayed data are representative of several is independent experiments. Values are expressed as in figure 1.

Panel And 6 shows the nucleotide sequence of the codon-optimized polynucleotide (hHER2ECDTM.opt, SEQ ID NO:9), which encodes a truncated human HER2 protein, and this protein contains extracellular and transmembrane domains of the protein HER2. Panel b shows the second polynucleotide, which encodes the extracellular and transmembrane domains of the protein HER2, and the second polynucleotide contains the nucleotide sequence of the wild type, which was not codon-optimized (hHER2ECDTM.wt, SEQ ID NO:10).

7 shows the result of analysis of the cell-mediated response induced in macaques-RH immunized with a mixture of three plasmids expressing human HER2 antigens, CEA and EpCAM, and these plasmids contain nucleotide sequences that are codon-optimized for high level expression in human cells. The same animals were then immunized with a mixture of three Ad5 vectors expressing the sequence of wild-type and each of the three antigens. Cell-mediated immune response directed against vysokogomogennogo (level 98.2% sequence similarity) of the protein HER2 macaque-rhesus, was measured using IFN-γ ELISPOT monthly for one year. Values are expressed as SFC/106PBMC minus nowych values, certain in the absence of peptides. Values that differed significantly (p<0,05) from the background, as measured in the control experiments without antigenic peptides, and exceeded arbitrarily selected threshold value 55 SFC/106PBMC is bold.

On Fig shows a comparison of cell-mediated immune response generated in mice by immunization with pV1J-hHER2.opt and pV1J-hHER2ECDTM.opt. The values refer to the frequency of IFN-γ-secreting spleen cells as measured by ELISPOT. Displayed data obtained from three animals and are representative of several independent experiments. Values are expressed as SFC/106the total number of spleen cells minus background values determined in the absence of peptides (usually less than 5 SFC/106the spleen cells). Values that differed significantly (p<0,05) from the background, as measured in the control experiments without antigenic peptides, and exceeded arbitrarily selected threshold 25 SFC/106the spleen cells, are shown in bold.

Detailed description of the invention

Human epidermal growth factor-2 (hHER2) is usually associated with several different types of tumors, including breast carcinoma, ovarian, stomach and colon. The present invention from OSISA to compositions and methods for producing or enhancing immunity to protein products, expressed gene hHER2, and impaired expression of hHER2 associated with carcinoma or its development. The Association violated the expression of hHER2 with carcinoma does not require protein hHER2 expressively in tumor tissue constantly during its development, because the abnormal expression of hHER2 may be present at the initiation of the tumor and not be detected later during tumor development or Vice versa.

To this end provided with a synthetic DNA molecules encoding human HER2 protein of full length or truncated human HER2 protein, referred to in this description as HER2ECDTM. Specified truncated HER2 contains the extracellular and transmembrane domains of the human HER2 protein. The codons of the synthetic DNA molecules designed to use codons that are preferred for the intended host cell, which in preferred embodiments is a human cell. The synthetic molecules may be used for the development of vaccines based on plasmid or recombinant adenovirus, which provide effective immunoprophylaxis against HER2-associated cancer by neutralizing antibodies and cell-mediated immunity. The synthetic molecules may be used in immunogenic compositions. The present invention provided yet polynucleotide, which by direct introduction in vivo vertebrate, including mammals such as primates and humans, induce the animal expression of the encoded proteins.

Was published nucleotide sequence of human wild-type HER2 (Coussens et al., Science 230:1132-39 (1985); King et al., Science 229:974-76 (1985)). The present invention provides a synthetic DNA molecules encoding human HER2 protein of full length or truncated human protein HER2, HER2ECDTM containing the extracellular and transmembrane domains hHER2. Synthetic molecules of the present invention contain a sequence of nucleotides, and at least one nucleotide modified to use codons that are preferred for human cells, thus providing an opportunity for the expression of high levels hHER2 or hHER2ECDTM in human host cells. The synthetic molecules may be used as a source of protein hHER2 or hHER2ECDTM, which can be used in anti-cancer vaccine to provide effective immunoprophylaxis against hHER2-associated carcinomas by neutralizing or antibody-based test and cell-mediated immunity. Alternatively, synthetic molecules may be used as the basis of the DNA vaccine or adenovirus vaccine.

“Triplet” codon of the four is possible nucleotide bases can exist in more than 60 different forms. Because these codons provide a message only 20 different amino acids (as well as the initiation and termination of transcription), some amino acids can be encoded by more than one codon - a phenomenon known as the degeneracy of the codon. By incompletely understood reasons, alternative codons are not represented equally in the endogenous DNA of different cell types. In fact, it turns out that there is a variable natural hierarchy or preference for certain codons in specific types of cells. As one example, the amino acid leucine is identified by any of the six DNA codons, including CTA, CTC, CTG, CTT, TTA and TTG. A comprehensive analysis of the genomic frequencies of codons for microorganisms revealed that endogenous E.coli DNA often contains CTG leucine-specific codon, while the DNA of yeast and myxomycetes often includes TTA leucine-specific codon. From the point of view of this hierarchy is usually assumed that the probability of obtaining high levels of expression of polypeptides rich in leucine, using the host E. coli to some extent will depend on the frequency of use of the codon. For example, there is a probability that the gene-rich codons TTA, is not sufficiently expressed in E. coli, whereas the gene rich CTG may be vysokoagressivnyh in this host. Similarly, prefer inim the codon for expression of the polypeptide, rich in leucine, in yeast host cells may be TTA.

The value of the phenomenon of codon preferences detected using methods of recombinant DNA, and this phenomenon may explain many of the early failures in achieving high levels of expression of exogenous genes in successfully transformed organisms owners - less “preferred” codon may be represented more than once in the gene of interest, and the mechanism of a host cell that implements the expression, may not work effectively. This phenomenon suggests that synthetic genes, which are designed in such a way that they comprise codons preferred for the intended host cell, provide the best form of alien genetic material to apply the techniques of recombinant DNA. Thus, one aspect of the present invention is a gene of the human HER2, which is codon-optimized for high level expression in human cells. In a preferred embodiment of the present invention, it was found that the use of alternative codons that encode the same protein sequence, can eliminate restrictions on the expression of exogenous protein hHER2 in human cells. Another aspect of the present invention is shortened gene the human is th HER2, hHER2ECDTM, which is codon-optimized for high level expression in a human host cell, and the specified truncated HER2 gene contains a nucleotide sequence that encode the extracellular and transmembrane domains of human HER2.

According to the present invention the sequence of the human gene and HER2 gene sequences for human HER2ECDTM were transformed into a polynucleotide sequence having identical to the transmitted sequence compared to the equivalent wild-type, but with the alternative usage of the codon, as described by Lathe “Synthetic Oligonucleotide Probes Deduced from Amino Acid Sequence Data: Theoretical and Practical Considerations” J. Molec. Biol. 183:1-12 (1985), which is incorporated into this description in its entirety by reference. Methodology in General consists of the identification of codons in the sequence of the wild type, which is usually not associated with vysokoagressivnyh human genes and replacing them with optimal codons for high expression in human cells. These optimal codons are referred to in the present description codons preferred for a person.” Then the new gene sequence is tested against unwanted sequences generated by substitutions codons (for example, the sequence “ATTA”, a random image is of the recognition sites of splicing intron, unwanted sites of restriction enzymes, high GC content etc). Unwanted sequences are removed by replacing the existing codons other codons encoding the same amino acid. Then the synthetic gene segments are tested for improved expression.

The methods described above were used to create synthetic gene sequences for human HER2 and human HER2ECDTM, resulting in a gene of full length and truncated gene containing codon-optimized for high level expression in human cells. Although the above procedure in General provides the methodology of the authors for the development of the codon-optimized genes for use in anti-cancer vaccines, specialists in the art it is clear that a similar vaccine efficacy and increased expression of genes can be achieved by small changes in the procedure or by small changes in the sequence. Specialists in the art also will understand that additional DNA molecules can be designed to provide high levels of expression of hHER2 or hHER2ECDTM in human cells, and only a portion of the codons of the DNA molecules are codon-optimized. The nucleic acid molecule of the present invention, with the society, do not have other nucleic acids.

Therefore, the present invention relates to synthetic polynucleotide containing the nucleotide sequence encoding human HER2 protein, such as human HER2 protein shown in SEQ ID NO:2, or a biologically active fragment or mutant form of the human HER2 protein, and a polynucleotide sequence contains codons optimized for expression in a human host. These mutated form of the protein hHER2 include, without limitation, conservative amino acid substitutions, amino-terminal truncation, carboxy-terminal truncation, deletion or insertion. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least essentially mimics the immunological properties of the protein hHER2 as shown in SEQ ID NO:2. Synthetic polynucleotide of the present invention encode mRNA molecules, which leads to the expression of functional human protein so that it is suitable for the development of therapeutic or prophylactic cancer vaccine.

Preferred polynucleotide of the present invention is polynucleotide containing a nucleotide sequence encoding a truncated human protein HER2CDTM (SEQ ID NO:14), moreover, the polynucleotide sequence contains codons optimized for expression in a human host. Particularly preferred polynucleotide of the present invention contains a nucleotide sequence as shown in SEQ ID NO:9.

The present invention also relates to a synthetic molecule of nucleic acid (polynucleotide)containing a nucleotide sequence that encodes an mRNA that leads to the expression of human HER2 protein, such as human HER2 protein full length, as shown in SEQ ID NO:2, or a shortened protein HER2ECDTM, for example, a sequence HER2ECDTM as shown in SEQ ID NO:14. Synthetic nucleic acid molecule of the present invention are codon-optimized for high level expression in a human host cell.

Also in the scope of the present invention included the codon-optimized polynucleotide containing a nucleotide sequence which encodes a variant HER2 polypeptide having at least 90% identity with the amino acid sequence SEQ ID NO:2, which may include up to Naamino acid substitutions along the entire length of SEQ ID NO:2, and Narepresents the maximum number of amino acid substitutions and is calculated by the formula

Na=Xa(XaY)

where Xandis own the th total number of amino acids in SEQ ID NO:2, and Y has a value of 0.90, and any work Xandand Y, which is not an integer, round to the nearest integer prior to subtracting it works from the Xand. Similarly, the present invention also considers the codon-optimized nucleotide sequences encoding variants of the polypeptide HER2CDTM as shown in SEQ ID NO:14.

The present invention also relates to recombinant vectors, and moreover, addition of the cells of the host, as prokaryotic and eukaryotic, which contain the nucleic acid molecule disclosed in the present description. Synthetic DNA molecules associated vector and the hosts of the present invention are suitable for development of anticancer vaccines.

Preferred DNA molecule of the present invention includes the nucleotide sequence disclosed in the present invention as SEQ ID NO:1 (shown in figure 1), which encodes the human HER2 protein shown in figure 2 and are shown as SEQ ID NO:2. The nucleotide sequence shown in SEQ ID NO:1, was codon-optimized for optimal expression in human cells. In order to avoid the difficulties associated with PCR amplification, in this embodiment of the present invention applied design with less hard optimization for sequence hHER2 positions 3601 and 3805, in which reduced GC content, while maintaining the same amino acid composition. Cm. example 5.

Another preferred DNA molecule of the present invention includes the nucleotide sequence disclosed in the present description as SEQ ID NO:9 (shown in figa), which encodes a human protein HER2ECDTM shown in SEQ ID NO:14. The nucleotide sequence shown in SEQ ID NO:9, was codon-optimized for optimal expression in human cells.

Specialist in the art understands that there may be a sequence of HER2, which will be codon-optimized for high level expression in a human cell, provided that one or more codons are replaced with codons preferred for a person. Preferably, at least 80% of the codons containing synthetic nucleotide sequence of the HER2 present invention was codons preferred for a person. More preferably, at least about 85% of the codons was preferred for a person, and even more preferably at least about 90% of the codons was preferred for a person.

The present invention also includes biologically active fragments or mutants of SEQ ID NO:1 that encode mRNA, th is leads to the expression of human HER2 protein. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of the protein hHER2, including, but not limited to, protein hHER2 as shown in SEQ ID NO:2. Any such polynucleotide includes, but is, certainly, without limitation: replacement of nucleotide deletions, insertions, amino-terminal truncation and carboxy-terminal truncation. The mutants of the present invention encode mRNA molecules, which leads to the expression of functional protein hHER2 in eukaryotic cells so that it is suitable for the development of therapeutic or prophylactic cancer vaccine.

The present invention also relates to a synthetic codon-optimized DNA molecules that encode a protein hHER2 or protein hHER2ECDTM, and the nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:9, but still encodes a protein hHER2 as shown in SEQ ID NO:2, or a protein hHER2ECDTM as shown in SEQ ID NO:14. Such synthetic DNA are within the scope of the present invention. Therefore, the present invention discloses the degeneracy of codons, which as a result can lead to a multitude of DNA molecules expressing an identical protein. In about the eating of the present invention also includes a mutation in the DNA sequence, which essentially do not change the basic physical properties of the expressed protein. For example, the replacement of valine with leucine, arginine, lysine or asparagine-glutamine may cause changes to the functionality of the polypeptide.

It is known that the DNA sequence encoding the peptide may be modified so as to encode a peptide, which has properties different from the properties of the natural peptide. How to change DNA sequences include, but are not limited to, site-directed mutagenesis. The modified examples of properties include, without limitation, changes in the affinity of the enzyme to the substrate or receptor ligand.

The present invention also relates to fused structures hHER2 and hHER2ECDTM, including, without limitation, fused constructs that Express a part of the human HER2 protein connected with various markers, including, but in no way limited to, GFP (green fluorescent protein), MYC epitope, GST and Fc. Any such merged design can be expressed in a certain cell lines and used for screening of modulators of the human HER2 protein disclosed in the present description. Also considered merged constructs that are designed to enhance the immune response against human HER2, including, without limitation: DOM, hsp70 and LTB.

Nastoyascheevremya also relates to recombinant vectors, which contain molecules of synthetic nucleic acids disclosed in the present description. Such vector may contain DNA or RNA. For most purposes, the preferred cloning vector DNA. Normal vectors include plasmids, modified viruses, baculoviruses, bacteriophages, Comedy, synthetic chromosomes of yeast and other forms episomal or integrated DNA that can encode a protein hHER2 or protein hHER2ECDTM. Specialists in the art can easily determine suitable vector for transfer or other application specific gene.

The expression vector containing the codon-optimized DNA encoding the protein hHER2, can be used for the expression of high levels hHER2 in the recombinant host cells. In addition, the expression vector containing the codon-optimized DNA encoding the protein hHER2ECDTM, can be used for the expression of high levels hHER2ECDTM in the recombinant host cells. Expression vector may include, without limitation, the cloning vector, the modified vector cloning, specifically designed plasmids or viruses. Also, if this is necessary for the expression of recombinant hHER2 or hHER2ECDTM in bacterial cells can be used a variety of bacterial expression vectors. In addition, the set of vectors ex is ressie fungi cells can be used for expression of recombinant hHER2, or hHER2ECDTM in the cells of fungi. In addition, many expression vectors of insect cells can be used for expression of recombinant protein in insect cells.

The present invention also relates to host cells transformed or transfitsirovannykh vectors containing the nucleic acid molecule of the present invention. Recombinant host cells may be prokaryotic or eukaryotic, including, without limitation, bacteria, such as E. coli, the cells of fungi, such as yeast, mammalian cells, including but not limited to, cell lines derived from cells of cattle, pigs, monkeys and rodents; insect cells, including, but not limited to, Drosophila, and cell lines derived silkworm mulberry. Such recombinant host cells can be cultured in suitable conditions for production of hHER2 or hHER2ECDTM, or a biologically equivalent form. In a preferred embodiment of the present invention the host cell is a human. As defined in the present description, the term “host cell” does not include the host cell in the body transgenic human, transgenic human fetus or human transgenic embryo.

As indicated above, the expression vector containing DNA encoding a protein hHER2 or protein hHER2ECDTM can be used for the expression of hHER2 or hHER2ECDTM in the recombinant host cells. Therefore, another aspect of the present invention is a method of expression of human HER2 protein or a human protein HER2ECDTM in the recombinant host cell, involving: (a) introducing a vector containing the codon-optimized nucleic acid which encodes a human HER2 protein or a human protein HER2ECDTM, into a suitable human host cell; and (b) culturing the host cell under conditions that allow expression of the indicated human HER2 protein or a specific human protein HER2ECDTM.

The preferred implementation of this aspect of the present invention provides a method of expression of human HER2 protein in the recombinant host cell, involving: (a) introducing a vector containing a nucleic acid as shown in SEQ ID NO:1, into a suitable human host cell; and (b) culturing the host cell under conditions that allow expression of the indicated human HER2 protein.

Another preferred implementation of this aspect of the present invention provides a method for expression of human protein HER2ECDTM in the recombinant host cell, involving: (a) introducing a vector containing a nucleic acid as shown in SEQ ID NO:9, a suitable human host cell; and (b) culturing the cells and the host in the conditions, which allow the expression of the indicated human protein HER2ECDTM.

After the expression of hHER2 or hHER2ECDTM in the host cell protein hHER2 or hHER2ECDTM can be allocated in the form of protein in active form. Some procedures protein purification are available and suitable for use. Recombinant protein hHER2 or protein hHER2ECDTM can be purified from cell lysates and extracts by various combinations of, or individual application of vysalivaniya, ion exchange chromatography, gel filtration, adsorption chromatography on a column of hydroxylapatite and hydrophobic chromatography. In addition, the recombinant protein can be separated from other cellular proteins by using immunoaffinity column made with monoclonal or polyclonal antibodies specific for the protein hHER2 full length, or polypeptide fragments of the protein hHER2.

Nucleic acids of the present invention can be assembled in an expression cassette, which contains sequences that are designed to ensure efficient expression of the protein in a human cell. In one of the embodiments of the present invention, the cassette contains a codon-optimized gene hHER2 full length with related sequences control transcription and translation, functionally related, such as a promotion is R and the sequence termination. In the second embodiment of the present invention, the cassette contains a truncated HER2 gene, HER2ECDTM, which encodes the extracellular and transmembrane domains of the human HER2 protein. In preferred embodiments, the implementation of the promoter is a cytomegalovirus promoter without intron And sequence (CMV), although specialists in the art it is known that can be used any number of other known promoters, such as a strong promoter of the immunoglobulin or other eukaryotic promoter of the gene. Preferred transcription terminator is a terminator of growth hormone in cattle, although there may be used other known transcription terminators. Combination with terminator CMV-BGH is especially preferred.

According to the present invention the expression cassette hHER2.opt or hHER2ECDTM.opt inserted into the vector. The vector is preferably an adenoviral vector, although can be used linear DNA associated with the promoter, or other vectors, such as adeno-associated virus and modified vaccinia virus, retrovirus and lentivirinae vector.

If the selected vector is an adenovirus, preferably, the vector was a so-called adenovirus, vecto the first generation. Such adenoviral vectors are characterized by the fact that they have a plot nonfunctional gene E1 and preferably deleteriously plot adenoviral E1 gene. In some embodiments, the implementation of the expression cassette is inserted into a position which is normally located in the adenovirus E1 gene. In addition, these vectors do not necessarily have a non-functional or deleteriously section E3. Preferably, in use adenovirus genome were deleterows both plots, as E1 and E3 (∆E1∆E3). Adenoviruses can be reproduced in the well-known cell lines, which Express the viral E1 gene, such as 293 cells, or PERC.6 cells, or cell lines derived 293 cells or PERC.6, temporarily or permanently transformed for the expression of an additional protein. For example, when using structures that have driven gene expression, such as adjustable tetracycline promoter system, the cell line can Express the components included in the regulatory system. One example of such cell lines is T-Rex-293; in the art of famous and other.

For ease of manipulation of adenovirus vectors adenoviruses can be in the form of a Shuttle plasmid. The present invention also relates to a Shuttle vector plasmid, which contains the plasmid portion and a portion of the adenovirus, and part of the adenovirus contains the adenoviral genome, which has depletirovannoi E1 and optional deletion of the E3, and has inserted expression cassette containing the codon-optimized human HER2 or codon-optimized hHER2ECDTM. In a preferred implementation options there are restriction sites flanking adenoviral part of the plasmid such that the adenoviral vector can be easily removed. The Shuttle plasmid can be replicated in prokaryotic cells or eukaryotic cells.

In a preferred embodiment of the present invention the expression cassette is inserted into the adenoviral plasmid pMRKAd5-HV0 (see Eminit et al., WO 02/22080, which is included in the present description in its entirety by reference). This plasmid contains the adenovirus Ad5 genome with deleteregvalue areas E1 and E3. The development of plasmid pMRKAd5-HV0 improved all early adenovector by extending the 5' CIS-acting packing area later in E1 gene for inclusion of items that were found, are important when optimizing viral packaging, resulting in increased amplification of the virus. Mainly, this reinforced an adenoviral vector capable of maintaining genetic stability after repeatedly passaged reproduction.

Standard techno is Ogii molecular biology of obtaining and purification of DNA structures give the possibility of adenoviruses, Shuttle plasmids and DNA immunogens of the present invention.

According to the present invention has determined that the molecules of the synthetic cDNA disclosed in the present description (for example, SEQ ID NO:1 and SEQ ID NO:9), which are codon-optimized for high level expression in human cell expressed with greater efficiency than the corresponding sequence of the wild type. In addition, in the present description shows that hHER2.opt is more immunogenic than hHER2, and more effective in developing both the cellular and humoral immune responses.

Therefore, the vectors described above can be used in immunogenic compositions and vaccines for the prevention of adenocarcinoma associated with impaired expression of HER2, and/or to treat existing cancers. Vector of the present invention provide an opportunity for the development and commercialization of vaccines by overcoming difficulties associated with obtaining high levels of expression of exogenous HER2 in successfully transformed organisms owners. Thus one aspect of the present invention is a method of prevention or treatment of HER2-associated cancer, involving the administration to a mammal a vaccine vector containing a synthetic codon-optimized molecule Amu is inovas acid, moreover, the synthetic codon-optimized nucleic acid molecule contains a nucleotide sequence which encodes a human HER2 protein as shown in SEQ ID NO:2, or human protein HER2ECDTM as shown in SEQ ID NO:14.

According to the method described above, the vaccine vector may be introduced to any mammal for the purpose of treatment or prevention of cancer. In a preferred embodiment of the present invention mammal is man.

In addition, specialists in the art can choose any type of vector for use in the described method of treatment and prevention. Preferably the vector is an adenoviral vector or a plasmid vector. In a preferred embodiment of the present invention, a vector is an adenoviral vector containing the adenoviral genome with a deletion in the area of adenovirus E1 and inserting in section E1 adenovirus, and the insert contains an expression cassette, comprising: (a) a synthetic codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (b) a promoter functionally linked with polynucleotides.

The present invention also relates to adenoviral vaccine vector containing the adenoviral genome with a deletion in the region E1 and arise the koi in the area E1, the insert contains an expression cassette, comprising: (a) a synthetic codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (b) a promoter functionally linked with polynucleotides.

In the preferred embodiment of this aspect of the present invention adenoviral vector is an Ad5 vector.

In other preferred embodiments, the implementation of the present invention, a vector is an Ad6 vector or RAD24 vector.

In another aspect, the present invention relates to vaccine plasmid, the plasmid containing part and the cassette expression, and part of the expression cassette contains (a) a synthetic codon-optimized polynucleotide encoding human HER2 protein; and (b) a promoter functionally linked with polynucleotides.

The present invention also relates to a vaccine plasmid, the plasmid containing part and the cassette expression, and part of the expression cassette contains (a) a synthetic codon-optimized polynucleotide encoding human protein HER2ECDTM; and (b) a promoter functionally linked with polynucleotides.

In some embodiments, implementation of the present invention the recombinant adenovirus vaccine disclosed in the present description, are used in various com is inatech, primary/booster, with a polynucleotide vaccine based plasmids in order to induce an enhanced immune response. In this case, two vectors injected into the primary and booster mode. For example, enter the first type of vector, then after a specified period of time, for example 2 weeks, 1 month, 2 months, six months, or another suitable interval, enter the second type of vector. Preferably the vector carrying the expression cassette encoding the same polynucleotide or combination of polynucleotides. In the embodiment, which is also used plasmid DNA, preferably, the vector contains one or more promoters, recognized by mammalian cells or insect. In a preferred embodiment, the plasmid may contain a strong promoter, such as, without limitation, the CMV promoter. A synthetic gene of the human HER2 gene HER2ECDTM, or other gene that is designed for expression can be associated with this promoter. An example of such a plasmid may be a plasmid expression V1Jns in mammals, as described (J. Shiver et al. in DNA Vaccines, M.Liu et al., eds. N.Y. Acad. Sci., N.Y., 772:198-208 (1996), which are included in the present description in its entirety by reference).

As stated above, the adenoviral vector plasmid vaccine and the vaccine can be introduced vertebral animal as part of a single t is repetitsionnogo mode to induce an immune response. Thus the present invention relates to a method of protecting a mammal from cancers, including: (a) introducing to a mammal a first vector comprising: i) a synthetic codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (ii) a promoter functionally linked with polynucleotides; (b) waiting for a predetermined period of time; and (C) introduction to the mammal a second vector comprising: i) a synthetic codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (ii) a promoter functionally linked with polynucleotides.

In one embodiment of the method of protection described above, the first vector is a plasmid, and the second vector is an adenoviral vector. In an alternative embodiment, the first vector is an adenoviral vector and the second vector is a plasmid.

The present invention also relates to a method of treatment of a mammal suffering from HER2-associated cancer, providing: (a) introducing to a mammal a first vector comprising: i) a synthetic codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (ii) a promoter functionally linked to polynucleotide is m; (b) waiting for a predetermined period of time; and (C) introduction to the mammal a second vector comprising: i) a synthetic codon-optimized polynucleotide encoding human HER2 protein or a human protein HER2ECDTM; and (ii) a promoter functionally linked with polynucleotides.

In one embodiment of the method of treatment described above, the first vector is a plasmid, and the second vector is an adenoviral vector. In an alternative embodiment, the first vector is an adenoviral vector and the second vector is a plasmid.

The number of expressed DNA or transcribed RNA, intended for introduction into the vaccine recipient, will depend in part on the strength of the used promoters and immunogenicity expressed gene product. In General, immunologically or prophylactically effective dose of about 1 ng to 100 mg, and preferably from about 10 μg to 300 μg of plasmid vaccine vector is injected directly into muscle tissue. Effective dose of recombinant adenovirus is approximately 106-1012particles and preferably about 107-1011particles. Also discusses subcutaneous injection, percutaneous injection, percutaneous injection and other modes is doing, such as intraperitoneal, intravenous or injection in the form of inhalation. It is also assumed that may be provided by the booster vaccination. Also is the predominant parenteral administration, such as intravenous, intramuscular, percutaneous, or other method of introduction of adjuvants, such as protein interleukin 12, simultaneously or sequentially with a parenteral vaccine of the present invention.

Vaccine vector of the present invention can be “naked”, i.e. unrelated to any proteins, adjuvants, or other substances that affect the immune system of the recipient. In this case, it is desirable that vaccine vector was in a physiologically acceptable solution, such as, without limitation, sterile saline or sterile buffered saline. Alternatively, priority may be the introduction of an immunostimulant, such as an adjuvant, a cytokine, a protein or other carrier, with the vaccines or immunogenic compositions of the present invention. Therefore, the present invention includes the use of such Immunostimulants in combination with the compositions and methods of the present invention. Immune, as used in the present description, in essence, refers to any substance to the / establishment, which amplifies or makes it possible immune response (or antibody-based test and/or cell-mediated) to an exogenous antigen. These Immunostimulants can be introduced in the form of DNA or protein. Any of a variety of Immunostimulants may be used in combination with vaccines and immunogenic compositions of the present invention, including, but without limitation, GM-CSF, tetanus toxin IFNα, IL12, B7.1, LFA-3 and ICAM-1. These Immunostimulants are well known in the art. Can also be used substances which promote cellular uptake of DNA, such as, without limitation, calcium ions. Such substances are usually of reagents that facilitate transfection, and pharmaceutically acceptable carriers. Experts in the art are able to identify specific immunostimulant or pharmaceutically acceptable carrier, as well as the appropriate time and mode of administration.

All publications mentioned in the present description, included in the present description by reference to describe and disclose methods and materials that can be used in conjunction with the present invention. Nothing in this description should not be construed as an admission that the present invention does not have the right to oppose such disclosure of the information source with an earlier priority based on earlier inventions.

Once described the preferred embodiments of the present from which retene with reference to the accompanying drawings, it should be clear that the present invention is not limited to this specific implementation options and that various changes and modifications can be carried out by specialists in the art without departure from the scope and essence of the present invention, as defined in the attached claims.

The following examples illustrate but do not limit the present invention.

EXAMPLE 1

The codon-optimized sequence of the human HER2.

Full coding hHER2.opt sequence is synthesized and assembled with the help of BIONEXUS (Bionexus Inc. Oakland CA.) and cloned in the vector pCR “blunt” ends (Invitrogen, The Netherlands). cDNA hHER2.opt designed using oligonucleotides and assembled using PCR. For many of the experiments disclosed in the present description, used nucleotide sequence hHER2.opt bearing optimized Kozak sequence at its 5'-end, the complete nucleotide sequence as shown in SEQ ID NO:11.

In addition, ATP-binding lysine residue 753 was replaced by alanine (CA) by replacing the codon AAA the codon GCA. This mutation suppresses tyrosinekinase activity of the corresponding protein and eliminates subsequent events alarm and, ultimately, the oncogenic activity of human (Messerle et al., Mol Cell Endocrino 105(1):1-10 (1994)) or rat HER2 (Ben Levi et al., see above). In addition, kinestetichnym mutant K can inactivate the activity signal coexpressing oncogene hHER2.wt.

EXAMPLE 2

Plasmid construction

pV1J-hHER2.wtthe sequence encoding human HER2 wild type, amplified using PCR from the plasmid pLTR-2/erb-B2 (courtesy of P.Di Fiore, European Institute of Oncology, Milan, Italy; Di Fiore et al., Science 237 (4811):178-82 (1987)) using primers hNeu.for1 (5'-CCAGTTTAAACATTTAAATGCCGCCACCATGGAGCTGGCGGCCT-3': (encoding SEQ ID NO:3 sequence is underlined)) and hNeu.rev2 (5'-GCCGTCGACTTTACACTGGCACGTCCAGACCCA-3': (SEQ ID NO:4)) and TaKaRa LA Taq polymerase (TaKaRa, Otsu, Shiga, Japan). The product of amplification, which includes optimized website start of translation (Kozak, M. J Mol Biol 196(4):947-50 (1987); Kozak, M., Nucleic Acid Res 15(20):8125-48(1987)), hydrolyzed restriction enzymes PmeI and SalI and cloned in the EcoRV sites and SalI digestion of plasmid expression pV1JnsA for mammals (see Montgomery et al., DNA Cell Biol 12(9):777-83 (1993)). Thus the generated plasmid pV1J-hHER2 contained the sequence of the human HER2 protein wild-type full length, the transcription of which was ruled by the early promoter of human cytomegalovirus with its intron And sequence. The sequence encoding human HER2 wild-type full length, followed by the signal sequence for polyadenylation of growth hormone large RoGator the cattle.

pV1J-hHER2.opt: fragment EcoRI-SalI from 3793 BP was cut out from plasmid pCR-hHER2.opt and cloned into the corresponding sites of plasmid pV1JnsB (Montgomery et al., see above), generating plasmid pV1J-hHER2.opt.

pV1J-hHER2ECDTM.opt: a fragment of 2168 BP amplified using PCR using TaKaRa taq with primers EcoRV-for (5'-CCAGATATCGAATTCTAGAGCCGCCACCATGGA-3' (SEQ ID NO:12)) and SalI-rev (5'-GCTGTCGACTTTATCAGATCAGGATGCCGAACACCACGCCC-3' (SEQ ID NO:13)) of pV1J-hHER2.opt. The resulting fragment hydrolyzed restriction enzymes EcoRV and SalI and cloned into the corresponding sites of plasmid pV1JnsB (Montgomery et al., see above), generating plasmid pV1J-hHER2ECDTM.opt.

EXAMPLE 3

The codon-optimized cDNA hHER2

Synthetic gene of human HER2 (hHER2.opt, figure 1) was developed to include codons preferred for a person (humanized), for each amino acid (hereinafter in the present description AA) residue. During Assembly of the gene by amplification by PCR was constantly deleterious sequence of 86 BP, starting from the position 3642, due to the high GC content of the sequence in this area. To solve this problem for the sequence hHER2 between the positions 3601 3805 and chose a design with less hard optimization that reduced GC content, while maintaining the same AA composition.

The codon-optimized cDNA was modified so that was maintained 83,9% identity on well leotides sequence with the original clone. The codon-optimized cDNA cloned into a vector pV1J (Montgomery et al., see above), placing it ahead Kozak-optimized sequence (5'-GCCGCCACC-3', SEQ ID NO:8), and under the control of the promoter of cytomegalovirus (CMV) human/intron a plus signal the termination of growth hormone in cattle (BGH). The design called pV1J-hHER2.opt (see example 2).

EXAMPLE 4

Expression in vitro plasmid constructions

Expression in vitro structures pV1J-hHER2.wt and pV1J-hHER2.opt was assessed by the time of transfection of cell lines with SOME kidney 293 human embryo and SS mouse cultured myoblasts and detection of expression of human HER2 flow cytometry. Everspiralling plasmid DNA pV1J-hHER2.wt without endotoxin encoding cassette expression of human HER2 used for immunization was purified from cells of E. coli DH12S (Invitrogen, Groningen, The Netherlands)using Qiagen endo-free plasmid Giga Kit (Qiagen, Hilden, Germany).

Plasmids pV1J-hHER2.wt or pV1J-hHER2.opt was transfusional using lipofectamine (Gibco BRL Invitrogen, Groningen, The Nrtherlands) in cells HEK-293. Similarly, cells SS mouse cultured myoblasts were transfusional a mixture of 1:1 or 10:1 plasmids pHygEGFP (BD Biosciences Clontech, PaloAlto, CA) and pV1J-hHER2.wt or pV1J-hHER2.opt.

Transfection of in vitro cells HEK-293 and SS showed that the codon-optimized sequence significantly enhances the expression of hHER2 compared to the wt sequence (figa and 3B).

EXAMPLE 5

Immunity is the situation mice

Six inbred female BALB/c mice (H-2dcourtesy G.Forni, University of Turin) were kept in standard conditions. Mice were treated according to the standards established by the European Union. In particular, hold a General anesthesia of mice with ketamine (Imalgene 500; Merial Italia, Milano, Italy) 100 mg/kg body weight and xylazine (Xilor, BIO 98; S.Lazzaro, Bologna, Italy) 5.2 mg/kg when it was necessary for the procedures.

Fifty micrograms of plasmid DNA was electroinactive in a volume of 50 µl in the quadriceps muscle of mice aged 6, 8 and 10 weeks, as described previously (Rizzuto et al., Proc. Natl. Acad. Sci. USA 96(11):6417-22 (1999)). 50 mcg optimized or unoptimized pCMV_hNeu were injected with without dissection of the skin in both the quadriceps muscle (25 μg in 50 μl saline/injection) and conducted electrical stimulation (ES), as described previously (Zucchelli et al., J.Virol. 74(24):11598-607 (2000); Rizzuto et al., see above). Briefly, an electric shock consisted of 10 sets of 1000 bipolar pulses (130, 75 mA, 200 μs/phase).

Ad injection was performed in the quadriceps muscle of mice in a volume of 50 ál. Serum was collected at week 7 (1 week prior to the first immunization, the preliminary drawing blood) and 12 week (two weeks after the last immunization).

EXAMPLE 6

ELISPOT analysis of IFN-γ mice.

Were detected murine splenocytes secreting IFN-γ, antigen-specific manner, and which uses standard sensitive enzyme-linked immunosorbent assay (ELISPOT) (Miyahira et al., J. Immunol Methods 181(1):45-54 (1995)). 96-well filter plates MultiScreen MAIP (catalogue number MAIPS4510; Millipore, Bedford, MA) were coated affinity-purified rat anti-mouse IFN-γ antibody (IgG1, clone R4-6A2, catalogue number 18181D, Pharmingen, San Diego, CA)diluted in sterile PBS. After incubation over night tablets were washed in PBST (0.005% tween in PBS) and incubated in the environment R10 for 2 hours at 37°C to block nonspecific binding.

Splenocytes were obtained by removing the spleen from Atanesyan mice under sterile conditions. The destruction of the spleen was carried out by grinding dissected spleen on a metal grid. Red blood cells were removed by osmotic lysis by adding 1 ml of 0,1X PBS to the cell sediment and shaking no more than 15 seconds. Then added one ml of 2X PBS and the volume brought up to 4 ml of 1X PBS. Cells were besieged by centrifugation at 1200 rpm for 10 min at room temperature and the precipitate resuspendable in 1 ml of medium R10 (RPMI 1640, supplemented with 10% fetal calf serum, 2mm L-glutamine, 50 units per ml of penicillin, 50 μg per ml streptomycin, 10 mm HEPES, 50 mkm 2-mercaptoethanol). Made the counting of living cells using staining Türks.

Splenocytes isolated from the spleens of two or more of immunized mice were incubated for 15 hours in the presence of 6 μg/ml of one or pool of proteins with density is 2.5 to 5·10 5cells/well. Of concanavalin a (ConA) was used as internal positive control for each mouse at 5 µg/ml After thorough washing PBST was added biotinylated rat anti-mouse IFN-γ antibody (catalogue number 18112D, PharMingen; San Diego, CA). The plates were incubated at 4°C overnight and then washed with PBST before addition of streptavidin-alkaline phosphatase (catalogue number E, PharMingen; San Diego, CA). After incubation for 2 hours at room temperature tablets thoroughly washed with PBST and was shown by incubation with one-step substrate by nitrosolobus tetrazole-5-bromo-4-chloro-3-indolylacetic (catalogue number 34042, Pierce, Rockford, IL) for 5-15 minutes to stains. The reaction was stopped by washing tablets with water. DMSO, concanavalin a (10 μg/ml) was the background and the positive control for each sample. Made calculations spots using computerized image analysis (AID ELR02 associated with software 2.6 AID ELISPOT, Strassberg, Germany).

The frequency of positive IFN-γ producing splenocytes in the total number of cells introduced into the hole, was calculated as the average of the spots obtained from iterations with two different concentrations of cells minus the average value obtained in a similar way spots, measured in control wells containing secretarysecretary. Changes in the frequency of IFN-γ producing cells was determined as the excess of the 95% confidence limit calculated from measurements of the controls. Differences with a p value<0.05 is considered significant.

EXAMPLE 7

Identification of immunodominant T-cell epitopes in the human HER2 protein.

Three hundred twelve 15-amino acid peptides overlapping by 11 amino acids, was developed to cover the full sequence of the human HER2. Those peptides, which also included seven peptides, designed to address the problems associated with the insolubility were synthesized by SynPep (Dublin, CA). All peptides have been shown by way of HPLC had a purity of >90% and were used without purification HPLC. The peptides of pererestorani in DMSO at 35 mg/ml of The peptides that did not dissolve immediately, was shaken at 37°C in order to accelerate the dissolution. If necessary, was added 1-3 additional amount(volume) DMSO for the complete dissolution of those peptides that have not yet passed into the solution after a few hours of shaking. Reconstituted peptides were combined so that each peptide was equally represented in the mixture. The final concentration of each peptide in the mixture was calculated so that it was 1 mg/ml of Each mixture was aliquot and stored at -80°C.

To identify immunodominant the T-cell epitopes of human gene HER2 in BALB/c mice (H-2 dgenetic background) 6-week-old female BALB/c mice were immunized by injection of 109vp Ad5-hHER2 in the quadriceps muscle. The second injection was performed three weeks later. The second group of mice were injected with the same method with saline as negative control. Three weeks after the second injection, animals were scored and assessed the frequency of IFN-γ-secreting T cells in splenocytes of mice using a sensitive enzyme immunoassay for interferon-γ (IFN-γ ELISPOT).

Three hundred eleven peptides, each with a length of 15 amino acids, overlapping by 11 residues and covering the entire sequence of the human HER2 protein, were United in eleven pools, designated by letters of the alphabet from a to K, from N-Terminus to the C-end. Each of these pools were tested for the ability to stimulate IFN-γ T cells in the spleen. For peptide pools a, b and M were measured using IFN-γ ELISPOT statistically significant production of IFN-γ mice immunized with Ad5-hHER2, in comparison with control in the absence of peptide. For identification of individual peptide responsible for the activity, peptides from pool a, and M were divided into three Padula, among which AndIIIand AIVBIIIand MImarked as positive. Single peptides from such positive podporou then tested on their ability starts the performance by the secretion of IFN-γ. Overlapping peptides hNeu-15 and hNeu-16 showed high and comparable reactivity. Significantly less reactivity demonstrated overlapping peptides hNeu-41 and hNeu-42. It was also shown that another peptide, hNeu-301, contained a T-cell epitope.

To confirm these data and to identify the subpopulations of T-cells CD4+or CD8+responsible for the production of IFN-γ, described IFN-γ-secreting T cells using intracellular staining (ICS). Murine splenocytes were incubated with single peptides for 12 hours in the presence of an inhibitor of secretion of brefeldin And recorded, was permeabilities and then stained with markers for intracellular IFN-γ, Cd3, CD4 and CD8 and analyzed by flow cytometry. ICS confirmed the reactivity of the peptide hNeu-15, identifying it as an epitope capable of activating cells CD8+. Peptides hNeu-15 and hNeu-16 were reactive equally in ELISPOT analysis, confirming that the epitope CD8+must contain at 11 AA residues that are common to the two peptides.

To identify the target nine-dimensional sequence, the authors tested three peptide length 9 AA, covering an area of overlap between hNeu-15 and hNeu-16. hNeu-15.3 contributed the highest reactivity, showing kaboolian reactivity in comparison with peptides hNeu-15 and hNeu-16 length 15 AA. Interestingly, the ome half reactivity was also detected with hNeu15.1, that indicates that in this 11 AA sequence coexist two overlapping, but distinct CD8+epitope.

IFN-γ ICS analysis also confirmed the reactivity hNeu301 and were identified by her as CD8+epitope. The analysis of such CD8+epitopes using IFN-γ ELISPOT confirmed the results obtained by using ICS. Finally, the low reactivity were detected for peptides hNeu41 and hNeu42, low response which was mainly CD4+.

EXAMPLE 8

Intracellular cytokine staining

Intracellular production of IFN-γ was measured according to the standard Protocol (BD Pharmingen. Briefly, 2×106the spleen cells were cultured for 15 hours in an environment R10 in the presence of 6 μg/ml of single or pooled peptides and brefeldin And, as an inhibitor of the transport protein (set Cytofix/Cytoperm Plus™ with GolgiPlug™; BD Pharmingen; San Diego, CA). Staphylococcal enterotoxin B (SEB), 10 µg/ml (catalog number S4881, SIGMA, Saint Louis, MI), and DMSO was tested using splenocytes as a positive control, and background, respectively.

Before staining of surface antigens Ab anti-mouse CD16/CD32 used to reduce nonspecific immunofluorescence signal (catalogue number 553142, BD PharMingen, San Diego, CA). Specific signal was obtained with APC-anti-mouse CD3e, PE-anti-mouse CD4 and PerCP-anti-mouse CD8a (catalog number 553066, 553653 and 53036, BD Pharmingen; San Diego, CA). Then cells were washed, fixed, permeability and stained for intracellular IFN-γ, using FITC-conjugated mAb (catalog number 554411, BD Pharmingen, San Diego, CA). IFN-γ by T lymphocytes was calculated as 1000×[(IFN-γ+, CD3+and CD4+or CD8+)/(CD3+and CD4+or CD8+)]. Usually, at least 50000 CD3+lymphocytes were collected using simultaneous selection event CD3+and small lymphocytes. All samples were obtained by staining within 24 hours using a flow cytometer FACSCalibur and CellQuest software (Becton Dickinson, San Jose, CA).

EXAMPLE 9

Titration and ittipiboon antibodies

Serum titration of antibodies were obtained by taking blood behind orbital plexus. ELISA plates (Nunc Maxisorp™, Roskilde, Denmark) were left covered with goat anti-human Fc-specific IgG (Pierce; catalogue number 31123) over night at 4°C at a concentration of 2 μg/ml in 50 mm NaHCO3(pH 9,6). Excess antibody was removed and nonspecific binding was blocked by incubation for 60 min at 37°C in PBBST5 buffer (BSA 5%, twin 0,05%). After washing was added to the supernatant IgB2-cells in the staining and incubated at room temperature for 2 hours (Chen et al., J Biol Chem 271(13):7620-9 (1996)). IgB2-cells (kindly provided by Dr. Y. Yarden, Weizmann Institute of Science, Rehovot, Israel) was a NECK-293 cell is, secreting dimeric protein, formed by the extracellular domain of HER2 and the Fc part of human Ig. The tablets were washed and serially diluted serum (1:4000 to 1:25600) in PBBST1 buffer (BSA 1%, tween 0.05%) is incubated over night at 4°C. Serum was collected before immunization was used as background. The leaching was performed in PBBST1. Secondary antibody (AP-conjugated goat anti-mouse IgG1 or IgG2a (Pharmingen, 557272 and 553389)) was diluted 1:40,000 in PBBST5 and incubated for 2-3 hours at room temperature on a shaker. After washing tablets was demonstrated by incubation with Sigma 106 a substrate for the phosphatase (Sigma; catalogue number A) diethanolamine. The tablets were read using an automated ELISA reader (Labsistems Multiskan Bichromatic, Helsinki, Finland) and the results were expressed as a=a405nm-A620nm. For each sample, subtract the background signal detected in serum collected before immunization.

The titers of anti-hHER2 serum was calculated as equal to the limiting dilution of serum that creates the absorbance, at least 3 times greater than the absorption autologous serum collected before immunization, at the same dilution.

EXAMPLE 10

Increased immunogenicity hHER2.opt

For the evaluation of immune responses induced by codon-optimized expression vector hHER2 and expression vector hHER2 wild-type mice BLB/c were immunized by intramuscular injection of plasmid DNA pV1J-hHER2.wt or pV1J-hHER2.opt with subsequent ES (as described in example 5). Mice were injected with three times, in 6, 8 and 10 weeks of age. Two weeks after the last immunization, each mouse was isolated splenocytes. For the quantitative determination of the frequencies of IFN-γ-secreting hHER2-specific CD8 T-cell precursor generated by immunization with plasmid DNA used ELISPOT analysis for H-2dcropped T-cell epitopes hNeu15.3 and hNeu42. Immunization with a HER2 sequence of the wild type caused barely detected CD8+response and reactivity of CD4+peptide was absent. In contrast, the optimized sequence of HER2 induced a 10-fold increase responses to peptide CD8+giving to 286 IFN-γ petrobrazi cells (SFC, average value), specific for the tested epitopes. He also detected a lower activity of CD4+. Peptide-specific IFN-γ SFC were not detected in mice immunized with pV1J-nsB (data not shown).

Sera from the same mice were tested by ELISA method, using IgB2 protein as substrate (pigv). Title hHER2-specific antibodies were detected in all mice immunized with pV1J-hHER2.opt, and the value of the geometric mean Ab titer was 46000 or 78000 for IgG1 or IgG2a. On the contrary, the group immunized with pV1J-hHER2.wt showed a titer of hHER2-specific antibodies, with the value of the geometric mean, is about 100 times lower is. Thus, these results demonstrate that the codon-optimized cDNA hHER2 is more effective in the development of cellular and humoral immune response than the sequence of the wild type.

EXAMPLE 11

Adenoviral vector

MRKAd5-hHER2.wt: DNA fragment SwaI-SalI from Pv1J-hHER2 containing cDNA of human HER2, cloned into the corresponding sites of Shuttle plasmids polyMRKΔE1 (Bett et al., Proc Natl Acad Sci USA 91(19):8802-04 (1994)). The obtained plasmid pMRKΔE1_hHER2 contained the CMV promoter of the person that controls the expression of the cDNA of the human HER2, followed by the polyadenylation signal of growth hormone in cattle. Spent the recombination plasmids pMRKΔE1_hHER2 the plasmid pAd5-HV0 with adenovirus basis for generating the pre-adenovirus plasmid pAd5-hHER2.wt.

MRKd5-hHER2ECDTM.opt: Plasmid pCR-hHER2.opt hydrolyzed EcoRI. Received an insert, length 2156 BP, purified and cloned in the EcoRI polyMRK-Ad5 Shuttle plasmid (see Emini et al., WO 02/22080 included in the present description in its entirety by reference).

Plasmid pAd5-hHER2.wt and pMRKAd5-hHER2.opt was linearizable hydrolysis PacI and transfusional in PerC6 cells to generate Ad5-hHER2 recombinant adenoviruses. Viruses were grown in large quantities by multiple amplification and purified by ultracentrifugation in a density gradient of cesium chloride (Fallaux et al., Hum Gene Ther 9(13):1909-17 (199)). Viral DNA was extracted by hydrolysis with proteinase K and the integrity of the genome was confirmed by restriction analysis.

Cells SOME infected 293 MRKAd5-hHER2.wt or MRKAd5-hHER2ECDTM.opt, using rates of infection (m.o.i.). Expression detected by Western-blot analysis revealed a more than 10-fold difference between the truncated protein expressed from codon-optimized sequence, in comparison with the full length protein, expressed from the wt sequence HER2 (data not shown).

EXAMPLE 12

Comparison of modes of immunization

The efficiency of adenovirus in the induction of cell-mediated immune response against human HER2 compared with the induction of the immune response plasmid DNA together with electrical stimulation, and adenovirus and plasmid DNA contained the expression cassette CMV-HER2. Fifty μg of plasmid pV1J-HER2 were injected with in the quadriceps muscle of wt mice BALB/c mice or transgenic BALB/c mice with overexpression of rat HER2 (specified in the form NeuT, see Lucchini et al., Cancer Lett 64(3):203-9 (1992)) with subsequent ES at the age of 6 and 9 weeks. Two weeks after the booster immunization, animals were scored, and spleen cells were collected and stimulated with peptides containing immunodominant R epitopes. Cells that make up a very small spot (SFC), were detected after with whom emulatie human peptides, as in BALB/c mice, and in mice neuT (figure 5). On average, the answer was 50 times lower than the response induced by immunization with Ad5-HER2. The above data show that immunization of mice with plasmid pV1J-HER2 induced a comparable response in BALB/c mice and neuT for each Protocol.

EXAMPLE 13

Immunization of rhesus monkeys with human HER2 in combination with human CEA and EpCAM antigens

To assess the effectiveness of immunization in rhesus monkeys (macaca mulatta) human tumor antigen HER2 in combination with other tumor antigens, a group of 4 rhesus monkeys (2 females and 2 males) were immunized with a mixture of plasmid DNA vectors expressing the codon-optimized sequence of the human tumor antigen Ep-CAM, CEA and HER2/neu.

Studies on immunization held in Biomedical Primate research Center (Biomedical Primate Research Centre (BPRC, Rijswijk, The Netherland). Studies on immunization were planned to assess T-cell responses induced by human antigens against homologues rhesus monkeys of the same antigens.

Macaques were vaccinated intramuscularly injection on 0, 2, 4, 6, 8, 10, 12, 14 and 16 weeks, with subsequent stimulation. Animals were injected with under anesthesia 1 ml solution (divided into two doses of 0.5 ml/dose)containing 6 mg of plasmid DNA for animals weighing 2-5 kg

For electrostim the regulation 2 of a series of 100 rectangular bipolar pulses each of duration 1 s) was applied every other second for the total processing time, comprising 3 seconds. The pulse length was 2 MS/phase with frequency and pulse amplitude 100 Hz and 100 mA (constant current mode), respectively.

Then the same macaques were vaccinated on 27 and 31 weeks with a mixture of three adenovirus 5 (Δ1-Δ3, “first generation”, P2 level), expressing the wt sequence of the human HER2, human CEA or human Arcam respectively.

To measure the immune response to homologues rhesus human HER2 using the above Protocol, collected the blood samples every four weeks for 1 year. Cell-mediated immune response was measured using IFN-γ ELISPOT analysis. The results, shown in Fig.7, indicate that the immunization Protocol, discussed above, was effective in inducing a specific immune response against endogenous homologues rhesus human HER2/neu.

EXAMPLE 14

Comparison R-specific T-cell response caused in mice by immunization pV1J-HER2.opt and pV1J-HER2ECDTM.opt

Evaluated the effectiveness of immunization mutant with a C-terminal deletion R preserved extracellular and transmembrane domains (HER2ECDTM). Plasmid DNA expressing the codon-optimized sequence of the full length (pV1J-HER2.opt) or shortened protein (pV1J-HER2ECDTM.opt), was electroadhesive 10 and 12 week and conducted analysis on the 14th week. Shortened protein HER2ECDTM induced anti-185 cell-mediated response, greater than the response induced by the protein p185 full length, both CD4+and CD8+ reactivity, as measured by IFN-gamma ELISPOT analysis (Fig). Analysis of gene expression in vitro in murine cultured myoblasts S showed no differences in expression between the two plasmids (data not shown).

1. Synthetic nucleic acid molecule, which is codon-optimized for expression in human cell high level of human protein antigen epidermal growth factor-2 (HER2/neu), where the synthetic nucleic acid molecule is determined by the nucleotide sequence presented in SEQ ID NO:1.

2. Synthetic nucleic acid molecule, which is codon-optimized for expression in human cell high level of truncated forms of the human protein antigen epidermal growth factor-2 (HER2/neu), which contains the extracellular and transmembrane domains of HER2/neu, where the synthetic nucleic acid molecule is determined by the nucleotide sequence, presented in SEQ ID NO:9.

3. Expression vector for mammalian cells, containing a nucleic acid molecule according to claim 1 or 2, which is functionally linked to a promoter.

4. The expression vector according to claim 3, which is an adenoviral vector.

5. The expression vector according to claim 4, which is an Ad5 vector.

6. The expression vector according to claim 4, which represents Ad6 vector or RAD24 vector.

7. The cell of the mammal host containing the vector according to claim 3, for the expression of the HER2/neu protein, or a shortened form of the human HER2 protein that contains the extracellular and transmembrane domains of the protein HER2.

8. The way of expression of human HER2/neu protein, or a shortened form of the human HER2 protein that contains the extracellular and transmembrane domains of the HER2 protein in recombinant cell of the mammalian host, including:
(a) introduction of the expression vector containing the nucleic acid according to claim 1 or 2, in a suitable cell of the mammalian host; and
(b) culturing the mammalian cells of the host under conditions which allow expression of the indicated human HER2 protein or specified shortened form of the human HER2 protein that contains the extracellular and transmembrane domains of the protein HER2.



 

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SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L.4 A2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Mus musculus L. 1C1 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The invention is also aimed at obtaining monoclonal antibodies 4A2 which are produced by the 4A2 hybridome, (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain) and are used as binding antigens in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and monoclonal antibodies 1C1 produced by the 1C1 hybridome (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain), used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The disclosed antibodies are used together in a "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain).

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5 cl, 3 dwg, 1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 1B2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the neucleoprotein of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Rattus Norvegicus 7B11 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the nucleoprotein of Ebola virus, Zaire subtype (Mainga strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain). The invention describes monoclonal antibodies 1B2 which are produced by the strain of hybrid animal cells Mus musculus L. 1B2, which relate to the subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain, and monoclonal antibodies 7B11 which are produced by the strain of hybrid animal cells Rattus Norvegicus 7B 11 related to the subclass of immunoglobulins IgG. The antibodies are used together in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: use of the invention enables to obtain results during "ВЭ" laboratory reseach and when designing a test system for highly reliable exposure of an antigen.

5 cl, 3 dwg, 2 tbl, 7 ex

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

SUBSTANCE: recombinant adenovirus apoptin-containing vectors are obtained by cotransfection into 911 helper cell line of p.Amb-VP3 adaptor plasmids (in case of VP3 protein expression) or pMAb-VP2 plasmids (in case of VP2 protein expression) and JM17 DNA. p.Amb-VP3 plasmids carry apoptin gene in 5'-3'-orientation, expressing under control of adenoviral main late promoter. Plasmid JM17 DNA contains complete adenoviral DNA excepted E1 and E2 regions. pMAb-°VP2 plasmids carry apoptin gene with two point mutation in limits of coding region. Cotransfections are carried out by calcium phosphate method. Recombinant adenoviral DNA is formed by homologous recombination between homologous viral sequences representing in p.Amb-VP3 (or pMAb-VP2) plasmid and in adenoviral DNA from plasmid JM17 DNA. Cell infection of various human tumors with gene delivery vectors causes to tumor cell apoptosis induction and sufficiently reduced normal, diploid, non-transformed or non-pernicious cell apoptosis.

EFFECT: new gene delivery vector capable to induce cell apoptosis.

8cl, 7 dwg

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