Plasmids coding versions of protein p185neu sequence, and their therapeutic application

FIELD: microbiology.

SUBSTANCE: invention represents plasmid vector for transfer of DNA, which comprises sequence coding various fragments of oncoprotein p185neu, which are able to induce immune response in respect to tumors, which hyperexpress p185neu. Invention is also related to pharmaceutical composition on the basis of vector for prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.

EFFECT: invention makes it possible to increase efficiency of prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.

10 cl, 14 dwg, 2 tbl, 2 ex

 

The invention relates to plasmid vectors containing DNA sequences encoding truncated and chimeric forms of the protein p185neuand for their use in DNA vaccination against Her-2/neu (ErbB-2)-positive tumors expressing p185 proteinneu. The plasmids according to the invention is able to induce a protective immune response, based on the induction of antibodies and/or T-lymphocytes against tumors expressing p185 proteinneu. Additionally, the invention relates to pharmaceutical compositions containing such plasmids, and to their use in the prevention and treatment of p185neu-positive tumors.

The background to the invention

The neoplastic cells often differ from normal cells that Express several of abnormal proteins. Due to this abnormal expression of some proteins can act as antigens associated with the tumor (TAA). This is because the immune system of the host body can recognize these anomalies and to induce an immune response that would protect the organism-owner from the appearance and development of the tumor. As a target in tumor immunotherapy, TAA must:

play pathogenetically role at a certain stage of neoplastic growth;

to determine the immune system, even when the tumor gives rise to clonal variations there, which no longer Express glycoproteins major histocompatibility complex (HLA);

be recognized by antibodies and T-lymphocytes.

In recent years, some TAA were detected in human carcinoma. Among them p185neuthe protein product of the oncogene Her-2/neu (ErbB2), is a particularly suitable target for immunotherapy (Lollini and Forni, 2003,TrendsImmunol.24:62). p185neuis a membrane receptor class I receptor family tyrosinekinase, which is also the receptor for epidermal growth factor (EGF-R / ErbB-1) and other related receptors (ErbB-3, ErbB-4), which play a key role in proliferation and differentiation of cells (Hynes and Stern, 1994,BBA1198:165).

p185neuthe receptor protein can be divided into three domains: extracellular domain (EC)domain, transmembrane domain (TM domain) and intracytoplasmic domain (IC domain). Was recently published crystallographic structure of the EC domain protein p185neuman and krysia described that this domain consists of four subdomains (I/L1, II/CR1, III/L2 and IV/CR2) a total of approximately 630 amino acids. Additionally, it was shown that protein p185neuhas a rigid structure, which allows him to interact with other ErbB receptors form dimers and call the transduction of proliferative signals, the even if the protein does not bind ligands directly (Cho et al., 2003,Nature421:756).

The oncogene Her-2/neu (ErbB2) is involved in normal processes embryonic organogenesis and epithelial growth, while in adults it is expressed only in small quantities (Presset al.,1990,Oncogene5: 953). In humans, overexpression of this oncogene is mainly caused by gene amplification. The oncogene Her-2/neu (ErbB2) hyperexpression approximately 30% of cases of breast carcinoma, and this overexpression is associated with more rapid tumor progression (Slamonet al.,1989,Science244:707). Among the various strategies that have been proposed, DNA vaccination, apparently, is an effective way to stimulate the immune response against Her-2/neu-positive tumors. Although protein p185neuis "self" antigen, i.e. a protein which is normally present in the body in patients with p185neu-positive carcinoma of the breast often is manifested as the cellular and humoral immune response (Signorettiet al.,2000,J. Natl. Cancer Inst.23: 1918; Disiset al.,1994,Cancer Res.54: 16; Peopleset al.,1995,Proc. Natl. Acad. Sci. USA92: 432). One of the objectives of antitumor immunotherapy aimed at p185 proteinncuis the increased intensity of existing immune response in patients, or generating an immune response in patients in whom this answer is not revealed. The FA is t, what p185 proteinneuis "self" antigen, means that the vaccine must be able to overcome the state of immunotolerance.

The authors of this patent application, first used and explained the efficacy of DNA vaccination in the activation of immune protection against spontaneous carcinomas of the breast, and transplanted Her-2/neu-positive tumors. These studies confirmed that the prevention and treatment of precancerous lesions is an achievable goal. In particular, in the experiments, the aim of which is to prevent the development of spontaneous mammary tumors arising in transgenic mice under the effect of Her-2/neu oncogene rats (mice FVB/neuT and mouse BALB-neuT), it was shown that a plasmid encoding EC and TM domains of the protein p185neurats can cause greater protection compared to the plasmid that encodes a full length protein p185neuor a plasmid, encoding only the EU domain (secretory antigen) (Amiciet al.,2000,Gene Ther. 7:703; Roveroet al.,2000, J.Immunol.165: 5133). Similar data were obtained by Chenet al.,(1998,Cancer Res.58: 1965). In addition, it was shown that the efficacy of vaccination with DNA plasmids increases, if it is followed by a very short electrical pulse intramuscular plasmid (Quaglinoet al.,2004,Dance Res. 64: 2858). Other authors have shown that plasmids encoding the full length protein p185neuif necessary modified in such a way that it does not possess tyrosinekinase activity, are effective for prophylaxis of tumours after transplantation p185neu-positive malignant cells (Wei-Zenet al.,1999,Int. J. Cancer81: 748). It was proved that the same plasmids as effective, even when not containing the leader signal peptide, which is responsible for protein processing in the endoplasmic reticulum, they lead to cytoplasmic localization of the antigen p185neu. In those cases, when using plasmids encoding the protein p185neu, which is located in the membrane due to the presence of the leader signal peptide, protective actions depend on humoral immune response. Conversely, the immune response mediated by T lymphocytes, was observed in those cases, if the vaccine does not contain the leader signal peptide, and therefore, p185 proteinneulocated in the cytoplasm transfection cells, but not on the plasma membrane (Pilonet al.,2001,J. Immunol.167: 3201). In addition, the combined vaccination obtained using plasmids with the leader signal peptide, and a plasmid in which the leader signal peptide has been removed, the two which is more effective for protection against tumor growth (Piechocki et al.,2001,J. Immunol,167;3367). This demonstrates the existence of a synergistic effect between humoral and cellular responses in the prevention p185neu-positive carcinomas (Reilly et al., 2001, Cancer Res. 61: 880).

It has been proven that vaccination with a plasmid that encodes EC and TM domains (EC-TM plasmid), is effective not only to prevent the development of spontaneous p185neu-positive carcinomas, but also for treatment of tumor mass with a diameter of 2 mm, involving a number of effector mechanisms of the immune system (T cells-helper and T-killer cells, antibodies, macrophages, neutrophils, natural killer cells, Fc receptors, IFN-gamma and perforin), which coordinated manner contribute to tumor rejection (Curcioet al.,2003,J. Clin. Invest.111:1161).

Description of the invention

Were designed special plasmids encoding full-TM domain, and decreasing areas of EC domain protein p185neurats. Truncated plasmids obtained by deletion of NH2-end 240 base pairs (bp) or multiples of the magnitude of this length, used in experiments aimed at preventing the growth of transplanted tumor cells that do overexpress p185 proteinneurats (TUBO cells). Moreover, a number of plasmids encoding chimeric form of the protein p185neuwere created by adding NH2-end sections ErbB2 cDNA person to serial is lnasty, encoding truncated forms of the protein in rats for the reconstruction of the full protein sequence.

Protective effect achieved by vaccination with a plasmid encoding full-sized domains of the EC and TM, mainly due to antigenic protection, while the protection achieved by the use of plasmids encoding truncated forms of the protein p185neurats, in many cases not associated with significant production of antibodies.

Based on the results of experimentsin vivowere selected plasmids capable of inducing a strong immune response, both humoral and mediated by T lymphocytes.

In the first aspect of the invention relates to a plasmid containing the coding sequence of a fragment of the protein p185neuthat is selected from the group consisting of SEQ ID NO: 1-5; or a sequence encoding a chimeric p185 proteinneuthat is selected from the group consisting of SEQ ID nos: 6-12 (the reference sequence for genes encoding proteins p185neuhuman and rat deposited in the Bank Gene Bank) under registration numbers M11730 and X03362 respectively).

DNA sequences encoding truncated and chimeric forms of the protein p185neuaccording to the invention can be inserted into any plasmid vectors suitable for use in mammals, particularly in humans. In addition to the above coding sequence is of elesta plasmids may contain functional elements to control transcription, in particular promoters, preferably the CMV promoter located in front of the coding sequence, the sequence of transcription initiation and stop sequences; selective marker, preferably the genes of resistance to ampicillin or kanamycin; CpG motifs; a polyadenylation site; and in case the enhancers or activators of transcription. Elements to control the transcription should be suitable for use vectors in mammals, particularly in humans.

In another aspect the invention relates to a pharmaceutical composition comprising a DNA plasmid as defined above, together with pharmaceutically acceptable carriers and excipients. Alternatively, the composition may contain a mixture of two or more different plasmids encoding both truncated and chimeric forms of the protein p185neu. For DNA vaccination preferably used in the pharmaceutical composition in a form suitable for parenteral administration, preferably in the form of injection solution. Principles and methods DNA vaccination known to specialists in this field and are described, for example, Liu, 2003;J. Int. Med.253: 402.

The use of plasmids encoding truncated and chimeric forms p185neufor prophylactic and therapeutic vaccination against p185neu-positive (Her-2/neu-, ErbB-2-positive) tumors have a number of the of roimushestvo, which increase their effectiveness. For plasmids encoding truncated forms of these benefits include:

1) the opportunity to receive the vaccine, encoding only certain sections of TAA in respect of which required the development of the immune response; in the use of this vaccine less likely the development of autoimmune processes;

2) exclusive induction of some selected forms of immune response, i.e. mediated by antibodies or T lymphocytes;

3) the opportunity to receive vaccines that combine multiple epitopes with specific immunogenicity, by binding together of fragments of cDNA that encode various truncated forms, not necessarily sequentially.

The use of chimeric plasmids obtained by a combination of truncated forms p185neuanimals of various kinds, allows you to:

a) vaccinated with plasmids encoding the protein determinants of the same species subjected to immunization, such as people that may cause specific high-affinity response;

b) combine plasmids encoding antigenic determinants of the same of the immunized species, cDNA sequences encoding antigenic determinants of other species, antigenic determinants, demonstrating significant similarity, but characterized in that the antigenic determinants of the other VI is s cause a more intense immune response, therefore, overcoming the state of tolerance. These allogeneic determinants that are recognized as partially exogenous, act as auxiliary determinants that facilitate the induction of a more intense response and cytokine release;

c) combine plasmids encoding antigenic determinants of the same species, cDNA sequences, which, by encoding the determinants of other species, some individuals may form heteroclites determinants that bind with high affinity to HLA molecules and induce a more intense immune response, with a higher affinity;

d) receiving the vaccine, which combines the advantages of a) advantages b) and (c).

DNA plasmids, properly obtained in accordance with the invention, used for the prevention and treatment of humans or animals with a high risk of developing p185neu-positive carcinoma or in patients with p185neu-positive primary tumors, recurrence or metastasis of these tumors. Prevention can be primary, when the tumor is still not expressed; secondary, when the tumor is in its early stages, such as pre-cancerous lesions; tertiary, if there is a relapse of a tumor or metastasis process.

Tumors treated plasmid and or compositions according to the invention, first of all, are tumors of epithelial origin, in particular, adenocarcinoma of lung, ovarian and breast cancer; squamous carcinoma tissues of the head and neck, and, more generally, tumors expressing p185 proteinneu.

Detailed description of the invention

Construction of plasmids encoding truncated forms of the proteinp185neurats

The backbone plasmid pCMV3.1 (obtained in the laboratory of the inventors on the basis of pcDNA3.1 from the company Invitrogen, Milan, Italy) was used to obtain DNA plasmids encoding full-TM domain, and decreasing areas of EC domain protein p185neuthe rats. pCMV3.1 contains the 5' UTR nucleotide sequence of the Her-2/neu rat (which is transcribed but not translated) and leader sequence (neuL). Secretory signal DNA fragment protein p185neurats were treated by enzymatic amplification of DNA using the vector pCMV-EC-TM (Amiciet al.,2000,Gene Ther.,7: 703; Roveroet al.,2000,J. Immunol.,165:5133) as the template, primers T7 as a sense oligonucleotide (oligonucleotide #1) and the oligonucleotide (oligonucleotide #2)having terminal siteEcoRI as of antisense oligonucleotide. After cleaning and processing enzymesHindIII andEcoRI, amplificatory fragment cloned in plasmid pCMV3.1, where R is swapnali by the same enzymes and thus, pCMV3.1-neuL. In a further seven different sequences that encode fragments of the EC domain and the full-size TM domain protein p185neurats were inserted in frame vector pCMV3.1-neuL, split by enzymesEcoRI andXbaI. New plasmid thus obtained was designated pCMV3.1-neuL-rEC1-TM (70 amino acids) (Fig 1),pCMV3.1-neuL-rEC2-TM (150 amino acids) (figure 2), pCMV3.1-neuL-rEC3-TM (-230 amino acids) (figure 3), pCMV3.1-neuL-rEC4-TM (-310 amino acids) (figure 4), pCMV3.1-neuL-rEC5-TM (-390 amino acids) (figure 5), pCMV3.1-neuL-rEC6-TM (-470 amino acids) (6) and pCMV3.1-neuL-rEC7-TM (-550 amino acids) (Fig.7). The fragment encoded by the first of these plasmids, 70 amino acids shorter, including secretory signal amino acid sequence. All other fragments progressively shortened to 80 amino acids.

These fragments were obtained by enzymatic amplification of DNA with seven different semantic oligonucleotides with terminal restriction siteEcoRI (oligonucleotides #3-#9), and of antisense oligonucleotide (oligonucleotide #10)that can recognize a website called "pcDNA3.1/BGH site reverse priming" (830-850 nucleotide on the 3' end of the multiple cloning site pCMV3.1. As DNA matrix for PCR used the vector pCMV-EC-TM (Amici A.et al.2000,Gene Ther.7: 703; Roveroet al.,2000,J. Immunol.165:5133). After enzymatic racepinephrine restriction EcoRI andXbaIamplification products were cloned in plasmid pCMV3.1-neuL.

Vaccination with plasmid pCMV3.1-neuL-rEC4-TM, as well as vaccination with plasmid pCMV3.I-neuL-rEC-TM, which encodes a full-sized domains of the EC and TM, in 100% of cases protects BALB/c mice against the development of tumors induced by inoculation of TUBO cells. On the other hand, vaccination with plasmids pCMV3.1-neuL-rEC1-TM, pCMV3.1-neuL-rEC2-TM and pCMV3.1-neuL-rEC3-TM, which encode the first three truncated forms of the protein p185neuprotects 70-80% of BALB/c mice. Plasmid pCMV3.1-neuL-rEC5-TM, which encodes the fifth truncated form, in 50% of cases protects BALB/c mice, whereas plasmids pCMV3.1-neuL-rEC6-TM and pCMV3.1-neuL-rEC7-TM, which encode the sixth and seventh truncated form, does not induce a protective response. The results show that cellular responses activated truncated form of the protein pI85neulocalized in the cytoplasm is sufficient to prevent tumors. However, concomitant activation of cellular and humoral response allows to obtain more effective treatment (Riellyet al.,2001,Cancer Res.61: 880). To achieve the production of antibodies vaccination must be performed by a plasmid that encodes a full-EC and TM domains of the protein p185neu. Vaccination with plasmid pCMV3.1-neuL-rEC4-TM, which encodes a fourth truncated form p185neudevoid of amino acids 1-310, retains the ability to provide full protection, but it is dlitelnyy answer 10 times lower than or antibody-based test response using plasmids pCMV3.1-neuL-rEC-TM (table 1).

Table 1
PlasmidsThe number of miceProtective actionAntibodies
pCMV3.1-neuL50%-
pCMV3.1-neuL-rEC-TM5100%+++
pCMV3.1-neuL-rEC1-TM580%-
pCMV3.1-neuL-rEC2-TM575%-
pCMV3.1-neuL-rEC3-TM570%-
pCMV3.1-neuL-rEC4-TM5100%+
pCMV3.1-neuL-rEC5-TM550%-
pCMV3.1-neuL-rEC6-TM50%-
pCMV3.1-neuL-rEC7-TM 50%-

Construction of chimeric plasmids containing the DNA of human and rat, are able to encode seven different fused forms of the protein p185neu(HuRT1-7)

Most of the epitopes presented by HLA, is located on the first subdomain (I/L1) protein p185neu. Therefore, for the induction of specific immune response against these epitopes were constructed chimeric plasmids encoding the sequence of ErbB2 protein man, who more and more extended, starting with NH2-the end (the outermost part of the EC domain). These new plasmids, designated HuRT (Human Rat Transmembrane), obtained by adding the missing sections ErbB2 cDNA person to sequences encoding truncated forms of rat p185 proteinneu.

The first five truncated plasmids encoding full-TM domain, and decreasing the fragments of the EC domain protein p185neurats were treated with enzymesHindIII andEcoRI. Five different fragments of the human cDNA obtained by PCR and split at the ends, cloned in these five truncated plasmids so that the reading frame is not moved. The cDNA fragments encoding the inserted parts of the protein p185neuperson, including the 5' UTR region and the secretory signal passing through endoplasmatic the reticulum, was obtained by amplification using the plasmid pcDNA3.1erbB2 as a matrix. Six oligonucleotides used as primers. Sense oligonucleotide is the same for all six primers and corresponds to the T7 primer (oligonucleotide #1), while five antisense oligonucleotides were split so that they recognize cDNA ErbB2 oncogene person in the ever-lengthening stretches of sequence and had the EcoRI restriction site at their 3' ends (oligonucleotides #11-#15). After purification and cleavage by enzymesHindIII andEcoRI amplificatoare fragments were inserted into the corresponding plasmids (pCMV3.1-rEC1-TM, pCMV3.1-rEC2-TM, pCMV3.1-rEC3-TM, pCMV3.1-rEC4-TM, pCMV3.1-rEC5-TM), which had previously been cleaved with the same enzymes. Thus, there were obtained five new plasmids (pCMV3.1-HuRT1-5)that encode chimeric proteins by the length of 689 amino acids, of which the 2 amino acids (Glu-Phe) lie in the restriction siteEcoRI used to join DNA molecules of human and rat. Proteins encoded by these chimeric plasmids differ from each other growing areas p185 proteinneuman and decreasing parts of the protein p185neurats.

To obtain chimeric plasmids encoding the sixth and seventh truncated form of the protein p185neurats were constructed two but what's the plasmid you can use the cloning sites, other thanEcoRI, as, for example, the restriction siteEcoRI in 1450 gene sequence of ErbB2 person. Plasmid pCMV3.1 modified by using a synthetic sequence consisting of the sense oligonucleotide (oligonucleotide #16) and antisense of the oligonucleotide (oligonucleotide #17), so that one of the two restriction sites for the enzymePmeIwas removed, and the restriction sites for enzymesHindIII andNheI located on their multiple cloning site were inverted. Thus obtained a new plasmid backbone outlined pCMV3.1H/N. Fragments for the sixth and seventh truncated forms of the protein p185neurats were obtained by amplification using the plasmid pCMV-EC-TM (Amiciet al.,2000,Gene Ther.,7: 703; Roveroet al.,2000,J Immunol.,165:5133) as a matrix and two different meaning of the oligonucleotide with the restriction siteNheI at their ends (oligonucleotides #18 and #19), and antisense oligonucleotide #10.

After enzymatic degradation by enzymesNheI andPmeI amplification products were cloned in plasmid pCMV3.1H/N, and thus, new plasmids pCMV3.1H/N-rEC6-TM and pCMV3.1H/N-rEC7-TM. The cDNA fragments encoding the inserted parts of the protein p185neuman, to create a chimeric p is asmid pCMV3.1H/N-HuRT6 and pCMV3.1H/N-HuRT7, was obtained by amplification using the plasmid pcDNA3.1erbB2 as template, primer T7 as a sense oligonucleotide (oligonucleotide #1), and two primers are designed so that they can recognize the human cDNA in the relevant provisions, and had on their ends of the restriction siteNheI (oligonucleotides #20 and #21), as antisense oligonucleotides.

After purification and cleavage by enzymesHindIII andNheI amplificatoare fragments were inserted into the corresponding plasmids (pCMV3.1H/N-rEC6-TM; pCMV3.1H/N-rEC7-TM), which was pre-digested with the same enzymes. Thus, we have obtained two new chimeric plasmids pCMV3.1H/N-HuRT6 and pCMV3.1H/N-HuRT7 that encode proteins by the length of 689 amino acids, of which the 2 amino acids (Val-Ser) lie in the restriction siteNheI used to join DNA molecules of human and rat.

As a result of these manipulations were obtained from the following plasmids:

plasmid pCMV3.1-HuRT1 (Fig)encoding a 70 amino acids of the EU-domain protein p185neuperson 2 amino acids belonging to the websiteEcoRI, and 618 amino acids protein p185neurats,

plasmid pCMV3.1-HuRT2 (Fig.9)encoding a 150 amino acids of the EU-domain protein p185neuman and 538 amino acids protein p185neurats,

plasmid pCMV3.1-HuRT3 (figure 10), 230 encoding amino acids EC domain protein p185neuthe man is a and 458 of the amino acids of the protein p185 neurats,

plasmid pCMV3.1-HuRT4 (11)encoding a 310 amino acid EC domain protein p185neuman and 378 amino acids protein p185neurats,

plasmid pCMV3.1-HuRT5 (Fig)encoding a 390 amino acids of the EU-domain protein p185neuman and 298 amino acid protein p185neurats,

plasmid pCMV3.1H/N-HuRT6 (Fig)encoding a 470 amino acids EC domain protein p185neuman and 218 amino acid protein p185neurats,

plasmid pCMV3.1H/N-HuRT7 (Fig)encoding a 550 amino acids EC domain protein p185neuman and 138 amino acid protein p185neurats.

Indirect proof membrane expression of a chimeric protein of human and rat encoded by these plasmids was obtained by immunization of mice with seven new plasmids (pCMV3.1-HuRT1-5 and pCMV3.1H/N-HuRT6-7). In all sera of vaccinated mice are specific antibodies to the chimeric p185 proteinneuhumans and rats. Moreover, animals vaccinated with plasmids encoding seven different chimeric proteins, are protected from lethal infection with TUBO cells and/or tumor cells that do overexpress p185 proteinneuman (D2F2-E2 cells).

EXAMPLES

Example 1: Construction of plasmids pCMV3.1-HuRT5

Plasmid pCMV3.1-rEC5-TM encoding a fifth truncated form of the protein p185neurats were digested with enzymesHindIII andEcoRI (BioLabs, Beverly,MA) to remove region 5' UTR and sequence neuL.

Band DNA 4794 P.N., corresponding plasmid pCMV3.1-rEC5-TM, devoid of 5' UTR region and the sequence neuL, was separated using agarose gel electrophoresis and eleirovania using the kit for gel extraction Qiaquick (Qiagen, Italy). cDNA for 5' UTR region, a leader sequence and the sequence encoding the missing part of the ErbB2 gene in human, were obtained using PCR. pcDNA3.1ErbB2 plasmid was used as matrix, the T7 primer (oligonucleotide #1) was used as the semantic of the oligonucleotide and primer containing the restriction siteEcoRI on the 5' end of the oligonucleotide #15) was used as the antisense of the oligonucleotide. For PCR used reagents and corrective Taq polymerase Finnzymes (CELBIO, Milan, Italy). After PCR amplified DNA was purified and precipitated by standard methods, resuspendable in 50 μl of H2O and were digested with enzymesHindIII andEcoRI. The cDNA fragment encoding the relevant part of ErbB2 person, and the linearized plasmid pCMV3.1-rEC5-TM cloned by ligation reaction using T4 DNA ligase (BioLabs, Beverly, MA).

Then the product of ligation was used to transform DH5α strain of the bacteriumE. colicompetent cells which were obtained by using calcium chloride.

The resulting clones were analyzed by alkaline lysis to determine the CL is new, containing chimeric plasmid pCMV3.1-HuRT5.

pCMV3.1-HuRT5 then analyzed by the method of sequencing by Sangero using an automated sequencing machine (ABI PRISM 310 Genetic Analyzer (Applied Biosystem) to confirm that the insert sequence human coding gene ErbB2, a plasmid encoding a fifth truncated form of the protein p185neurats, was done correctly and without shifting the reading frame.

List of oligonucleotides:

#1 T7 primer (SEQ ID No: 13)

#2 neu leader antisense EcoRI (SEQ ID No: 14)

#3 rECD1 semantic EcoRI (SEQ ID No: 15)

#4 rECD2 semantic EcoRI (SEQ ID No: 16)

#5 rECD3 semantic EcoRI (SEQ ID No: 17)

#6 rECD4 semantic EcoRI (SEQ ID No: 18)

#7 rECD5 semantic EcoRI (SEQ ID No: 19)

#8 rECD6 semantic EcoRI (SEQ ID No: 20)

#9 rECD7 semantic EcoRI (SEQ ID No: 21)

#10 pcDNA3.1/BGH reversionary website priming (SEQ ID No:22)

#11 His-Myc semantic EcoRI mut (SEQ ID No: 23)

#12 His-Myc antisense EcoRI (SEQ ID No: 24)

#13 70 erbB2 antisense EcoRI (SEQ ID No: 25)

#14 150 erbB2 antisense EcoRI (SEQ ID No: 26)

#15 230 erbB2 antisense EcoRI (SEQ ID No: 27)

#16 310 erbB2 antisense EcoRI (SEQ ID No: 28)

#17 390 erbB2 antisense EcoRI (SEQ ID No: 29)

#18 HindΙΙΙ-NheΙ semantic (SEQ ID No: 30)

#19 HindΙΙΙ-NheΙ antisense (SEQ ID No: 31)

#20 rECD6 semantic HheΙ (SEQ ID No: 32)

#21 rECD7 semantic HheΙ (SEQ ID No: 33)

#22 470 erbB2 antisense HheΙ (SEQ ID No: 34)

#23 550 erbB2 antisense HheΙ (SEQ ID No: 35)

Example 2: testin vivo

Animals

In all experiments we used female mice of BALB/c mice aged 7 weeks. The animals came from the lab Charles River Laboratories (Calco, Milan, Italy), where they were removed under aseptic conditions and in accordance with rules established by the European Union.

Intramuscular injection followedin vivoby electroporation

In order to avoid pain and undesirable reduction of tibial muscles of each mouse was anestesiologi intraperitoneal injection of 300 μl of Avertin, a solution consisting of 0,58 g tribromoethanol (Sigma-Aldrich) and 310 μl of tert-amyl alcohol (Aldrich) 39.5 ml of deionized H2O. Tibial muscle shot of mice were shaved and every muscle was injected 20 μl of a solution containing 25 μg DNA. The solution containing DNA was prepared directly before use in accordance with the instructions Dr. F. Pericle (Valentis, Inc., The Woodlands, Texas, USA). This solution contained plasmid DNA at a concentration of 1.25 mg/ml poly-L-glutamate in the form of sodium salt (Sigma-Aldrich, S.r.l., Milan, Italy) at a concentration of 6 mg/ml, sodium chloride at a concentration of 150 mm (Fluka, BioChemika, Buchs, Switzerland), distilled water, free of endotoxin (No Free Water, Promega Corporation) to a final volume of 1 ml to About 5 minutes after inoculation of two electrical pulse, the intensity of 375 V/cm2and a duration of 25 MS each, generated with the help of the device is Electro Square Porator electroporator (T820, BTX, San Diego, CA, USA) was applied to both the tibial muscle of mice, using two steel electrode located at a distance of 3 mm in a square arrangement on the lateral tibia. Genetic immunization using electroporation was performed twice for each animal for 21 and 7 days before inoculation of tumor cells.

Inoculation of tumor cells

On the left side of each mouse was inoculable 0.2 ml suspension containing 2×105of TUBO cells.

Evaluation of tumor growth in vivo

Tumor growth was assessed weekly by palpation, and the tumor size was measured in two perpendicular diameters measured with a ruler. The tumor is considered a tumor larger than 1 millimeter. Tumor growth was monitored for 100 days from the inoculation of the tumor or exceeding the size of the tumor 10 mm in diameter, the time at which animals were scarificial. The results showed that the chimeric plasmid pCMV3.1-HuRT5 has the ability to protect vaccinated BALB/c mice from lethal inoculation of TUBO cells in 100% of cases (table 2).

Table 2
PlasmidsThe number of miceProtectionSurvival (days)
pCMV3.1-neuL50%+35
pCMV3.1-neuL-rEC-TM5100%+100
pCMV3.1-HuRT55100%+100

Evaluation of anti-p18Sneuantibodies present in the serum of vaccinated animals

The day before inoculation of tumor cells in animals vaccinated with the chimeric plasmid pCMV3.1-HuRT5 took blood. Serum was analyzed to determine whether rat p185 antibodiesneu. Sera were incubated for 45 minutes at 4°C with cells that do overexpress p185neurats. After washing solution, called the wash buffer, which consists of a phosphate-saline buffer (PBS)containing 0.2% bovine serum albumin (BSA, Sigma, Milan, Italy) and 0.1% of sodium azide (NaN3, Sigma, Milan, Italy), samples were incubated for 20 minutes at 4°C with antibody to mouse immunoglobulin, conjugated with FITZ, washed with wash buffer and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, California, USA). Simultaneously, the same cells were incubated with decreasing concentrations of monoclonal antibodies to c-ErbB2/c-neu (Ab4, Oncogene) in affect, the, to show the dependence between the intensities of fluorescence resulting from cytofluorimetric analysis, and concentrations of anti-p185neuantibodies in the sera of animals. The data obtained show that all vaccinated animals have high levels of antibodies to p185neurats and, therefore, the chimeric plasmid pCMV3.1-HuRT5 are effective for induction of rejection of the transplanted p185neu-positive tumors and specific activation or antibody-based test response.

1. The plasmid vector to transfer DNA containing a sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, encoding a fragment of the protein p185neuor a sequence selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, encoding a chimeric p185 proteinneu.

2. The plasmid vector according to claim 1, additionally containing a promoter transcription.

3. The plasmid vector according to claim 2, where the specified promoter is a CMV promoter.

4. The plasmid vector according to claims 1 and 2, which is suitable for use in mammals, particularly in humans.

5. Pharmaceutical composition for prevention or treatment of patients at risk of developing p185neu-positive tumors, or patients with primary tumors, metastases or recurrences p185neu-positive tumors containing plasmid vector according to claims 1-3, together with pharmaceutically acceptable carriers and the excipients.

6. The composition according to claim 5, suitable for parenteral administration.

7. The composition according to claim 6 in the form of injection solution.

8. The composition according to claim 5 in the form of DNA vaccines.

9. The use of plasmid vector according to claims 1 to 4 to obtain a therapeutic agent used for the prevention or treatment of patients at risk of developing p185neu-positive tumors or patients with primary tumors, metastases or recurrences p185neu-positive tumors.

10. The use according to claim 9 to produce DNA vaccines.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to genetic therapy and concerns nucleotide sequence that codes insulin-like human growth factor IGF-1 presented with a synthetic gene including a sequence SEQ ID NO:1, recombinant plasmid DNA, containing this sequence, an eukaryotic cell containing recombinant plasmid DNA, construction for genetic therapy and a pharmaceutical composition for genetic therapy with regenerative and wound healing action.

EFFECT: advantage of the invention consists in decreased doses and introduction rate of injected preparations.

5 cl, 2 ex, 2 tbl, 4 dwg

FIELD: pharmacology; biotechnology.

SUBSTANCE: invention concerns biotechnology area, in particular to the gene-engineering way providing mass production multimeasured recombinant human Mannan-Binding Lectin (MBL), also can be used in the biomedical industry. The offered way includes a) a cultivation stage of cells-owners lines CHO, transfectant with a recombinant vector pMSG-MBL, containing sequence of human MBL coding area, and b) a clearing stage of the recombinant protein. Thus at the first stage preferably use new cellular line CHO MBL D1-3, deposited with the Korean collection of sample cultures at number KCTC 10472BP which is cultivated in a protein-free medium with bioreactor use, and on the second selective clearing of high-molecular form MBL of medium cultivation is spent, providing separation of samples with the help anion exchange chromatography, their drawing on a column with an immobilised MBL-binding protein on it , in particular with glycosilated protein of a cover of a virus pre-S, in the presence of ions of calcium and the subsequent elution using a buffered solution with EDTA or EGTA.

EFFECT: high output of functionally active product opens possibilities of its application by working out of therapeutic agents for treatment of virus, bacteriemic or fungoid infections.

6 cl, 11 dwg, 9 ex

FIELD: bioengineering.

SUBSTANCE: novel polynucleotide is invented which is produced from the nucleotide sequence of the IFNα-17 gene, containing the single nucleotide polymorphism SNP g771c. Also, the novel polynucleotide is invented which is derived from the natural protein IFNα-17 of the wild type containing SNP G45R.

EFFECT: can be used for producing effective therapeutic agent with antiviral, antiproliferative and/or immunomodulating activity.

13 cl, 5 dwg, 6 ex

FIELD: genetic material-containing medicines.

SUBSTANCE: invention relates to gene engineered VEGF-IBMed construct representing expression vector plasmid having cloned DNA insertion encoding vascular endothelial growth factor in three forms thereof. Said construct is claimed as VEGF-IBMed.

EFFECT: plasmid vector providing increased effectiveness and safety.

FIELD: medicine, gene engineering, in particular production of new variants of gamma-interferon polypeptide having gamma-interferon activity.

SUBSTANCE: new polypeptide variants include S99T replacement in contrast with huIFNG amino acid sequence, may be shortened on C-end and are capable of enhancing utilization of natural N-glycosilation site in 97 place. Mutant forms of gamma-interferon containing threonine in 99 place may include from 1 to 10 modifications without losses polypeptide activity. Polypeptide is obtained by expression in host cells transformed with vector including nucleic acid encoding mutant IFNG. Conjugant of mutant with PEG is obtained by pegilation, wherein said conjugant has increased half-life time in serum and reduced immunogenicity. Obtained IFNG polypeptide is useful in pharmaceutical composition for treatment and prevention of interstitial pulmonary diseases.

EFFECT: mutant gamma-interferon with enhanced utilization of natural N-glycosilation site in 97 place in contrast with natural form of huIFNG.

43 cl, 3 dwg, 4 tbl, 11 ex

FIELD: genetic engineering, biochemistry, pisciculture.

SUBSTANCE: invention relates to the determined nucleotide sequence encoding glycoprotein of the Russian isolate ZL-4. Based on this sequence DNA-plasmids are constructed that provide expression of glycoprotein of the Russian isolate ZL-4 in fishes. DNA-vaccine constructed on basis of indicated DNA-plasmids provides the development of protective immunity in fishes against the following infection with ZL-4 isolate of carp spring viremia virus. Using the invention provides protection of carp in Russian aquaculture from the following infection with carp spring viremia virus.

EFFECT: valuable properties plasmid DNA and glycoprotein gene.

3 cl, 4 dwg, 3 ex

FIELD: molecular biology, gene engineering, medicine.

SUBSTANCE: disclosed is part of nucleotide sequence disposed upstream from encoding sequence of CARP gene having promoter activity providing specific expression of gene operably linked therewith in cardiac cells in vivo. Detected promoter sequence is accepted to mouse CARP gene sequence from nucleotide in (-2266) site to nucleotide in (+92) site. Also disclosed are cassette and vectors for expression of therapeutically valuable target protein in cardiac tissue containing therein said regulatory sequence.

EFFECT: genetic constructs useful in pharmaceutical compositions.

19 cl, 10 dwg, 10 ex

FIELD: gene engineering, in particular purification and isolation of polynucleotides.

SUBSTANCE: invention relates to purification and isolation of polynucleotides regulating mammalian gene transcription and is useful in regulation of heterologous polynucleotide expression, obtaining transgene animals, and identification of affined regulatory DNA sequences. DNA containing transcriptional regulatory DNA of hamster gene EF-1α was isolated by screening of genome library to Chinese hamster ovary (CHO-K1). Chimeric polynucleotide including isolated regulatory DNA of hamster gene EF-1α operably bonded to gene sequence encoding target protein product other than protein encoded by hamster gene EF-1α was constructed. Obtained chimeric polynucleotide is used as component of expression plasmid for transformation or transfection of host cell. To increase target gene transcription in host cell DNA containing regulatory DNA of hamster gene EF-1α was integrated into host cell genome DNA in site operably bonded to target gene. Method of present invention make it possible to increase mRNA expression level for operably bonded heterologous polynucleotides by 3-11 times.

EFFECT: increased mRNA expression of operably bonded heterologous polynucleotides.

31 cl, 3 tbl, 7 ex

FIELD: genetic engineering, immunology, medicine.

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

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

33 cl, 5 dwg, 1 ex

FIELD: microbiology.

SUBSTANCE: glia cells are extracted, seeded, and cell is cultivated to produce monomolecular layer. Then cells are extracted and washed. Total RNA is extracted from them, reverse transcription is carried out, as well as amplification of produced DNA. Genes expression is assessed under action of tested compound.

EFFECT: invention makes it possible to accelerate screening of compounds, to search for new compounds with highest activity, which are potential neuroprotectors.

4 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there is described method for preparing a polymorphous region of gene PAR1 which can contain T to C replacement in position 3090 and/or A to C replacement in position 3329 of the polynucleotide sequence of wild type gene (NM_001992) with applying a pair of specific primers, and also the method for observing said region prepared of a DNA-containing biological sample for the presence or absence of the specified replacements. There are offered complete sets of the components application of which provides both amplification of the polymorphous region of gene PAR1 under the invention, and if needed, further analysis for genetic modifications in positions 3090 and/or 3329.

EFFECT: higher accuracy of estimating risk of cardiovascular diseases.

7 cl, 17 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology and immunology. Separated and cleaned DNA is presented, which codes receptor CTLA-4 (CD 152) of cat. The following is also suggested - diagnostic oligonucleotide, cloning vector, vaccine, methods of induction, strengthening and suppression of immune response in cat.

EFFECT: creation of model cat for research of retroviral infection.

24 cl, 10 dwg, 6 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.

EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.

16 cl, 3 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: as a result of substitution of part of amino-acid sequence of human protein PRG-4 coded with exon 6 with artificial polypeptide, including 4-15 sequences KEPAPTT or KEPAPTT-like sequences, recombinant proteins are produced with considerably lower number of glycolised repetitions compared to natural lubricin, which at the same time preserve its lubricating properties. It is suggested to use new recombinant proteins in pharmaceutical compositions and in methods of treatment, where natural lubricin is traditionally used.

EFFECT: application of the present invention for medical purposes has certain advantages related to simplification, compared to native protein.

28 cl, 1 dwg, 3 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of genetic engineering and medicine. Described is animal, non-human, having sequence of nucleic acid encoding presenilin 1, carrying mutations, corresponding to M233T and L235P mutations in PS1 protein of mouse. Animal also contains sequence of nucleic acid, encoding whole gene or part of gene, encoding APP. APP protein represents APP751, originates from human and carries mutations Swedish and London. Animal is intended for application in fight against Alzheimer's disease. Also described are PS1 protein and encoding it nucleic acid.

EFFECT: invention can be used in medicine for discovering compounds intended for Alzheimer's disease treatment.

20 cl, 50 dwg, 1 tbl, 8 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to production of versions of the Gla domain of human factor VII or human factor VIIa, and can be used in medicine. Amino acid sequence of the FVII or FVIIa version is obtained, which differs on 1 to 15 amino acid residues with amino acid sequence of the human factor VII (hFVII) or human factor VIIa (hFVIIa), in which a negatively charged amino acid residue is introduced by substitution in position 36. Obtained variants of FVII or FVIIa are used in a composition for treating mammals with diseases or disorders, where blood clotting is desirable.

EFFECT: invention allows for producing versions of FVII or FVIIa with high clotting activity and/or high activation of factor X, compared to natural form of hFVIIa.

42 cl, 3 dwg, 5 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to genetic therapy and concerns nucleotide sequence that codes insulin-like human growth factor IGF-1 presented with a synthetic gene including a sequence SEQ ID NO:1, recombinant plasmid DNA, containing this sequence, an eukaryotic cell containing recombinant plasmid DNA, construction for genetic therapy and a pharmaceutical composition for genetic therapy with regenerative and wound healing action.

EFFECT: advantage of the invention consists in decreased doses and introduction rate of injected preparations.

5 cl, 2 ex, 2 tbl, 4 dwg

FIELD: biotechnology.

SUBSTANCE: thrombin derivative is described that includes chain A and chain B where chain B has an aminoacid sequence whose aminoacids of the series active center in position 205 and histidine in position 43 in the aminoacid sequence of chain B of thrombin are substituted and where: the specified thrombin derivative decomposes thrombin substrate to the extent of 10% or less when interacting with thrombin substrate in 50 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl at the temperature of 37°C for 3 hours and the specified thrombin derivative retains the ability of getting combined with C-terminal hirudin peptide immobilised in gel.

EFFECT: pharmaceutical formula is disclosed that contains the thrombin derivative described.

54 cl, 48 dwg, 2 tbl, 31 ex

FIELD: medicine.

SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.

EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.

29 cl, 5 dwg, 6 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: there is offered molecule of nucleic acid inducing CEA immune response, containing a nucleotide sequence that codes a fused protein on a basis of carcinoembryonal antigen (CEA) or its functional version fused with a subunit B of thermolabile enterotoxin E coli. There are described versions thereof, as well as the related purified protein. There is disclosed an expression vector containing said molecule of nucleic acid, and a host-cell containing specified vector. There are described adenoviral vaccinal vector for inducing the immune response and a vaccinal plasmid on the basis of the specified molecule.

EFFECT: application of the invention allows to inducing the immune response in a mammal which is stronger, than that induced with natural CEA that can find application in medicine for cancer treatment.

20 cl, 62 dwg, 20 ex

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