Fused proteins of carcinoembryonal antigen

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

 

The technical field to which the invention relates

The present invention relates generally to the treatment of cancer. More specifically, the present invention relates to polynucleotides coding for fused proteins, and fused proteins contain at least part of the tumor-specific polypeptide carcinoembryonic antigen. The present invention also relates to recombinant vectors and hosts containing these polynucleotide, peeled fused proteins and methods of enhancing the immune response against CEA using these compositions and molecules, are described here.

The level of technology

The immunoglobulin superfamily (IgSF) consists of mnogochislennyh genes that encode proteins with diverse functions, one of which is intercellular adhesion. IgSF proteins contain at least one Ig-associated domain, which is important for maintaining the correct interactions intermolecular binding. Because these interactions are essential for diverse biological functions of members of the IgSF, impaired or aberrant expression of many IgSF adhesion molecules correlated with many human diseases.

The carcinoembryonic antigen (CEA) belongs to the subfamily of the Ig superfamily, consisting of glycoproteins, known as CEA-associated mol is coli cell adhesion (SEACAM). It was shown that SEASAM act as molecules as gomotopicheskoi and heterotypic intercellular adhesion (Benchimol et al., Cell 57: 327-334 (1989)). In addition to cell adhesion, CEA (also known as SEASON) inhibits cell death occurring in the separation of cells from the extracellular matrix, and may contribute to cellular transformation (rebirth cells)associated with certain proto-oncogene, such asBcl2andC-Myc(see Bernstein, J. Clin. Oncol. 20(8): 2197-2207 (2002)). Sequence encoding CEA man, were cloned and characterized (U.S. patent No. 5274087; U.S. patent No. 5571710 and U.S. patent No. 6843761. Cm. also Beauchemin et al., Mol. Cell. Biol. 7:3221-3230 (1987); Zimmerman et al., Proc. Natl. Acad. Sci. USA 84:920-924 (1987); Thompson et al., Proc. Natl. Acad. Sci. USA 84(9):2965-69 (1987)).

Normal expression of CEA was detected during embryonic development and in the mucosa of the colon in adults. Overexpression of CEA was first detected in tumors of the colon of a man more than thirty years ago (Gold and Freedman, J. Exp. Med. 121:439-462 (1965)and since then has been found in almost all colorectal tumors. In addition, overexpression of CEA is found in a large percentage of adenocarcinomas of the pancreas, liver, breast, ovary, cervix, and lung. Due to its prevalence in these types of tumors and limited expression in normal tissue is x CEA is considered a tumor-specific autoantigens and a target for active and passive immunotherapy. Recent clinical data have established that different approaches to vaccination can form b - and T-cells specific against CEA, providing additional evidence that CEA is a target for molecular and immunological intervention to treat these types of cancer.

Therapeutic approaches targeting CEA include the use of anti-CEA antibodies (see Chester et al., Cancer Chemother. Pharmacol. 46 (Suppl): S8-S12 (2000)), and vaccines based on CEA (for review see Berinstein, supra). The development and industrial implementation of a number of vaccines has been hindered by difficulties in obtaining high levels of expression of exogenous genes. The success of vaccines based on DNA also was hampered by the inability to obtain an immune response sufficient amount of individuals. Although there have been developed a DNA vaccine targeting different proteins, the immune responses were relatively weak compared with conventional vaccines.

The ease of manipulation of DNA is a favorable opportunity for the development of vaccines, including strategies merged structure of genes, in which the antigens are associated with different immunobiologii elements. Increasing the immune response to antigens of the target has been demonstrated in animal models using vectors encoding antigens fused with heat shock protein (HSP) 70(Liu et al., J. Virol. 74: 2888-94 (2000); Cheng et al., J. Immunol. 166: 6218-26 (2001); Chen et al., Cancer Res. 60: 1035-42 (2000)) with the Fc part of IgG1 (You et al., J. Immunol. 165: 4581-92 (2000)), with the associated with lysosomal membrane protein (LAMP) (Su et al., Cancer Res. 62: 5041-48 (2002)) and universal Th-epitope from tetanus toxin (Renard et al., J. Immunol. 171:1588-95 (2003); King et al., Nature Med. 4: 1281-86 (1998); Lund et al., Cancer Gene Ther. 10: 365-76 (2003); Padua et al., Nature Med. 9(11): 1413-17 (2003); Savelyeva et al., Nature Biotechnol. 19: 760-64 (2001); Wahren et al., WO 2004/092216). The enhancement of immune responses to antigens of the target is particularly important for cancer vaccines because of the limited immunogenicity of tumor antigens and the necessity of overcoming tolerance to exercise effective anticancer effects.

Thus, despite the identification of the nucleotide sequences of the wild-type protein-coding CEA, described above, would be highly desirable to develop a vaccine that can induce enhanced CEA-specific immune response in relation to the full-size cDNA CEA wild type when administered to a mammal. Would also be desirable to develop methods of treatment or prevention of CEA-related cancers, using molecules of nucleic acids or proteins that safely and effectively strengthen CEA-specific immune response.

The invention

The present invention relates to polynucleotides coding for fused proteins, being the m these fused proteins contain at least part of the tumor-specific polypeptide carcinoembryonic antigen, merged with a significant part immunoprivileged item, such as a bacterial toxin. In preferred embodiments, the implementation of CEA-part encoded fused protein CEA demeterova-the end of the anchor domain. In preferred embodiments, the implementation immunocellular element is A - or b-subunit of thermolabile enterotoxinE. colior a substantial part thereof. In other preferred embodiments, the implementation immunocellular element is minimized domain fragment of tetanus toxin (DOM) or a substantial part thereof. The present invention also relates to recombinant vectors, including, but not limited to, adenovirus and plasmid vectors containing the specified polynucleotide, and the cells of the host containing the recombinant vectors. Presents pure fused proteins encoded by polynucleotide of the present invention.

Additionally, the present invention relates to methods of inhibiting or preventing the development of cancer in a mammal by the induction of an immune response to CEA protein by introduction of a vaccine or pharmaceutical compositions containing the described here merged design CEA or fused proteins. In the preferred embodiments described here, this immune response is enhanced in comparison with the response, inducir the private vaccine CEA wild-type.

In the context of the entire description of the invention and the accompanying claims, the following definitions and abbreviations:

The term "promoter" refers to the website recognition on the DNA chain, which binds RNA polymerase. The promoter initiating forms a complex with RNA polymerase to initiate and run transcriptional activity. This complex can be modified by activating sequences, called "enhancers"or any abscopal sequences, called "silencers".

The term "cassette" refers to a nucleotide or gene sequence, which is expressed from the vector, for example, the nucleotide or gene sequence encoding a fused design hCEA-LTB. Typically, the cassette contains a sequence of a gene, which can be embedded in a vector, which in some embodiments, the implementation has a regulatory sequence for expression of the nucleotide or gene sequence. In other embodiments, the implementation of this nucleotide or gene sequence has some regulatory sequences for expression. In additional embodiments, the implementation, the vector has some regulatory sequences, and the nucleotide or gene sequence has other regulatory sequence is. For example, the vector may be a promoter for transcription of the nucleotide or gene sequence, and the nucleotide or gene sequence has the sequence termination of transcription. Regulatory sequences that may be present in the vector include, but are not limited to, enhancers, sequences, transcription termination, acceptor and donor sequences, splicing, introns, sequences binding of ribosomes and sequencing of poly(A)-add.

The term "vector" refers to a means by which DNA fragments can be introduced into the body-the owner or fabric-owner. There are different types of vectors, including plasmid, a virus (including adenovirus), bacteriophages and Comedy.

The term "first generation", as applied to adenoviral vectors, describes adenoviral vectors, which are replication defective. Adenoviral vectors of the first generation usually have deleteriously or inactivated area E1 gene and, preferably, have deleteriously or inactivated area E3 gene.

The abbreviation "DOM" typically refers to the N-terminal domain fragment With tetanus toxoid.

The abbreviation "LT" usually refers to a thermolabile enterotoxinE. coli. "LT" can refer to a full enterococ is in, containing subunit a and b or a substantial part of subunit A, or a substantial part of subunit C. the Abbreviation “LTA” means the subunit And thermolabile of enterotoxinE. colior its significant part, including subunit, which is shortened at the C-end N-end, but retain biological activity, as well as subunits, which contain internal amino acid insertions, deletions or substitutions, but retain biological activity. The abbreviation "LTB" means In thermo-labile subunit of enterotoxinE. colior its significant part, including subunit, which is shortened at the C-end N-end, but retain biological activity, as well as subunits, which contain internal amino acid insertions, deletions or substitutions, but retain biological activity.

Under the designation "pV1J/hCEAopt" see plasmid construction described herein, containing pedrani (IE) promoter with CMV intron a full size optimized in terms of codons gene CEA person, derived from the bovine growth hormone polyadenylation sequence and termination of transcription and the minimum frame pUC (see example 2). Under the designation "pV1J/hCEA" understand the design, essentially the same as that described above, except that this design contains a gene CEA wild type of man instead optimized in respect of the codons of the gene CEA person.

Under the designation "pV1J/hCEA-LTB" see plasmid construction described herein, containing pedrani (IE) the CMV promoter with intron a, CEA gene of the person deprived of his encodes a GPI anchor sequence fused at its C-end with subunit In thermolabile of enterotoxinE. coliderived from the bovine growth hormone polyadenylation sequence and termination of transcription and the minimum frame Fig.

Under the designation "pV1J/hCEAopt-LTB" understand the design, essentially the same as described immediately above, except that this design contains optimized in terms of codons gene CEA person deprived of his encodes a GPI anchor sequence, instead of the corresponding part of the CEA gene wild-type person.

Under the designation "pV1J/hCEAopt-LTBopt" understand the design, essentially the same as described immediately above, except that the sequence of CEA, and the LTB sequence are optimized in terms of codons for high level expression in human cells.

Under the designation "pV1J/rhCEAopt-LTBopt" understand the design, essentially the same as described above except that are optimized in terms of codons gene CEA man replaced the genome of CEA macaque-rhesus, optimized in terms of codons for expression high, Ural branch of the nya in human cells.

Under the designation "pV1J/hCEA-LT" see plasmid construction described herein, containing pedrani (IE) the CMV promoter with intron a, CEA gene of the person deprived of his encodes a GPI anchor sequence fused at its C-end with subunit And thermolabile of enterotoxinE. coliderived from the bovine growth hormone polyadenylation sequence and termination of transcription and the minimum frame Fig. Construction of plasmid vectors containing different slit design CEA-LT, described in example 2.

Under the designation "pV1J/hCEA-DOM" see plasmid construction described herein, containing pedrani (IE) the CMV promoter with intron a, CEA gene of the person deprived of his encodes a GPI anchor sequence fused at its C-end with N-terminal domain fragment With tetanus toxoid (DOM)derived from bovine growth hormone polyadenylation sequence and termination of transcription, and the minimum frame pUC (example 2).

Under the designation "pV1J/rhCEAopt-DOMopt" understand the design, essentially the same as described above except that are optimized in terms of codons gene CEA man replaced the genome of CEA macaque-rhesus, optimized in terms of codons for high level expression in human cells.

Under the designation "pV1J/hCEA-FcIgG" see plasmid construction, opican what Yu here containing pedrani (IE) the CMV promoter with intron a, a gene CEA person, devoid of encoding the GPI anchor sequence fused at its C-end c heavy fragment constant chain immunoglobulin G1 derived from the bovine growth hormone polyadenylation sequence and termination of transcription, and the minimum frame pUC (example 2). pV1J/hCEAopt-FcIgGopt denotes the design is essentially the same as described, except that the nucleotide sequence encoding CEA and FcIgG were optimized in terms of codons for high level expression in human cells.

Under the designation "Ad5/hCEAopt and Ad5/hCEA" understand two designs described herein, which contain adenovirus Ad5 genome, in which deleterows areas E1 and E3. In design "Ad5/hCEAopt area E1 replaced optimized in terms of codons gene CEA person in the E1 parallel orientation under control of the CMV promoter without intron a, followed by the polyadenylation signal of bovine growth hormone. Design "Ad5/hCEA is essentially the same as that described above, except that the area E1 of the Ad5 genome is replaced by a sequence of CEA wild-type person. Under the designation "Ad5/hCEAopt-LTB" understand the design Ad5 essentially the same as described above except that are optimized in terms of consecutive codons is required CEA person deprived of encoding the GPI anchor sequence and fused at its C-end with subunit In thermolabile of enterotoxin E. coli.Construction of adenoviral vectors containing different slit design CEA-LT, described in example 3.

"Immunobiology element" means a portion of the slit CEA protein of the present invention, which can stimulate or enhance the immune response to a protein CEA, related full-CEA wild type. Immunobiologii elements of the present invention is selected from the group consisting of heat shock protein (HSP) 70, associated with lysosomal membrane protein (LAMP), a fragment With tetanus toxoid (FrC), N-terminal domain of FrC (DOM), heaviest fragment constant chain immunoglobulin G1 (FcIgG), the glycoprotein of vesicular stomatitis virus (VSV-G), cholera toxin (CT) fromVibrio choleraeand thermolabile of enterotoxinE. coli(LT). The term "Immunobiology element" is used interchangeably with the term "adjuvant".

As used here, "protein" means a protein that has at least two covalently associated polypeptide, where the polypeptide is from a single protein sequence or domain, and the other polypeptide is from the second sequence of the protein or domain. Slit proteins according to the invention contain the CEA polypeptide or its fragment or variant and a second polypeptide, which contains a significant portion immunoprivileged element, which, in kotoryj cases, is a bacterial toxin. The CEA polypeptide, fragment or variant may be CEA person or CEA-homolog other species. The polypeptides that make up protein, preferably, are connected by the N-end - To-end. The CEA polypeptide and a subunit of the toxin can be merged in any order. In some embodiments, implementation of the present invention, With the end of CEA polypeptide fused to the N-end of the b subunit of the toxin, as shown, for example, in figure 1A. However, it is also considered fused proteins, which Immunobiology element fused to the N-end of the polypeptide CEA. It is assumed that the term "fused protein CEA" is a General term that refers to the merged structure described above, which contains a CEA polypeptide or its fragment or variant fused with a polypeptide that contains Immunobiology element.

The term "fused design CEA-LT" refers to a sequence of nucleic acid, in which at least part of the CEA gene fused with a substantial part or subunit of the LTA or LTB subunit of thermolabile enterotoxinE. coli.The term "fused protein CEA-LT" refers to the polypeptide encoded by the merged design CEA-LT. It is assumed that the terms "fused design CEA-LT" and "protein CEA-LT" also denote their fragments, their homologues and their functional equivalents (the place called "options"), such as fragments, homologues and equivalents, in which one or several amino acids insertion, deleterow or replaced by another amino acid (other amino acids). Fused design CEA-LT according to the invention when administered to a mammal, for example, a person can stimulate the immune response by helper T cells or cytotoxic T-cells or can stimulate the packaging antibodies are at least as good as the sequence of CEA "wild type". In preferred embodiments of the invention fused design CEA-LT may enhance immune response compared with CEA wild-type.

The term "fused design CEA-DOM" refers to a sequence of nucleic acid, in which at least part of the CEA gene fused with an essential part of lean domain fragment of tetanus toxin, unless the context clearly indicates that the term refers to this protein sequence. The term "fused protein CEA-DOM" refers to the polypeptide encoded described slit design CEA-DOM. It is assumed that the terms "fused design CEA-DOM" and "protein CEA-DOM" also denote their fragments, their homologues and their functional equivalents (collectively "options"), such as fragments, homologues and equivalents, in which one or several amino acids inser iravani, deleterows or replaced by another amino acid (other amino acids). Fused design CEA-DOM according to the invention when administered to a mammal, for example, a person can stimulate the immune response by helper T cells or cytotoxic T-cells or can stimulate the production of antibodies are at least as good as the sequence of CEA "wild type". In preferred embodiments of the invention fused design CEA-DOM may enhance immune response compared with CEA wild-type.

The abbreviation "AD" refers to the anchor domain of the gene or protein CEA. Anchor domain of CEA wild-type person located from about amino acids to approximately 679 702 amino acids of the sequence SEQ ID NO:20.

The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Individuals in need of treatment include both individuals with existing disabilities and individuals who are predisposed to the occurrence of the violation, or individuals, who should be prevented this violation.

"Violation" is any condition that would benefit from treatment with molecules according to the invention, including nucleic acid molecules described herein, and fused proteins, which are encoded pointed to by the mi molecules of nucleic acids. The term "violation" includes chronic and acute disorders or diseases including those pathological conditions which make the mammal is predisposed to the disorder. Assuming the molecules of the invention for treating disorders or conditions characterized by pathological proliferation, including, but not limited to, breast cancer, colorectal cancer and lung cancer.

The term "effective amount" denotes an amount of the vaccine composition, sufficient for its introduction to obtain adequate levels of the polypeptide, so that the induced immune response. The person skilled in the art it will be clear that this level may vary.

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

"hCEA and hCEAopt" refers to the carcinoembryonic antigen human and optimized in terms of codons the carcinoembryonic antigen person, meet the but.

"rhCEA" and "rhCEAopt" refers to the carcinoembryonic antigen macaque-rhesus and optimized in terms of codons the carcinoembryonic antigen macaque-rhesus, respectively.

"Essentially similar" means that a particular sequence of nucleic acid or amino acid sequence identical to at least 75%, preferably 85%, more preferably 90%, even more preferably, 95% of the reference sequence. In the invention, the reference sequence can be a relevant part of the nucleotide or amino acid sequence of wild-type CEA person or nucleotide or aminokislota sequence of the wild-type bacterial toxin or subunit, for example, subunits LTA LTB or thermolabile of enterotoxinE. colias indicated in the description. A reference sequence may be, for example, the sequence of CEA wild-type macaque-rhesus. Thus, the protein sequence of CEA, which is "essentially similar" protein CEA wild-type human or its fragment is identical to at least 75% of the relevant fragment of CEA wild type person for the length of this fragment, identical, preferably 85%, more preferably 90%, even more preferably 95%. Whether a particular serial is inost protein or the nucleotide sequence of CEA, LTB or LTA "essentially similar" to the reference sequence may be determined, for example, by comparing sequence using the programs for sequence analysis, such as a computer program GAP, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program uses a method of mapping Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), certified Smith and Waterman (Adv. Appl. Math. 2:482, 1981).

"A significant portion" of the gene, its variant, fragment or subunit refers to the part of at least 50%, preferably 75%, more preferably 90% or, more preferably, 95%, a reference sequence.

"Gene" refers to a nucleic acid molecule, the nucleotide sequence which encodes a polypeptide molecule. Genes may be contiguous nucleotide sequences or they can include built-in segments as introns, promoter regions, splicing sites and repeating sequences. The gene can be either RNA or DNA. Preferred gene is a gene that encodes the peptide according to the invention.

The term "nucleic acid" or "nucleic acid molecule" refers to ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)probes, oligonucleotides, fragments or portions thereof, and primers. DNA can be either complementary is Oh DNA (cDNA), or genomic DNA, e.g. a gene coding for a protein CEA.

"CEA wild-type"or "protein wild-type"or "wt-protein" means a protein containing natural amino acid sequence, or its variant. Amino acid sequence of CEA wild-type human is shown in figure 7E (SEQ ID NO:20). Amino acid sequence of CEA wild-type macaque-rhesus was described previously (WO 2004/072287, see figures 7A-7B).

"CEA gene wild-type" refers to a gene containing a nucleotide sequence which encodes a natural protein CEA, including proteins derived from humans, or proteins derived from another organism, including, but not limited to, other mammals, such as rat, mouse and rhesus. The nucleotide sequence of the gene CEA person is available in this area (supra). Cm. also Beauchemin et al., Mol. Cell. Biol. 7:3221-3230 (1987); Zimmerman et al., Proc. Natl. Acad. Sci. USA 84:920-924 (1987); Thompson et al., Proc. Natl. Acad. Sci. USA 84(9):2965-69 (1987). The nucleotide sequence of the gene of the wild type macaque-rhesus shown in figures 7C-7D.

The term "mammal" refers to any mammal, including humans.

The abbreviation "Ag" refers to the antigen.

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

The abbreviation "ORF" refers to the open reading frame of the gene.

Brief description of the figures

Figure 1 schematically presents the vectors developed in this study. Shows the main characteristics of plasmid vectors and Ad-vectors encoding the merged design CEA-LTA and CEA-LTB. It is also shown inverted terminal repeats (ITR) of the Ad5 genome.

The figure 2 shows the nucleotide (SEQ ID NO:7, panel A) and amino acid (SEQ ID NO:8, panel B) sequence fused design hCEA-LTA shown as an example. The nucleotide sequence of LTA are shown in bold.

The figure 3 shows the nucleotide (SEQ ID NO:9, panel A) and amino acid (SEQ ID NO:10, panel B) sequence fused design hCEA-LT shown as an example. The nucleotide sequence LT shown in bold.

The figure 4 shows the nucleotide sequence (SEQ ID NO:11) fused design hCEAopt-LTB shown as an example. The nucleotide sequence LTB shown in bold.

The figure 5 shows the nucleotide (SEQ ID NO:12, panel A) and amino acid (SEQ ID NO:13, panel B) sequence fully optimized merged design hCEA-LTB, denoted here hCEAopt-LTBopt shown as an example. Nucleotide and amino acid sequences LTB shown in bold. Sequence of connections created by the cloning strategy used for the merged design is the work of the sequences of CEA and LTB, underlined.

The figure 6 shows the nucleotide (SEQ ID NO:14, panel A) and amino acid (SEQ ID NO:15, panel B) sequence fully optimized merged design CEA-LTB macaque-rhesus indicated here rhCEAoptLTBopt shown as an example. Nucleotide and amino acid sequences LTB shown in bold. Sequence of connections created by the cloning strategy used for the merged design sequences of CEA and LTB, underlined.

The figure 7 shows the nucleotide sequence of the genes of wild-type, encoding CEA macaque-rhesus (panels a and b, SEQ ID NO:16 and 17) and amino acid sequences of the corresponding proteins (panels C and D, SEQ ID NO:18 and 19), as described previously (U.S.S.N. 60/447203). Panel E shows the amino acid sequence of CEA wild-type human (SEQ ID NO:20), which has been described previously (see, for example, U.S. patent No. 5274087).

The figure 8 shows a comparison of the efficiency of expression in cells transfected with the various constructs of the CEA. Panel And depicts the efficiency of expression in HeLa cells, transfected with 3 μg of the plasmid carrying the sequence of the wild type hCEA, hCEA-LTA and hCEA-LTB, together with 0.2 μg of plasmid pV1J/mEPO as an indicator. Panel b shows the results of a similar experiment by transfection using pV1J/hCEAopt and pV1J/hCEAopt-LTB. Efficiency the efficiency of expression was determined 3 days after transfection by measuring the amount of protein CEA, present in cell extracts and normalized this value in relation to the expression of EPO. Shows the results refer to the average values of expression of CEA two independent transpency.

Figure 9 shows a comparison of the efficiency of expression of various recombinant adenoviral vectors expressing CEA. HeLa cells were infected at multiplicity of infection (moi) of 100 and 1000 Ad/hCEAopt and Ad/hCEAopt-LTB. The efficiency of expression was determined by measurement after 3 days after infection an effective amount of the protein CEA released in cell extracts. Shows the results represent the average expression of CEA two independent infections.

The figure 10 shows the analysis of cell-mediated immune response induced by different plasmid vectors encoding CEA person. Three groups of mice 57BL/6 intramuscularly was electroadhesive 50 μg of the indicated plasmids (CEA, merged design CEA-LTA or merged design CEA-LTB) at 0-3 weeks. A fourth group of mice were immunized with a mixture of 25 µg pV1J/hCEA-LTA and 25 µg pV1J/hCEA-LTB. Panel A. two weeks after stimulation, the number of IFNγ-secreting T cells specific against CEA was determined by ELISPOT analysis on splenocytes of individual mice (white circles) using pools of peptides, which include all of this is protein. Specified geometric mean values (black diamonds). The panel depicts intracellular staining of IFNγ United splenocytes from immunized mice with pooled peptides D. For each group are shown non-specific production of IFNγ (DMSO).

The figure 11 shows the antibody titers of mice immunized with plasmid DNA vectors encoding the CEA. Individual titers against purified protein CEA person was measured using ELISA in serum from individual mice immunized with plasmids pV1J/hCEA, pV1J/hCEA-LT and pV1J/hCEA-LT. Also shown are average values (black diamonds).

The figure 12 shows the analysis of cell-mediated immune response induced by different plasmid vectors encoding the CEA. Groups of 4 BALB/c mice were electroadhesive specified by the plasmid, as described above (figure 4). Two weeks after the last injection, the number of IFNγ-secreting T cells specific against CEA was determined by ELISPOT analysis on splenocytes from individual mice (white circles) using pools of peptides, which include the whole protein. Also indicated are average values (black diamonds).

The figure 13 shows the analysis of the response of CEA-specific CD8+T cells induced by various plasmid vectors encoding the CEA. Mice C/DR4 electr who were injected with the indicated plasmid, as described above (see figure 4). Two weeks after the last injection of IFNγ-secreting T cells were stained with pooled peptides D. For each group are shown non-specific production of IFNγ (DMSO).

The figure 14 shows the analysis of the response of CEA-specific CD8+T cells induced by various plasmid vectors encoding the CEA. HHD mice were electroadhesive specified by the plasmid, as described above (see figure 4). Two weeks after the last injection was performed intracellular staining of IFNγ using pools of peptides and D. For each group shows a nonspecific packaging IFNγ (DMSO).

The figure 15 shows cell-mediated and humoral immune response to CEA-transgenic mice (N=9)immunized with 5 electroinjection once a week these plasmids. The total number of 50 μg of plasmid DNA were injected with intramuscularly at each vaccination. Panel A. two weeks after the last injection, the number of IFNγ-secreting T cells specific against CEA was determined by intracellular staining on splenocytes from individual mice (circles) with pooled peptides D. also Shown are values of the geometric mean (triangles). Panel C. Individual titles against purified protein CEA person was measured using ELISA at each with the holder of the mice, immunized with plasmids pV1J/hCEAopt and pV1J/hCEA-LT. Also shown is the geometric mean values (black diamonds). These results indicate that the merged design CEA-LTB destroys tolerance to CEA in transgenic mice.

The figure 16 shows the analysis of the response of CEA-specific CD8+T cells induced by various adenoviral vectors encoding the CEA. CEA-transgenic mice were immunized with different doses of Ad/hCEAopt and Ad/hCEAopt-LTB at 0 and 2 weeks. Two weeks after the last injection was performed intracellular staining of IFNγ PBMC from each individual mouse with pooled peptides D (black circles). Also shown is the geometric mean values (black diamonds). Non-specific production of IFNγ (DMSO) each injected group was less than or equal to 0.01%.

The figure 17 shows the results of studies on protection against tumors immunized with CEA-transgenic mice, provoked cells MS-CEA. A group of 10 CEA-transgenic mice were immunized 5 weekly electroinjection indicated plasmid DNA (50 μg/injection). Two weeks after the last injection mice were immunized again a single injection of 1·1010RF (viral particles) of the corresponding Ad-vector. 14 days after the second immunization with adenovirus mice were immunized subcutaneously the injection of 5·10 5cells MS-CEA. Panel a shows the percentage of not having tumors of mice at that point in time. Panel b shows the average volume of tumors in each immunized group. These results show that immunization CEA-transgenic mice CEA-LTB protects mice from tumor development.

Figure 18. Panel And a schematic of a representative fused proteins CEA, used in this study. Vectors expressing the fused protein CEA were derived from plasmids pV1Jns as described in example 2. These constructs contain the nucleotide sequence of CEA nucleotide (nt) 1 - nucleotide (nt) 2037 with a total deletion of 64 amino acids (AA)corresponding to the GPI-anchor sequence, and Express CEA amino acid (AA) 1 - amino acid (AA) 679. Specify the coordinates of the sequence of each protein, merged with CEA. Panel b shows the expression of pV1J-derived constructs in transfected cells. HeLa cells were transfusional plasmids pV1J/CEA-VSV-G, pV1J/CEA-FcIgG, pV1J/CEA-DOM, pV1J/CEA-HSP70, pV1J/CEA-LAMP or pV1J/CEA and processed for Western blot analysis as described in example 5. Indicated the specificity of the antibody used for the Western blot. Shows protein CEA (black arrow). Also shown is the position of molecular mass standards (in kilodaltons).

Figure 19 shows a comparison of the efficiency of expression of f the th designs CEA. HeLa cells were transfusional the indicated plasmids and made of CEA protein present in cell lysates (A) and supernatant (C), were measured using ELISA as described in example 8. The results obtained are representative results of two independent experiments.

The figure 20 shows the comparison of the immunogenicity of different designs, coding fused proteins CEA. Mice 57BL/6 was electroporative intramuscularly using 5 or 50 μg per dose of the indicated plasmids. Injections were performed on days 0 and 14. The number of IFNγ-secreting T cells in PBMC in each individual mouse was determined using a pool of peptides covering amino acids 497-703 (pool D), as described in examples 6 and 15. Also shown is the average number of IFNγ-secreting T cells (black circles). The SFC values (forming spots of cells) pV1J/CEA-DOM and pV1J/CEA-FcIgG significantly different from the values of SFC pV1J/CEA. Panel C. the Titer of the antibodies were measured using ELISA using purified CEA as a substrate. Shows the average values of each group immunized with a dose of 50 μg of the indicated plasmids. Titers that are significantly different from the titles of mice injected pV1J/CEA, are indicated by an asterisk.

The figure 21 shows the induction of CEA-specific immune responses in CEA-transgenic mice. A group of 12 CEA-transgenic mice were immunized is plazmidnoi DNA (50 μg/dose, electroinjection in the quadriceps muscle) or adenoviral vectors (109RF/dose), bearing optimized with regard to the use of codons cDNA CEA, CEA-DOM or CEA-FcIgG. CEA-specific CD8+T cells induced by immunization scheme DNA/DNA (a) and Ad/Ad (C)was measured by intracellular IFNγ staining on PBMC of each immunized mouse. Also shown are averages for each group (black circles). Group CEA-DOM and CEA-FcIgG immunized with immunization schemes DNA/DNA and Ad/Ad significantly different from CEA-vaccinated group. The titles of CEA-specific antibodies for each individual mouse immunized according to the immunization scheme DNA/DNA (C) or Ad/Ad (D), were measured using ELISA. The titers induced by the vectors CEA-DOM and CEA-FcIgG, significantly different from the titers induced by CEA.

The figure 22 shows the immunogenicity schemes DNA/Ad. A group of 12 CEA-transgenic mice were immunized plasmid DNA (50 μg/dose, electroinjection in the quadriceps muscle) or adenoviral vectors (109RF/dose), bearing optimized with regard to the use of codons cDNA CEA, CEA-DOM or CEA-FcIgG. CEA-specific CD8+T cells were measured by intracellular IFNγ staining on PBMC of each immunized mouse (A). Also shown are averages for each group (black circles). Group CEA-DOM and CEA-FcIgG C is acima differed from CEA-vaccinated group. The titles of CEA-specific antibodies of each individual mouse were measured using ELISA (B). The titers induced by the vectors CEA-DOM and CEA-FcIgG, significantly different from the titers induced by CEA. Shown are average values (black circles).

The figure 23 shows the detection of CD4+T-cell response to the protein is tetanus toxoid. CEA-transgenic mice were immunized pV1J/CTF-DOMopt as described in example 16. Intracellular staining of IFNγ in the United PBMC from individual mice was performed with peptide P30. Shows the discrimination of whole cells and discrimination for CD8+(R3) and CD4+T cells (R4).

The figure 24 shows the antitumor effects of vaccination vectors bearing optimized in terms of codons cDNA CEA, CEA-DOM or CEA-FcIgG. A group of 10 CEA-transgenic mice were immunized by vaccination schedules DNA/DNA (A), Ad/Ad (b) and DNA/Ad (C) using plasmid DNA vectors and Ad-vectors bearing optimized with regard to the use of codons cDNA CEA, CEA-DOM or CEA-FcIgG as described in example 18. Two weeks after the last injection mice were immunized sc-inoculation 5·105tumor cells MS-CEA. The percentage does not have tumors in mice vaccinated groups were determined with one-week intervals and compared with the percentage of untreated controls. Mice vaccinated vectors With The A-DOM (scheme DNA/Ad), significantly different from control mice (logarithmic rank criterion p<0,05).

The figure 25 shows the effect of elimination of CD4, CD8, or NK on the induction of antitumor activity induced by immunization CEA-DOM schema DNA/Ad. CEA-transgenic mice were immunized by repeated weekly injections of 50 μg pV1J/CEA-DOMopt followed by stimulation of 1·109HF Ad/CEA-DOMopt (example 19). One week after the last injection mice or overburden, or do not overburden in relation to CD4+T cells, CD8+T cells or NK cells. Two weeks after the last injection mice were immunized sc-inoculation 5·105tumor cells MS-CEA. The percentage does not have tumors in mice vaccinated groups were determined with one-week intervals and compared with the percentage of untreated controls. These results show that the percentage of no tumors in mice vaccinated group was significantly different from untreated controls and depleted groups.

The figure 26 shows the nucleotide sequence (SEQ ID NO:21) approximate fully optimized merged design hCEA-DOM, here called hCEAoptDOMopt. Amino acid sequence of the encoded protein is also shown (SEQ ID NO:45). CEA is part of the nucleotide sequence of this particular merged design CEA consists of nucleotides 1-2037, kotoryaaya optimized in terms of codons for high level expression in the cell is the master of man. DOM-a portion of this nucleotide sequence is shown in bold and is also optimized in terms of codons for high level expression in human cells. The sequence of the junctions created by the cloning strategy used for the merged design sequences of CEA and LTB, underlined.

Figure 27 shows an exemplary nucleotide (SEQ ID NO:25) sequence fused design hCEA-FcIgGopt called here hCEAoptFcIgGopt. Also shown is the sequence of the encoded protein (SEQ ID NO:46). CEA is part of the nucleotide sequence of this particular merged design CEA consists of nucleotides 1-2037, which are optimized in terms of codons for high level expression in the cell is the master of man. FcIgG-a portion of this nucleotide sequence is shown in bold and is also optimized in terms of codons for high level expression in human cells. The sequence of the junctions created by the cloning strategy used for the merged design sequences of CEA and LTB, underlined.

The figure 28 shows the nucleotide sequence of part of the CEA cDNA wild-type person from nucleotide 1 to nucleotide 2037 (SEQ ID NO:22, panel A), the coding portion of the protein hCEA from amino acid 1 to amino acid 679 (SEQ ID NO:23, panel B).

The figure 29 shows a non-optimized nucleotide sequence is minimized domain fragment of tetanus toxin (DOM) cDNA from nucleotide 1 to nucleotide 825 (SEQ ID NO:47)encoding a protein DOM, is also shown (SEQ ID NO:48).

The figure 30 shows a non-optimized nucleotide sequence of approximate merged design CEA-DOM human (SEQ ID NO:49). CEA is part of the nucleotide sequence of this particular merged design CEA consists of nucleotides 1-2037. DOM-a portion of this nucleotide sequence is shown in bold.

Figure 31 shows an exemplary nucleotide sequence (SEQ ID NO:50) merged design CEA-DOM macaque-rhesus, here called rhCEA-DOMopt. The sequence of the encoded protein is also shown (SEQ ID NO:51). CEA is part of the nucleotide sequence of this particular merged design CEA consists of nucleotides 1-2037, which are optimized in terms of codons for high level expression in the cell is the master of man. DOM-a portion of this nucleotide sequence, which is also optimized in terms of codons for high level expression in human cells shown in bold.

The figure 32 shows the nucleotide sequence (SEQ ID NO:52) merged design CEA-STV macaque-rhesus, here called rhCEA-opt. Posledovatel the activity of the encoded protein is also shown (SEQ ID NO:53). CEA is part of the nucleotide sequence of this particular merged design CEA consists of nucleotides 1-2037, which are optimized in terms of codons for high level expression in the cell is the master of man. STW-a portion of this nucleotide sequence, which is also optimized in terms of codons for high level expression in human cells shown in bold.

Detailed description of the invention

The carcinoembryonic antigen (CEA) is usually associated with the development of adenocarcinoma. The present invention relates to compositions and methods for the induction or enhancement of immunity to the protein product expressed by the tumor-specific antigen CEA, and aberrant expression of CEA is associated with carcinoma or its development. The Association of aberrant expression of CEA with carcinoma does not require protein CEA expressively in tumor tissue at all time points of its development, because aberrant expression of CEA may be present in the tumor and not to be determined later in the progression of the tumor, or Vice versa.

For this purpose, the present invention relates to polynucleotides, vectors, cells, owners and encode proteins containing a sequence of CEA or a version thereof, for use in vaccines and pharmaceutical HDMI is tion for the treatment and/or prevention of cancer. Polynucleotide according to the invention contain a nucleotide sequence encoding a CEA protein or variant, fused with a nucleotide sequence that encodes at least subunit immunoprivileged item, such as a bacterial enterotoxin or a substantial part thereof, which can effectively promote the immune response to the associated CEA.

The nucleotide sequence of CEA according to the invention can be obtained in person or may be a homologue of CEA another kind. The nucleotide sequence of CEA wild-type person described (see, for example, U.S. patent No. 5274087; U.S. patent No. 5571710 and U.S. patent No. 6843761). The sequence of CEA macaque-rhesus has recently been described (WO 2004/072287). CEA is part of the slit structure of CEA may be full or may be any option that is sufficient for the induction of CEA-specific immune response in a mammal. Options CEA according to the invention include, but are not limited to, sequences that are truncated at the C - or N-end sequences with conservative substitutions, and consistency with internal deletions or insertions.

In preferred embodiments, the implementation of the present invention CEA-part of the merged design CEA CEA is the person or its functional equivalent. In other FAV is titeling options implementation CEA-part is CEA macaque-rhesus or its functional option.

Thus, the present invention relates to synthetic polynucleotide containing a nucleotide sequence encoding a protein of CEA, and the specified protein contains protein CEA or its biologically active fragment or mutant form of the protein CEA, merged with immunocellular element or its subunit, which can effectively enhance the immune response to CEA protein. These mutant forms of CEA protein include, but are not limited to, conservative substitutions of amino acids shorter aminocore form, shortened on carboxylic form, deletions or additions. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least essentially repeats the immunological properties of the protein CEA SEQ ID NO:20. Synthetic polynucleotide according to the invention encode mRNA molecules that Express a functional protein CEA so that it can be used to develop therapeutic or prophylactic cancer vaccine.

In preferred embodiments of the invention CEA-part encoded fused protein CEA represents CEA human (SEQ ID NO:20) or its functional equivalent is, for example, CEA person with a deletion of its C-terminal anchor domain (AD) (SEQ ID NO:23), which is located from about amino acid 679 to about amino acid 702 full CEA person. Not communicating with theory, the authors present invention believe that the deletion of the anchor domain increases the secretion of the fused protein CEA, increasing by cross-priming of the immune response CEA-LTB. In other preferred embodiments, the implementation of CEA-part is CEA macaque-rhesus (SEQ ID NO:18 and 19) or its functional equivalent.

Part immunoprivileged element merged CEA protein of the invention can stimulate or enhance the immune response to a protein CEA and selected from the group consisting of heat shock protein (HSP) 70, associated with lysosomal membrane protein (LAMP), a fragment With tetanus toxoid (FrC), N-terminal domain of FrC (DOM), heaviest fragment constant chain immunoglobulin G1 (FcIgG), the glycoprotein of vesicular stomatitis virus (VSV-G), cholera toxin (CT) fromVibrio choleraeand thermolabile of enterotoxinE. coli(LT). In preferred embodiments of the invention, this adjuvant part of the merged design CEA contains N-terminal domain of FrC (DOM), which has been shown to effectively enhances the immunogenicity simultaneously delivered antigens. The following pre is reverent versions of the implementation, adjuvant part of the merged design CEA contains a subunit of LT or its substantial part. In other preferred embodiments, the implementation of the adjuvant part of the merged design CEA is an essential part FcIgG.

Was merged described the design of the CEA, containing truncated CEA person, merged with a single epitope of tetanus toxin (Q830-L844) (Lund et al., Cancer Gene Therapy 10: 365-376 (2003)). In contrast to this merged structure with a single epitope, fused design CEA according to the present invention contain a substantial part of immunoprivileged element or its subunit, as described above, which can enhance the immunogenicity of CEA protein or variant. A significant part of immunoprivileged element, which is used to described are compositions and methods, does not include parts that are less than 50% full-subunit of the toxin. Used here is a strategy that uses a full-sized subunit adjuvant or a substantial part, was applied to ensure greater immune response to the sequence of the slit CEA. Not based on any theory, the authors present invention believe that if a bacterial toxin selected as adjuvant that contains more than one helper epitope, a restriction sequence Tox is on the fused protein, the only epitope would prove to reduced effect on the immunogenicity of the studied protein. In addition, the inventors believe that, if mediated adjuvant enhancing the immune response depends on the interaction of this adjuvant with specific receptors of the cells and not based on universal epitope, the interaction with the receptor could depend on specific structural configuration, which would require substantial part immunoprivileged element for signs of adjuvant. In this case, a short sequence adjuvant containing a single epitope, was insufficient for the mediation of increasing the immune response.

In the framework of the present invention also covers nucleotide sequences encoding variants or mutants described here Immunobiology elements, including, but not limited to, substitutions, deletions, additions of nucleotides, aminobenzene shortening and carboxykinase shortening. In some cases it may be advantageous to add specific point mutations to a nucleotide sequence that encodes adjuvant or its subunit, to reduce or eliminate the toxicity of the encoded protein. Examples of variants of this aspect of the invention subunit of LT merged with CEA sequence fused design CEA, and subunit LT shortened by the removal of its signal sequence. No basis is as on any theory the inventors believe that the deletion of the signal sequence of the toxin, for example, the signal sequence LTB, provides start posttranslational processing of this merged structure of CEA signal sequence of CEA.

Immunobiology element, it subunit or a substantial part thereof may be fused with aminocom.com or carbonsilicon sequence of CEA. Additionally, the sequence immunoprivileged element and the sequence of CEA can be merged by type N-end N-end-end - end-end N-end N-end - To-end. In preferred embodiments of the invention With the end of CEA polypeptide fused to the N-end immunoprivileged element.

The present invention relates to a synthetic molecule of nucleic acid (polynucleotide)containing a nucleotide sequence which encodes mRNA which expresses a new protein CEA; for example, the nucleotide sequence encoding the fused protein represented in SEQ ID NO:8, 10, 13, 15, 45, 51 and 53. The nucleic acid molecule of the present invention do not contain other nucleic acids.

The present invention also relates to recombinant vectors and recombinant cell host as prokaryotic and eukaryotic, which contain m is likely nucleic acids, described in the description of the present invention. These synthetic DNA molecules associated vectors and hosts according to the invention can be used for the development of anticancer vaccines.

Examples of nucleic acid molecules of the present invention contain a nucleotide sequence selected from the group consisting of SEQ ID NO:7, 9, 11, 12, 14, 21, 25, 49, 50 and 52, shown in figures 2-6, 26-27, 30-32, which encode the examples of the fused protein of the present invention CEA-LT, CEA-LTB, CEA-DOM, CEA-FcIgG and CEA).

The present invention also includes biologically active fragments or mutants of SEQ ID NO:7, 9, 11, 12, 14, 21, 25, 49, 50 and 52, which encode mRNA expressing examples of fused proteins CEA. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least essentially mimics the pharmacological properties of the protein hCEA, including, but not limited to, protein hCEA presented in SEQ ID NO:20. Any such polynucleotide includes, but is not limited to, substitutions, deletions, additions of nucleotides, aminobenzene shortening and carboxykinase shortening. Mutations of the invention encode mRNA molecules that Express a functional protein CEA in eukaryotic cells so that it was applicable in the development of anticancer vaccines.

In volume nastojasih the invention also includes a mutation in the DNA sequence, which essentially do not change the final physical properties of the expressed protein. For example, the substitution of leucine for valine, lysine to arginine, or glutamine for asparagine may not cause changes to the functionality of the polypeptide.

As stated above, the present invention additionally relates to recombinant vectors that contain nucleic acid molecule described in this description of the invention. These vectors can be composed of DNA or RNA. In most cases, when cloning preferred are vectors based on DNA. Typical vectors include plasmids, modified viruses, baculovirus, phage, Comedy, artificial chromosomes in yeast and other forms episomal or integrated DNA that can encode a protein of CEA. The person skilled in the art can determine a suitable vector for the transfer of a specific gene or other uses.

The present invention relates to purified fused proteins CEA encoded by the nucleic acids described in the present description. In exemplary embodiments of this aspect of the invention, the slit CEA protein contains an amino acid sequence selected from the group consisting of SEQ ID NO:8, 10, 13, 15, 45, 46, 51 and 53.

The present invention relates to DNA sequences which hybridize with SE ID NO:7, 9, 11, 12, 14, 21, 25, 49, 50 or 52 in tough conditions. As an example, but not limited to, the procedure using conditions of high stringency, is the following. Prehybridization of filters containing DNA is carried out in a period of approximately 2 hours during the night at about 65°C. in buffer composed of 6×SSC, 5× denhardt's solution and 100 μg/ml denatured DNA salmon sperm. Filters hybridized for about 12-48 hours at 65°C in a mixture to prehybridization containing 100 μg/ml denatured DNA salmon sperm and 5-20×106pulse/min32P-labeled probe. Washing of filters is performed at 37°C for approximately 1 hour in a solution containing 2×SSC, 0,1% SDS. Followed by washing in 0.1×SSC, 0,1% SDS at 50°C for 45 minutes before radioautography. Other procedures using conditions of high stringency, include either phase hybridization carried out in 5×SSC, 5x denhardt's solution, 50% formamide at about 42°C for about 12-48 hours, or stage of the washing cycle carried out in 0.2×SSPE, and 0.2% SDS at about 65°C. for about 30-60 minutes. The reagents mentioned in the above procedures for conducting hybridization high rigidity, is well known in this field. The detailed composition of these reagents can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York (1989) or Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rdEdition; Cold Spring Harbor Laboratory Press, Plainview, NY (2001). In addition to the above conditions, other conditions of high stringency which may be used are also well known in this field.

Expressing a vector containing a nucleic acid molecule encoding a protein CEA can be used for the expression of high level of the slit CEA protein in a recombinant cell host. Expressing vectors can include, but is not limited to, cloning vectors, modified cloning vectors specifically designed plasmids or viruses. Can also be used in a variety of bacterial expressing vectors for the expression of recombinant fused sequences of CEA in bacterial cells, if this is desirable. In addition, various expressing vectors of fungal cell can be used for the expression of the fused sequences of CEA in the cells of fungi. Further, the various expressing vectors in insect cells can be used for expression of recombinant protein in insect cells.

The invention relates also to the cell host transformed or transfitsirovannykh vectors containing the nucleic acid molecule of the present invention. Recombinant kladionica can be prokaryotic or eukaryotic, including, but not limited to, bacteria, such asE. colithe cells of fungi, such as yeast, mammalian cells, including, but not limited to, cell lines derived from bulls, pigs, monkeys, and rodents; and insect cells, including, but not limited to, cell lines derived fromDrosophilaand silkworms. Such recombinant cell host may be cultured in suitable condition for receiving the slit CEA protein or a biologically equivalent form. In a preferred embodiment, a host cell is a human cell. As defined here, the term "a host cell" is not intended to include the host cell in the body transgenic human fetal human or human embryo.

As noted above, expressing a vector containing a DNA encoding a protein CEA can be used for the expression of the slit CEA protein in a recombinant cell host. Thus, another aspect of the present invention is a method of expression of the slit CEA protein in the recombinant cell host, providing for (a) introducing a vector containing a nucleic acid containing a nucleotide sequence which encodes a protein CEA, in a suitable cell host, where this protein contains a CEA protein or variant, merged the significant part immunoprivileged element or its subunit, where this Immunobiology element or its subunit selected from the group consisting of heat shock protein (HSP) 70, associated with lysosomal membrane protein (LAMP), a fragment With tetanus toxoid (FrC), N-terminal domain of FrC (DOM), heaviest fragment constant chain immunoglobulin G1 (FcIgG), the glycoprotein of vesicular stomatitis virus (VSV-G), cholera toxin (CT) fromVibrio choleraeand thermolabile of enterotoxinE. coli(LT); and where this protein is able to induce an immune response in a mammal; and (b) culturing this host cell under conditions which allow expression of the specified fused protein CEA.

Preferred immunobiologii elements for use in this aspect of the invention is selected from the group consisting of LTB, LTA, DOM and FcIgG.

In the following preferred embodiment of this aspect of the present invention the nucleotide sequence of CEA-part of this merged structure and/or part immunoprivileged element of this merged designs are optimized in terms of codons for high level expression in human cells.

The present invention relates also to method of expression of a fused protein CEA-LT in the recombinant cell host, providing for (a) introducing a vector containing a nucleic acid containing a sequence nucleate is s, which encodes a protein CEA-LT, in a suitable cell host, where this protein contains a CEA protein or variant, merged with a significant part of the subunit of the LT, and where this protein is able to induce an immune response in a mammal; and (b) culturing this host cell under conditions which allow expression of the specified fused protein CEA-LT.

In a preferred variant of the method of expression of a fused protein CEA-LT described above, a subunit of LT is an essential part of the LTB, and in the sequence LTB demeterova its signal sequence. In other embodiments, implementation subunit of LT is LTA or its essential part.

The present invention relates also to method of expression of a fused protein CEA-DOM in the recombinant cell host, providing for (a) introducing a vector containing a nucleic acid containing a nucleotide sequence which encodes a protein CEA-DOM, in a suitable cell host, where this protein contains a CEA protein or variant, merged with a substantial part of the N-terminal domain fragment of tetanus toxin (DOM) and where this protein is able to induce an immune response in a mammal; and (b) culturing this host cell under conditions which do possible the expression pointed to by the th fused protein CEA-DOM.

In preferred embodiments of the method of expression of a fused protein CEA-DOM, as described above, the DOM part is optimized in terms of codons for high level expression in human cells. In other preferred embodiments, the implementation of CEA-part of the merged design CEA is optimized in terms of codons for high level expression in human cells. In other preferred embodiments, the implementation as CEA-part, and the DOM part are optimized in terms of codons for high level expression in human cells.

After the expression of the slit structure of CEA in the cell-host protein CEA can be selected with the receiving slit CEA protein in active form. Several procedures protein purification are available and suitable for use. Recombinant protein 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 hydroxyapatite and hydrophobic chromatography. In addition, protein CEA may be separated from other cellular proteins by use of immunoaffinity column made with monoclonal or polyclonal antibodies specific against CEA protein or polypeptide fragments the clients CEA protein.

Were designed molecules of nucleic acids containing fused design CEA, and merged encoded proteins according to the invention for amplification of CEA-specific immune response, akin to the full-size cDNA that encodes a CEA, for use in the development of vaccines. For additional strengthening of the immunogenic properties of merged sequences of CEA according to the invention in some embodiments described herein implement polynucleotide encoding the fused protein CEA contain optimized codons for subsequent high expression level in the cell host as described above. In these variants of implementation, at least a portion of the codons fused structures CEA is designed to use codons that are preferred for the intended host cell, which in preferred embodiments is a human cell. These optimized merged design CEA can be used to develop recombinant DNA vaccines based on adenovirus or based on plasmids, which provide effective prevention against CEA-induced cancer through immune system, provide neutralizing antibodies and cell-mediated immunity. These synthetic molecules can be used as immunogenic compositions. The present image is the buy treats are optimized in terms of codons merged polynucleotides CEA, which, by direct injection spinal animalin vivoincluding mammals such as primates and man, induce expression of the encoded proteins in the animal.

As indicated above, in some embodiments of the present invention, these synthetic molecules containing a nucleotide sequence in which some of the nucleotides were modified so that was used codons preferred for human cells, which makes possible the expression of the high protein level in the cell is the master of man. These synthetic molecules may be used as the source of the fused protein CEA, for example, fused protein CEA-LT, which can be used in anti-cancer vaccine to provide effective immunoprophylaxis against CEA-induced carcinomas through immunity provided neutralizing antibodies and cell-mediated immunity. These molecules are nucleic acids can also serve as a basis for anticancer vaccines based on DNA.

"Triplet" codon of the four possible nucleotide bases can exist in more than 60 variant forms. Because these codons provide a transcript (information) only 20 different amino acids (as well as the initiation and termination of transcription), some of aminox the slots can be encoded by more than one codon, what is known as the phenomenon of degeneracy of codons. For reasons not fully understood, alternative codons heterogeneously present in the endogenous DNA of different cell types. Indeed, there appears to be a variable natural hierarchy or "preference" for certain codons in some types of cells. For example, the amino acid leucine is encoded by any of the six DNA codons, including CTA, CTC, CTG, CTT, TTA and TTG. An exhaustive analysis of the frequency of codons in the genome of microorganisms revealed that endogenous DNAE. coliusually contains the codon CTG defining leucine, whereas DNA of yeast and myxomycetes most often involves codon TTA defining leucine. Due to this hierarchy, one would expect the probability of obtaining high levels of expression of leucine-rich polypeptide hostE. coliwill depend to some extent on the frequency of use of the codon. For example, there is a possibility that gene-rich codons TTA, is weakly expressed inE. coliwhile gene rich CTG, it may be highly expressed in the host. Similarly, the preferred codon for expression of leucine-rich polypeptide in yeast cells, the owners will be TTA.

The involvement of the phenomenon of preference codons is manifested in the ways of recombinant DNA, and this f is a nomen may explain many of the previous failures in achieving high levels of expression of exogenous genes in successfully transformed organisms hosts: "less preferred" codon can repeatable way be present in embedded gene, but the apparatus of this expression host cell can not work effectively. This phenomenon suggests that the synthetic genes were designed in such a way that they include the preferred codons of the intended host cell, provide the best form of alien genetic material for use in practice of the methods of recombinant DNA. Thus, one aspect of the present invention is a gene fused design CEA, which are optimized in terms of codons for 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 overcome the limitations of the expression of exogenous merged CEA protein in human cells.

In accordance with some of the options for implementation of the present invention, molecules of nucleic acids that encode fused proteins of CEA, turns into a polynucleotide sequence having identical to the transmitted sequence, but using alternative codons, as described in the work of the Lathe, “Synthetic Oligonucleotide Probes Deduced from Amino Acid Data: Theoretical and Practical Considerations” J. Molec. Biol. 183:1-12 (1985), cited here as reference. This method however is but involves the identification of codons in the sequence of the wild type, which are not usually associated with highly expressed human genes, and the replacement of optimal codons for high level expression in human cells. Then this new sequence of the gene is examined for undesirable sequences generated by these substitutions codons (for example, the sequence "ATTTA", inadvertent creation of sites of recognition splicing introns, unwanted sites restricts etc). Unwanted sequence helps eliminate the replacement of the existing codons different codons encoding the same amino acid. Then these synthetic gene segments have improved the expression.

Described here above methods are used for the generation of synthetic sequences of genes that encode fused proteins CEA, obtaining gene containing codons optimized for expression of the highest level. Although the above procedure ensures that the summation of the methodology of the authors to construct optimized in terms of codons of genes for use in anti-cancer vaccines, the person skilled in the art it is clear that a similar vaccine efficacy or increased expression of genes can also be achieved minor variations in this procedure, or minor variations in the sequence.

Specialists in this region the STI is also clear what can be designed with additional molecules of nucleic acids, which can provide high levels of expression of the slit structure of CEA in human cells, and only a portion of the codons in these DNA molecules are optimized in terms of codons. For example, in some embodiments, implementation of the present invention, the codons that make up CEA-part of the merged design CEA, are optimized for high level expression in human cells, and codons comprising the adjuvant of this merged structure of CEA are essentially the same as the nucleotide sequence of the adjuvant of the wild type. In other embodiments, implementation of the present invention codons comprising the adjuvant part of the merged design CEA, are optimized for high level expression in human cells, and the codons that make up CEA is part of this merged structure of CEA are essentially the same as the CEA gene of the wild type. In other embodiments, implementation of the present invention as CEA-part and part adjuvant merged design CEA are optimized in terms of codons for high level expression in human cells. Fused design CEA, in which only a subpopulation of codons is optimized in CEA-parts and/or adjuvant merged con is e.g. CEA, also discusses invention.

Nucleic acids according to the invention can be assembled in expressing cassette, which contains sequences that are designed to ensure efficient expression of the protein in the cell. The cassette preferably contains a gene that encodes a protein CEA, with appropriate regulatory sequences for transcription and translation, functionally related, such as the promoter and the termination sequence. In the preferred embodiment, this promoter can be a promoter of cytomegalovirus without the sequence of the intron And (CMV), although specialists in this field will understand that any of a number of other known promoters, such as a strong promoter of the immunoglobulin or the other promoters of eukaryotic genes, can be used. Preferred transcription terminator is a terminator of bovine growth hormone, although it can also be used other known transcription terminators. Especially preferred is a combination of CMV-BGH terminator.

In accordance with the invention, the expression cassette fused design CEA built into the vector. This vector is preferably an adenoviral or plasmid vector, although can be also used linear DNA associated with promotora, or other vectors, such as adenopathy a virus or a modified vaccinia virus, retroviral or lentiviral transfer vector.

If the selected vector is an adenovirus, preferably, to them was the so-called adenoviral vector of the first generation. These adenoviral vectors have non-functional region of the gene E1 and preferably deletionism region of the E1 gene of adenovirus. In some embodiments, the implementation of the expression cassette is inserted into position in which is usually located adenoviral E1 gene. In addition, these vectors do not necessarily have non-functional or deleteriously area E3. Preferably used genome of adenovirus had deleteregvalue areas E1 and E3 (Δ1Δ3). These 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 produced from 293 cells or PERC.6, which transtorno or stably transformed to Express an additional amount of protein. For example, using designs that are regulated gene expression, for example, regulated by the promoter of the tetracycline system, this cell line can Express the components involved in this regulatory system. One example of this cleoc the second line is the T-Rex-293; in this area there were many other examples.

For ease of manipulation of adenovirus vector, the adenovirus may be in the form of a Shuttle plasmid. The present invention relates also to a Shuttle plasmid vector, which contains the plasmid portion and an adenoviral part, and an adenoviral part contains adenoviral genome with a deletion of E1 and, optionally, a deletion of E3, and has an integrated expression cassette containing the coding protein CEA nucleotide sequence. In preferred embodiments, the implementation has a restriction site flanking adenoviral part of this plasmid, so that the adenoviral vector can be easily removed. This Shuttle plasmid can replicate in prokaryotic cells or eukaryotic cells.

In the preferred embodiment of this invention the expression cassette is integrated into an adenoviral plasmid pMRKAd5-HVO (see Emini et al., WO 02/22080, which is incorporated herein by reference). This plasmid contains the gene of adenovirus Ad5 with a deletion of the areas E1 and E3. Construction of plasmids pMRKAd5-HV0 was improved relative to the previous adenovectors lengthening of the 5'CIS-acting packaging area additionally in the E1 gene for inclusion of items that were found, are important in optimizing packaging VI the USA, leading to enhanced amplification of the virus. Preferably, this enhanced adenoviral vector capable of maintaining genetic stability after breeding with a high number of passages.

Standard methods of molecular biology for obtaining and purifying DNA structures allow you to get the adenovirus Shuttle plasmid and DNA immunogen of the present invention.

In accordance with the present invention it was determined that molecules encoding a protein CEA-LT described herein (e.g., SEQ ID NO:12), which contain a significant portion subunits LTA or LTB thermolabile of enterotoxinE. coliexpressed with equivalent efficiency in comparison with the corresponding sequence of CEA wild type (see example 4). It was also shown that plasmids pV1J/hCEA-LTA and pV1J/hCEA-LT induce a higher response in the form of antibodies than pV1J/hCEA, confirming adjuvant effect shown subunits LT on CEA-specific immune response (see example 11). Thus, the described results demonstrate that the merged design coding CEA sequence with cDNA LTA or LT leads to an increase in CEA-specific immune response. Apparently, LT has more to intensify the effect of this immune response with a predominant induction of CD8+T cells, whereas LTA inducir the t predominant CD4 +reaction.

In accordance with the present invention it was also shown that tolerance to autoantigens CEA can be destroyed more effectively relative to the full-size cDNA CEA wild type due to increased immunogenic properties of the merged design CEA-LT. Amplifying effect on LTB immunogenic properties of CEA was also observed when injecting plasmids carrying fully optimized codons cDNA fused design CEA-LT. Finally, the described results using adenoviral vectors carrying the slit design CEA-LT, suggest that the enhanced immunogenicity of the slit structures CEA-LT is not limited to immunization with plasmid DNA (see example 13).

Additionally, in accordance with the present invention it was shown that plasmids pV1J/hCEA-DOM and pV1J/hCEA-FcIgG induce greater CEA-specific cell-mediated and humoral response than CEA (see example 15). In accordance with the present invention it was also shown that tolerance to autoantigens CEA can be destroyed more effectively described herein fused designs CEA with DOM and FcIgG relative to the full-size cDNA CEA wild type due to increased immunogenic properties of these merged structures CEA. Enhanced immunogenic properties of these fused proteins was observed after immunization with the NK - or Ad-vectors, that suggests that the enhanced immunogenicity of the slit structures CEA-LT is not limited to immunization with plasmid DNA (see example 16).

Thus, the above-described vectors can be used in immunogenic compositions and vaccines for the prevention of adenocarcinoma associated with aberrant expression of CEA and/or to treat existing cancers. The vectors according to the invention make possible the development and production of vaccines to eliminate difficulties of obtaining high levels of expression of exogenous CEA in successfully transformed organisms owners and providing the slit CEA protein that can induce an enhanced immune response when administered to a mammal, such as man.

For this purpose, one aspect of the present invention is a method of prevention or treatment of CEA-related cancer, involving the administration to a mammal a vaccine vector containing polynucleotide containing a nucleotide sequence which encodes a protein CEA, and this protein CEA contains a CEA protein or variant, merged with a significant part immunoprivileged element selected from the group consisting of heat shock protein (HSP) 70, associated with lysosomal membrane protein (LAMP), a fragment With tetanus toxoid (FrC), N-terminal house is on FrC (DOM), heavy fragment constant chain immunoglobulin G1 (FcIgG), the glycoprotein of vesicular stomatitis virus (VSV-G), cholera toxin (CT) fromVibrio choleraeand thermolabile of enterotoxinE. coli(LT); moreover, this protein is able to induce an immune response in a mammal.

In the preferred embodiments described here Immunobiology element selected from the group consisting of LTA, LT, DOM and FcIgG.

As described above, the vaccine vector may be administered for treatment or prevention of cancer in any mammal, including, but not limited to, lung cancer, breast cancer and colorectal cancer. In a preferred embodiment of the present invention this mammal is man.

Additionally, the person skilled in the art can choose any type of vector for use in the described method of treatment and prevention. Preferably, this vector is an adenoviral vector or a plasmid vector. In a preferred embodiment of the present invention this vector is an adenoviral vector containing the adenoviral genome with a deletion in the region of adenovirus E1 and insertion in the area E1 adenovirus, and the insertion contains an expression cassette containing (a) a nucleotide sequence which encodes a fused white the CEA, where this protein CEA contains a CEA protein or variant, merged with immunocellular element or its essential part; where this Immunobiology element selected from the group consisting of heat shock protein (HSP) 70, associated with lysosomal membrane protein (LAMP), a fragment With tetanus toxoid (FrC), N-terminal domain of FrC (DOM), heaviest fragment constant chain immunoglobulin G1 (FcIgG), the glycoprotein of vesicular stomatitis virus (VSV-G), cholera toxin (CT) fromVibrio choleraeand thermolabile of enterotoxinE. coli(LT); and where this protein is able to induce an immune response in a mammal; and (b) the promoter is functionally associated with this polynucleotides.

Additionally, the present invention relates to adenoviral vaccine vector containing the adenoviral genome with a deletion in the region of adenovirus E1 and insertion in the area E1 adenovirus, and the insertion contains an expression cassette containing (a) a nucleotide sequence which encodes a protein CEA, where this protein CEA contains a CEA protein or variant, merged with a significant part immunoprivileged element; where this Immunobiology element selected from the group consisting of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT, LTA and LTB; and where this protein is capable of to induce an immune response in a mammal; and (b) the promoter in respect of the material associated with this polynucleotides.

In the preferred embodiment of this aspect of the invention, the adenoviral vector is a vector Ad5.

In another preferred variant of the invention, the adenoviral vector is a vector Ad6.

In another preferred variant of the invention, the adenoviral vector is a vector RAD24.

For use in the present invention is also considered adenoviral vaccine vector containing the adenoviral genome, which is a natural infect species other than humans, including, but not limited to, adenoviral vectors chimpanzees. The preferred embodiment of the present invention is a vaccine vector chimp Ad3.

In another aspect, the present invention relates to vaccine plasmid, the plasmid containing part and the expression cassette, where this part of the expression cassette contains (a) a nucleotide sequence which encodes a protein CEA, where this protein CEA contains a CEA protein or variant, merged with immunocellular element or its substantial part selected from the group consisting of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT, LTA and LTB; and where this protein is able to induce an immune response in a mammal; and (b) the promoter is functionally associated with this polynucleotides.

In some embodiments, the implementation of this is th invention the recombinant adenovirus or polynucleotide vaccines based on plasmid, described here are used in various combinations priming/stimulation for the induction of enhanced immune response. In this case, these two vectors impose a schema priming and stimulation". For example, the first type of vector injected once or a few times, then after a predetermined period of time, for example, 2 weeks, 1 month, six months, or other appropriate interval enter the second type of vector once or several times. Preferably, these vectors are expression cassettes encoding the same polynucleotide or combination of polynucleotides. In the embodiment, in which the use of plasmid DNA, preferably, this vector contains one or more promoters, recognized by mammalian cells or insect. In the preferred embodiment, this plasmid contains a strong promoter, such as, but not limited to, the CMV promoter. Such promoter may be attached synthetic fused gene CEA or another expressed gene. An example of such a plasmid may be expression plasmid V1Jns mammals described in J. Shiver et al., in DNA Vaccines, M. Liu et al., eds., N.Y. Acad. Sci., N.Y., 772: 198-208 (1996), which is incorporated herein by reference.

As stated above, the vaccine in the form of an adenoviral vector and plasmid vaccine can be administered spinal animal in the form of cha is ti single therapeutic schemes for induction of the immune response. For this purpose, the present invention relates to a method of protecting a mammal from CEA-caused cancer, providing an introduction to the mammal a first vector containing (i) a nucleotide sequence which encodes a protein CEA, where protein CEA contains a CEA protein or variant, merged with a significant part immunoprivileged element selected from the group consisting of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT, LTA and LTB; and where a protein is able to induce an immune response in a mammal; and (ii) a promoter functionally linked to this polynucleotides; (b) maintaining within a predetermined period of time; and (C) introduction to the mammal a second vector containing (i) a nucleotide sequence which encodes a protein CEA, where this protein CEA contains a CEA protein or variant, merged with a significant part immunoprivileged element selected from the group consisting of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT, LTA and LTB; and where this protein is able to induce an immune response in a mammal; and (ii) a promoter functionally associated with this polynucleotides.

In one embodiment, the protection method 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 is th vector is a plasmid.

In the above-described method, the first type of vector may be entered more than once, with each introduction of this vector is separated from the previous specified period of time. For such a number of introductions of the first type of vector can be followed by the introduction of a second type of vector once or several times after the passage of a specified period of time. Like processing the first type of vector, the second vector may also be given once or more than once with predetermined time intervals.

In addition, the present invention relates to a method of treatment of a mammal suffering from CEA-related adenocarcinoma involving (a) introduction to the mammal a first vector containing (i) a nucleotide sequence which encodes a protein CEA, where this protein CEA contains a CEA protein or variant, merged with a significant part immunoprivileged element selected from the group consisting of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT and LTB; and where this protein is able to induce an immune response in a mammal; and (ii) a promoter functionally linked with this polynucleotides; (b) maintaining within a predetermined period of time; and (C) introduction to the mammal a second vector containing (i) a nucleotide sequence which encodes a protein CEA, where this protein CEA contains Bel is to CEA or its variant, merged with a significant part immunoprivileged element selected from the group consisting of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT and LT; and where this protein is able to induce an immune response in a mammal; and (ii) a promoter functionally associated with this polynucleotides.

In one embodiment, the above method of treatment of 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.

In preferred embodiments, the methods described above, these vectors contain nucleotides that encode a protein CEA-LT, where this protein CEA contains a CEA protein or variant, merged with a significant part of the subunit of the LT. In additional preferred embodiments, this vector contains a nucleotide sequence which encodes a protein CEA-LTB. In additional preferred embodiments described here, the vectors contain a nucleotide sequence which encodes a protein CEA-DOM, where this protein CEA contains a CEA protein or variant, merged with a significant part of subunit DOM. In additional preferred embodiments, this vector contains a nucleotide sequence which encodes a protein CEA-FcIgG.

If estvo expressed DNA or transcribed RNA, which must be entered in the vaccine recipient, will depend on the power used promoters and immunogenicity expressed gene product. Usually, immunologically or prophylactically effective dose of about 1 ng to 100 mg, and preferably about 10-300 μg of plasmid vaccine vector is administered directly into muscle tissue. Effective dose of recombinant adenovirus of approximately 106-1012particles and preferably approximately 107-1011particles. Also considered subcutaneous injection, intradermal injection, the penetration through the skin and other routes of administration such as intraperitoneal, intravenous, intramuscular or inhalation delivery.

In preferred embodiments, the implementation of the present invention, the vaccine vector is administered to the recipient via intramuscular injection.

Vaccine vectors of the present invention can be "naked", i.e. not associated with any proteins or other agents that affect the immune system of the recipient. In this case, it is desirable that these vaccine vectors were in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Alternatively, it may be preferred that call for the giving agent, which promotes cellular uptake of DNA, such as, but not limited to, calcium ion. These agents are usually called enhance the transfection reagents and pharmaceutically acceptable carriers. Specialists in this field will be able to define a specific reagent or pharmaceutically acceptable carrier, as well as the right time and the scheme of introduction.

All cited publications are listed here as a reference to describe and disclose the methods and materials that can be used in conjunction with the present invention. Nothing should be construed as recognition that the present invention may be contrasted with the information source with an earlier priority.

After the description of the preferred embodiments of the present invention with reference to the accompanying figures should be understood that this invention is not limited to the above specific implementation options and that they can be made various changes and modifications to the person skilled in the art without deviating from the scope or concept of the present invention defined in the attached claims.

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

EXAMPLE 1

Design fused proteins CEA

To determine the immunogenicity of the cast proteins CEA designed a number of vectors, coding for amino acids (hereinafter referred to as AA) 1-679 protein CEA person, merged with the panel selected polypeptide (see example 2). These sequences were chosen because of their reported immunobiologii properties that have been demonstrated in various experimental systems. Fused design CEA designed connection cDNA CEA protein with a deletion of the anchor sequence GPI alien polypeptides (examples of the structures depicted in figure 18A). Tumor antigen associated with the HSP70 sequences, FcIgG or LAMP to determine whether the increased absorption of the antigen or re-targeting endosomal compartment to lead to increased immune response. Similarly, the slit design fragment of tetanus toxin (FrC) or with minimal domain, deprived of potentially competitive binding epitopes of MHC class I (DOM, see figure 29) (Rice et al., J. Immunol. 168: 3908-13 (2002)), designed to enhance humoral and CD4+T-cell reactions. CEA also linked with the encoding VSV-G sequence to determine whether the merged design with viral glycoprotein on the immunogenic properties of the CEA.

Coding sequences of these merged structures CEA cloned in vectors pV1Jns under control of the CMV promoter/intron a plus signal polyadenylic the project for bovine growth hormone (BGH) (example 2). Plasmids pV1J/hCEA-FrC, pV1J/hCEA-DOM, pV1J/hCEA-FcIgG, pV1J/hCEA-LAMP, pV1J/hCEA-VSV-G and pV1J/hCEA-HSP70 are CEA cDNA wild type, fused with the coding sequences of these alien polypeptides. Examples of nucleotide and amino acid sequences fused structures hCEA-DOM and hCEA-FcIgG shown in figures 26, 27 and 30.

To assess the actions of subunits LTA and LT thermolabile of enterotoxinE. colion the immunogenicity of CEA generated a number of additional fused constructs encoding amino acids 1-679 CEA protein, fused with the coding or LTA (amino acids 18-259)or LT (amino acids 21-125) sequence. Schematic representation of the structure examples of the slit structures CEA-LTA and CEA-LT obtained for this study, shown in figure 1. Examples of nucleotide and amino acid sequences fused structures CEA-LT shown in figures 2-6.

Fused design CEA-LT was designed by the connection cDNA CEA protein with a deletion of the anchor sequence with sub-LT, which was removed the coding sequence of the signal peptide. Coding sequences of these merged structures CEA cloned in vectors pV1Jns under the control of the promoter of the cytomegalovirus (CMV)/intron a person plus the polyadenylation signal of bovine growth hormone (BGH). Plasmids pV1J/hCEA-LTA and pV1J/hCEA-LT are CEA cDNA wild type, merged skomorowski sequences LT and LT, respectively (see example 2).

All designs, bearing merged design CEA-LT, generate a merged design CEA cDNA (nucleotides 1-2037) with cDNA-fragment LT comprising nucleotides 64-375. Encoding LT sequence received PCR amplification of genomic DNAE. coliusing sequence-specific primersLTB-S15'- T A T T C T A G A T G C T C C C C A G A C T A T T A C A G A A -3' (SEQ ID NO:1) andLTB-A15'- T A T G C G G C C G C C T A G T T T T C C A T A C T G A T T G C C G C-3' (SEQ ID NO:2). Amplified DNA was introduced at the 3'end of the coding sequence of CEA to obtain plasmids.

EXAMPLE 2

Plasmid construction

pV1J/CEAoptand pV1J/CEA:These two designs are optimized with regard to the use of codons cDNA and cDNA CEA wild type, respectively. The coding sequence of the CEA is located between pregradnim the CMV promoter/intron a cytomegalovirus and a polyadenylation signal of bovine growth hormone. To generate pV1J/hCEAoptplasmid pCR-hCEAopt uncoupledEcoRI for 1 hour at s. The resulting insertion 2156 BP was purified and cloned in the siteEcoRI plasmids pV1JnsB ((Montgomery et al., DNA Cell Biol. 12(9): 777-83 (1993)).

To generate pV1J/hCEA, plasmid pCR-hCEA (Song et al., Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumour immunity by combined DNA-based vaccination and nonviral cytokine gene transfer. Gene Therapy 7: 481-492 (2000)) were digestedEcoRI. Polucen the th insertion 2109 BP cloned in the siteEcoRI plasmids pV1JnsA (Montgomery et al., supra).

pV1J/hCEA-LT and pV1J/hCEAopt-LT:Optimized in terms of codons cDNA LT synthesized by assembling oligonucleotides (Geneart GmbH, Regensburg, Germany) and cloned in the vector pCR-script (Stratagene, La Jolla, CA). To generate pV1J/hCEAopt-LTopt, LToptamplified via PCR using the following PCR primers:LTopt-5'XbaI(5'end) and 5'- G C T C T A G A G C C C C C C A G A G C A T C A C C G A G C T G T G C - 3' (SEQ ID NO:3) andLTopt-3'BglII(3'end) 5'- G C T C T A G A A C C C C T C A G A A C A T C A C C G A T C T G T G C G C C - 3' (SEQ ID NO:4). Then this amplificatory product was embedded in the siteXbaI/BglII plasmids pV1J/hCEAopt.

pV1J/hCEA-LTA:Encoding LTA sequence corresponding to nucleotides 54-774 that encode amino acids 18-259, amplified using PCR from genomic DNA ofE. coliusing sequence-specific primersLT-S15'- T A T T C T A G A T A A T G G C G A C A A A T T A T A C C G - 3' (SEQ ID NO:5) andLT-A15'- T A T G C G G C C G C T C A T A A T T C A T C C C G A A T T C T G T T - 3' (SEQ ID NO:6). Amplified DNA was digested with appropriate restrictase and was built in plasmid pV1J/hCEA.

pV1J/rhCEAopt-LT: 3'-fragment cDNA CEA macaque-rhesus (nucleotides 1641-2026), which was optimized in terms of codons for high level expression in human cells, amplified using PCR from pV1J-rhCEAopt. Amplificatory cdntral devoid encodes a GPI-anchor sequence and carried the restriction enzymes cut sites XbaI/BglII. This fragment was embedded in the sitePstI pCR-blunt-rhCEAopt obtaining thus an intermediate plasmid pCR-blunt-rhCEAopt XbaI/BglII. rhCEAopt were extracted in the form ofBglII/SalI fragment and cloned into the same sites in pV1J-nsB obtaining thus pV1J-rhCEAopt XbaI/BglII. LTBopt amplified using PCR from the pCR-script-LTBopt with adding sitesXbaI andBglII at the 5'- and 3'-ends, respectively, and cloned in pV1J-rhCEAopt XbaI/BglII obtaining thuspV1J-rhCEA-LTopt.

pV1J/CEA-FrC, pV1J/CEA-DOM, pV1J/CEA-FcIgG, pV1J/CEA-LAMP, pV1J/CEA-HSP70 and pV1J/CEA-VSV-G:All these constructs encoding these fused proteins CEA, generate a merged design CEA cDNA from nucleotides 1-2037 (SEQ ID NO:22, figure 28A), corresponding to amino acids 1-679 (SEQ ID NO:23, figure 28C), with the cDNA fragment corresponding to the following fragments: fragment With tetanus toxoid (CEA-FrC, SEQ ID NO:24), N-terminal domain of FrC (CEA-DOM, SEQ ID NO:21 and 49), the heavy fragment constant chain immunoglobulin G1 (CEA-FcIgG, SEQ ID NO:25), associated with lysosomal membrane protein (CEA-LAMP, SEQ ID NO:26), heat shock protein 70 (CEA-HSP70, SEQ ID NO:27) or the glycoprotein of vesicular stomatitis virus (CEA-VSV-G, SEQ ID NO:28).

Coding sequences FrC and DOM were obtained using PCR amplification from a plasmid pRep-TeT.C, as described in Rice et al. (J. Immunol. 169: 3908-13 (2002)). FcIgG was obtained from total RNA of human PBMC. VSV-G and HSP70 were obtained from p-FAST-VSV-G and PL is smidi pY3111, respectively. LAMP1 was obtained by Assembly of genes. Amplification was performed using the following primers: FrC sense (5'-T A TT C T A G AT T C A A C A C C A A T T C C A T T T T C T T A T T C -3' (SEQ ID NO:29), FrC antisense (5'-G C G G C C G C T A G A A C T A T T T G T C C A T C C T T C A T C -3' (SEQ ID NO:30), DOM sense (5'-T A TT C T A G AT T C A A C A C C A A T T C C A T T T T C T T A T T C -3' (SEQ ID NO:31), DOM antisense (5'- T T A G C G G C C G C T A G T T C T G T A T C A T A T C G T A A A G G G -3' (SEQ ID NO:32), FcIgG sense (5'-T C T A G AT A A A A C T C A C A C A T G C C C A -3' (SEQ ID NO:33), FcIgG antisense (5'- G C C G A C T C A T T T A a C C C G G A G A C A G G G A G -3' (SEQ ID NO:34), LAMP sense (5'-T C T A G AT T T G A T C C C C A T T G C T G T G G G C G G T G C C C T G -3'(SEQ ID NO:35), LAMP antisense (5'- G G C G T G A C T C C T C T T C C T G C C A A T G A G G T A G G C A A T G A G -3' (SEQ ID NO:36), VSV-G semantic (5'-A T AT C T A G AT T T C A C C A T A G T T T T T C C A C A C A A C C -3' (SEQ ID NO:37), VSV-G antisense (5'-G C G G C C G C C T T C C T T C C A A G T C G G T T C A T C T C T A T G -3' (SEQ ID NO:38), HSP70 sense (5'-G CT C T A G AT A T G G C T C G T G C G G T C G G G A T C G A C C -3' (SEQ ID NO:39)) and HSP70 antisense (5'-G C C G C G G C C G C T C A C T T G G C C T C C C G G C C G T C G T C G -3' (SEQ ID NO:40). Amplified DNA was introduced at the 3'end of the coding CEA sequence to obtain plasmids pV1J/CEA-FrC, pV1J/CEA-DOM, pV1J/CEA-FcIgG, pV1J/CEA-LAMP, pV1J/CEA-HSP70 and pV1J/CEA-VSV-G.

pV1J/CEA-DOMopt and pV1J/CEA-FcIgGopt:Optimized with regard to the use of codons cDNA DOM and FcIgG synthesized by assembling oligonucleotides (Geneart GmbH, Regensburg, Germany) and cloned in the vector pCR-script (Stratagene, La Jolla, CA). To generate pV1J/CEA-DOMopt, DOMopt/sub> amplified via PCR using the following primers: DOMoptsemantic DOM (5'- G T T AT C T A G AA G C A C C C C C A T C C C -3' (SEQ ID NO:41)) and DOMoptreverse (5'-T T A A G A T C T C T A A G A T C T G G T G T C G T A T C T C A G G G G -3' (SEQ ID NO:42). Then amplificatory product was embedded in the siteXbaI/BglII plasmids pV1J/CEAopt. To generate pV1J/CEA-FcIgGopt, FcIgGoptamplified via PCR using the following primers: FcIgGoptsense (5'- T T AT C T A G AA A G A C C C A C A C C T G C C C C C C T T G C -3' (SEQ ID NO:43)) and FcIgGoptreverse (5'- T A T A G A T C T T A G G G T A C C T T A C T T G C C G G G G -3' (SEQ ID NO:44)), amplificatory product was embedded in the siteXbaI/BglII plasmids pV1J/CEAopt.

EXAMPLE 3

Adenoviral vectors

Ad5/hCEAopt: Plasmid pCR-hCEAopt uncoupledEcoRI. The resulting insertion 2156 BP was purified and cloned inEcoRI Shuttle plasmids polyMRK-Ad5.

Ad5/CEA: Shuttle plasmid pMRK-hCEA to generate Ad5 vector was obtained by cleavage of the plasmid pDelta1sp1B/hCEASspI andEcoRV. Then the fragment 9,52 TPN ligated with 1272 BPBglII/BamHI-restricciones processed by the fragment maple product from plasmid polyMRK.PacI/StuI-fragment of pMRK-hCEA and pMRK-hCEAopt containing the expression cassette for hCEA and E1 flanking regions Ad5, recombinable linearized withClaI plasmid pAd5 in cellsE. coliBJ5183. The obtained plasmids b is whether pAd5-hCEA and pAd5-hCEAopt, respectively. Both plasmids cutPacI for release ITR Ad and transfusional cells PerC-6. Amplification of Ad5 vectors were performed using serial passage. MRKAd5/hCEA and MRKAd5/hCEAopt was purified using standard purification in CsCl gradient and intensively were dialyzed against buffer A (5 mm Tris-Cl pH 8.0, 1 mm MgCl2, 75 mm NaCl, 5% sucrose, 0.005 to tween-20).

Ad5/hCEAopt-LTB:Plasmid pMRK-hCEAopt-LTB designed cutting Shuttle plasmids polyMRK-Ad5SwaI and legirovaniem this linearized vector with a DNA fragment 2300 BP, obtained from pV1J/hCEAopt-LTB, which was restricionEcoRI,BglII and processed by the fragment maple. This pMRK-hCEAopt-LTB was linearizable and recombinable in the Ad genome, as described above.

Ad5/CEA-DOMopt and Ad5/CEA-FcIgGopt:Plasmids pMRK-CEA-DOMopt and pMRK-CEA-FcIgGopt designed cutting Shuttle plasmids polyMRK-Ad5SwaI and legirovaniem this linearized vector with a DNA fragment of 2.9 TPN obtained from pV1J/CEA-DOMopt, or legirovaniem this linearized vector with a DNA fragment 2700 BP, obtained from pV1J/CEA-FcIgG1opt that were restrictivelyEcoRI,BglII and processed by the fragment maple. pMRK-CEA-FcIgGopt and pMRK-CEA-DOMopt was linearizable and recombinable in the Ad genome, as described above.

EXAMPLE 4

Comparative efficiency of expression of various fused structures CEA-LT

It was shown that the application of optimizer the offer in respect of cDNA codons for genetic vaccination against viral diseases induced a higher immune response partially due to increased expression of the protein target. To check whether the coding LT sequence to enhance the immunogenic properties of the CEA cDNA, designed to enable the preferred for a person (humanized) codons for each amino acid residue, was also designed plasmid pV1J/hCEAopt-LTB. Finally, designed also fully optimized in terms of codons variant fused design CEA-LT using synthetic optimized in terms of codons cDNA LT to generate plasmids pV1J/hCEA-LTBopt.

For definitions not limited to whether the action LT on the immunogenicity of CEA immunization with plasmid DNA, was also designed vector adenovirus type 5 encoding merged design opt-LTB, flanked by the CMV promoter/intron a and the BGH polyadenylation signal. The molecular weight of these fused proteins CEA expressed as plasmid and adenoviral vectors, did not differ from the molecular mass derived from the corresponding vectors encoding full-size form CEA cDNA (data not shown).

To compare the efficiency of expression vectors encoding the merged design CEA-LTA and CEA-LT, and the efficiency of expression of cDNA full-CEA, HeLa cells were transfusional plasmids pV1J/hCEA-LTA and pV1J/hCEA-LTB. The expression of CEA those to whom instructions were compared with the corresponding expression plasmids bearing CEA cDNA wild type, pV1J/hCEA. Similarly, the efficiency of expression plasmids pV1J/hCEAopt-LT compared with the efficiency of expression pV1J/hCEAopt. The efficiency of expression of these constructs was determined two days after transfection by monitoring the number of CEA protein in cell extracts.

Transfection of plasmids pV1J/hCEA-LTA and pV1J/hCEA-LT gave about two times higher amount of protein CEA (183 and 139 ág/l, respectively, figure 8A), detected in the culture supernatant, in comparison with plasmid pV1J/hCEA (91 μg/l). Similarly, the efficiency of expression constructs pV1J/hCEAopt and pV1J/hCEAopt-LT was also comparable (113 and 136 ág/l, respectively; figure 8B). Finally, the efficiency of expression of Ad/hCEAopt and Ad/hCEAopt-LTB were also compared by infection of HeLa cells at different moi (multiplicity of infection). The efficiency of expression of CEA of these two vectors was comparable at moi 1000 (1790 and 1400 µg/l, respectively, figure 9), while at moi 100 vector Ad/hCEAopt-LTB were given approximately four times lower amounts of protein CEA, detected in the culture supernatant than Ad/hCEAopt (390 and 1500 µg/l, respectively).

Thus, these results indicate that the cDNA encoding a fused protein CEA-LTA and CEA-LT, is expressed with equivalent efficiency relative to the efficiency of expression is relevant to the respective cDNA, coding a full-sized protein CEA. In addition, comparable expression of CEA of these cDNA did not depend on the type of vector is a vector of genes used for their delivery.

EXAMPLE 5

Detection of expression of CEA

The expression of CEA plasmid and Ad-vectors were monitored using Western blot analysis and ELISA. Plasmids were transfusional HeLa cells using Lipofectamine 2000 (Life Technologies). Adenoviral infection of HeLa cells was performed in serum-free medium for 30 minutes at 37aboutAnd then added a fresh environment. After 48 hours of incubation were collected lysates of whole cells. Protein CEA present in these cell lysates, were detected using Western blot analysis using rabbit polyclonal antisera. This protein is detected in the form of a strip 180-200 kDa. The number of expressed CEA was detected in cell lysates using a set of Direct Elisa CEA (DBC-Diagnostics Biochem Canada Inc.).

The expression of the fused proteins in transfected cells was investigated by Western blot analysis using antibodies specific against CEA, VSV-G, FcIgG, tetanus toxin or HSP70. HeLa cells or transfusional the indicated plasmid or infected chosen Ad-vector. After 48 hours of incubation were collected lysates of whole cells and culturalinstitute.

The expression of CEA in a cellular lysate or supernatant was also subjected to monitoring using a set of Direct Elisa CEA (DBC-Diagnostics Biochem Canada Inc.). Protein CEA detected with an antibody specific against this fused polypeptide in transfected cell lysates, while not observed in the expression of the target antigen in monocrystalline control samples (figure 18B). The molecular weight of these fused proteins did not differ significantly from the molecular weight CEA. This apparent lack of differences in molecular weight between the different polypeptides CEA owing, perhaps, to a high degree of glycosylation of this tumor antigen.

To compare the efficiency of expression vectors encoding these merged design CEA, with the efficiency of expression pV1J/CEA, HeLa cells were transfusional different plasmids and the expression of CEA of these structures was determined two days after transfection, using ELISA. Plasmids pV1J/CEA-FrC, pV1J/CEA-DOM, pV1J/CEA-FcIgG, pV1J/CEA-LAMP, pV1J/CEA-VSV-G and pV1J/CEA-HSP70 expressed CEA with comparable efficiency relative to pV1J/CEA (figure 19A). Most of the fused proteins secretarials and detected in the cell supernatant; however, CEA-LAMP was not released from transfected cells, possibly due to its transition into the lysosomal compartment (figure 19C). Thus, these results what you shows that cDNA, codereuse fused proteins CEA-FrC, CEA-DOM, CEA-VSV-G, CEA-FcIgG, CEA HSP70 and CEA-LAMP, expressed with an efficiency equivalent to the efficiency of the cDNA encoding the full-size protein CEA.

EXAMPLE 6

Peptides

Lyophilized peptides hCEA purchased from Bio-Synthesis and resuspendable in DMSO at 40 mg/ml Pools of peptides with a length of 15 amino acids, 11 overlapping residues were collected as described (Facciabene et al., J. Virol. 78: 8663-72 (2004). Final concentrations were as follows: pool a=1.2 mg/ml, Poole 0.89 mg/ml, pool 0,89 mg/ml, Poole D 0.8 mg/ml of the Peptides were stored at -80aboutC. Immune response against DOM was subjected to monitoring using peptide tetanus toxoid P30 (F947NNFTVSFWLRVPKVSASHLE967(SEQ ID NO:54) (Rice et al., J. Immunol. 167: 1558-65 (2001)).

EXAMPLE 7

Immunization of mice and stimulation of tumor antigen

All animal studies were approved by the IRBM institutional animal care and use committe. Female mice 57BL/6 (H-2b) were purchased from Charles River (Lecco, Italy). Mouse HLA-A2.1 (HHD) were kindly provided by F. Lemmonier (Institute Pasteur, Paris, France). Mice 57BL/DR4 were purchased from Taconic (Germantown, NY). Mouse .tg (transgenic) (H-2b) were kindly provided by J. Primus (Vanderbilt University) and they were kept under standard conditions (Clarke et al., Cancer Res. 58:1469-77 (1998)). Fifty micrograms of plasmid DNA was electroinactive in a volume of 50 µl in the quadriceps muscle of mice, as described previously (Rizzuto et al., Proc. Natl. The Aca. Sci. USA 96(11): 6417-22 (1999)). Injection of Ad spend in the quadriceps muscle of mice in a volume of 50 μl. Humoral and cell-mediated immune response was analyzed in the time specified.

Of C57BL/6 mice were subjected to two injections of DNA in the quadriceps muscle with subsequent electric stimulation, as described above (Rizzuto et al., supra). Injection was carried out at three-week intervals. CEA-transgenic mice were subjected to either 5 weekly injections of plasmid DNA (50 μg/injection), 2 injections of Ad-vectors (1×109viral particles/injection), or 5 weekly injections followed by stimulation of Ad. Two weeks after the last injection were analyzed humoral and cell-mediated reaction. Mice were also administered subcutaneous (s.c) injection of 5×105cells MS-CEA (Clarke et al., supra). At intervals of one week, mice were examined for tumor growth.

EXAMPLE 8

Detection and titration of antibodies

Serum titration of antibodies received retrogression krovoisliania. Tablets ELISA (Nunc maxiprep) were coated with 100 ng per well of purified protein CEA (Fitzgerald), diluted in buffer for sensitization of the surfaces (50 mm NaHCO3pH 9.4) and incubated overnight at 4aboutSince, as described previously (Facciabene et al., supra). Then the plates were blocked by PBS containing 5% BSA for 1 hour at 37°C. Serum mice Rabba who ranged in PBS with 5% BSA (dilution 1/50 to assess the extent of seroconversion; the dilution 1:10 - 1:31,2150 for the evaluation of the title). Preimmunization serum was used as background. Diluted sera were incubated overnight at 4°C. Washing was performed using PBS with 1% BSA, 0.05% Tween-20. Secondary antibodies (goat antibodies against mouse IgG conjugated to peroxidase, Sigma) diluted in PBS, 5% BSA and incubated for 2-3 hours at room temperature on a shaker. After washing tablets showed using 100 μl per well of TMB substrate (Pierce Biotechnology, Inc., Rockford, IL). The reaction was stopped 25 ál per well of 1 M solution of H2SO4and the tablets were read at 450 nm/620 nm. The titers of anti-CEA-sera was calculated as the reciprocal limiting dilution of serum giving an optical density of at least 3 times higher than the optical density of autologous preimmunization serum at the same dilution.

EXAMPLE 9

IFN-γ-ELISPOT-analysis

The analysis was performed using mouse splenocytes and CEA-specific peptides, as described previously (Facciabene et al., supra). 96-well tablets MAIP (Millipore Corp., Billerica, MA) was senzibilizirani 100 µl per well of purified rat antibody against mouse IFN-γ (IgG1, clone R4-6A2, Pharmingen)diluted to 2.5 μg/ml in sterile PBS. After washing PBS were blocking tablets 200 µl per well of the environment R10 for 2 hours at 37°C.

Splenocytes were sown at 5×105and 2.5×105cells per well in duplicate and incubated for 20 hours at 37°C With 1 µg/ml suspension of each peptide. Of concanavalin a (ConA) was used as a positive internal control for each mouse at a concentration of 5 µg/ml After washing PBS, 0.05% Tween 20, the plates were incubated overnight at 4°C With 50 µl per well conjugated with Biotin rat antibody against mouse IFNγ (rat RatIgG1, clone XMG 1.2, PharMingen)diluted 1:2500 in the buffer for analysis. After intensive washing tablets showed by adding 50 μl per well NBT/B-CIP (Pierce Biotechnology, Inc., Rockford, IL), until the manifestation of the spots did not become clearly visible. The reaction was stopped by thorough washing tablets with distilled water. The tablets were air-dried and then counted using automated ELISPOT reader.

EXAMPLE 10

Intracellular staining of cytokines

Od the h-two million mouse splenocytes or PBMC in 1 ml RPMI with 10% FCS were incubated with a pool of peptides (final concentration of each peptide 5-6 µg/ml) and brefeldin A (1 μg/ml; BD Pharmingen cat. #555028/2300kk) at 37°C and 5% CO2within 12-16 hours, as described previously (Facciabene et al., supra). Then cells were washed in FACS buffer (PBS-1% FCS, 0.01% of NaN3), and incubated with purified anti-CD16/CD32 Fc-specific antibody (blocking reagent) (BD Pharmingen cat # 553142) for 15 min at 4°C. Then cells were washed and stained with surface antibodies: CD4-PE-conjugated antimurine antibody (BD Pharmingen cat. #553049), CD8 PercP-conjugated antimurine antibody (BD Pharmingen cat. #553036) and APC-conjugated antimurine antibody against CD3 (BD Pharmingen cat. #553066) for 30 min at room temperature in the dark. After washing the cells were fixed and increase their permeability solution Cytofix-Cytoperm (BD Pharmingen cat. #555028/2300kk) for 20 min at 4C in the dark. After washing solution PermWash (BD Pharmingen cat. #555028/2300kk) cells were incubated with IFNγ-FITC-antibodies (BD Pharmingen). Then cells were washed, fixed with 1% formaldehyde in PBS and analyzed on a flow cytometer FACS Calibur using CellQuest program (Becton Dickinson, San Jose, CA).

EXAMPLE 11

The immunogenicity of the slit structures CEA-LT

For testing immune responses induced by plasmid encoding the merged design CEA-LTA and CEA-LT, groups of 9 mice 57BL/6 were immunized with two injections i.m. 50 μg each of the plasmids pV1J/hCEA, pV1J/hCEA-LTA and pV1J/hCEA-LT. Additionally, to test whether coexpressed merged Belko is CEA-LTA and CEA-LT to have an additional effect on the immunogenicity of the protein CEA, a group of mice were immunized joint injection of 25 μg each of the plasmids pV1J/hCEA-LTA and pV1J/hCEA-LT. Immunization was performed with a three-week intervals. Plasmid DNA routinely have electroinactive in the skeletal muscle of the mouse due to enhanced transduction and immunogenicity associated with this particular procedure (Zucchelli et al. J. Virol. 74: 11598-11607 (2000); Widera et al. J. Immunol. 164: 4635-4640 (2000)).

Cell-mediated immunity induced by various plasmids was measured using ELISPOT analysis 2 weeks after the last injection. Antigen-specific secretion of IFNγ stimulated of splenocytes was measured using four pools of 15-dimensional peptides, overlapping by 11 amino acids and includes full CEA glycoprotein. Pool a includes amino acids 1-147, the pool includes amino acids 137-237, pool comprises amino acids 317-507 and pool D includes amino acids 497-703. As negative control the production of cytokines was measured after stimulation of splenocytes DMSO at the same concentration, which was used to solubilize the peptides CEA.

The immune response induced by DNA vaccination in mice 57BL/6, was primarily shifted to the With-end of the protein, because the value of SFC (forming spots of cells), detected with a pool And peptides were slightly above background with all structures (figure 10). With the EMA vaccination pV1J/hCEA-LTB surpassed scheme of vaccination pV1J/hCEA, as shown by higher values of the geometric mean SFC, detektirovanie pools b, C and D (pV1J/hCEA-LTB: 482, 1436 and 2054 SFC/106the splenocytes, respectively; pV1J/hCEA: 45, 350 and 264 SFC/106the splenocytes, respectively). Similarly, plasmid pV1J/hCEA-LTA also had a synergistic effect on CEA-specific immune response in comparison with pV1J/hCEA. However, the increase in immune response was observed only from pools C and D peptides (925 and 528 SFC/106the splenocytes, respectively), whereas the immune response, measured with a pool of peptides was low (15 SFC/106the splenocytes). In addition, co-injection of plasmids pV1J/hCEA-LTA and pV1J/hCEA-LT did not show synergistic action on the immune response to CEA in comparison with the immune response, measured in the treated pV1J/hCEA-LTB group, but rather led to a decrease in SFC values, detected with peptide pool b and D (528 210 and SFC/106the splenocytes, respectively).

To determine T-cell specificity, induced after vaccination with different designs CEA, intracellular IFNγ staining was performed on pooled splenocytes from injected mice using pool D peptides. CD8+-specific response was detected in mice injected pV1J/hCEA-LTB, superior (4,5%) CD8+-specific response detected with pV1J/hCEA-LTA and pV1J/hCEA (0,14% and 0.8%, respectively, Fi is ur 10V). In contrast, pV1J/CEA-LTA induced a strong CD4+-specific reaction, higher (1,21%)than CD4+-specific response observed with pV1J/hCEA-LT and pV1J/hCEA (0,55% 0,58%, respectively).

The induction of humoral immune response to CEA was tested by measuring antigen-specific antibodies (figure 11). Both plasmids pV1J/hCEA-LTA and pV1J/hCEA-LT induced a higher response of antibodies than pV1J/hCEA, confirming adjuvant effect exhibited by sub-LT, CEA-specific immune response. Thus, these results demonstrate that the merged design coding CEA sequence with cDNA LTA and LT leads to an increase in CEA-specific immune response. However LT, apparently, has a greater reinforcing effect on this immune response with predominant induction of CD8+T cells, whereas LTA induces a predominant CD4+reaction.

EXAMPLE 12

The immunogenicity of the slit structures CEA-LT in different strains of mice

For definitions not limited to whether a synergistic effect subunits LT on CEA-specific immune response to only a single genetic background of the mice, immunization with DNA-based was conducted in mice BALB/c, C57/DR4 and HLA-A2.1 (D). Mouse BALB/c mice were chosen because of their immunocompetent, because this strain of mice is extremely reactive scheme immunizationbasics varieties. HHD transgenic mice Express the genes of the MHC class I human. Similarly, transgenic mice C/DR4 genes are MHC class II human. Thus, these two strains of transgenic mice may provide information regarding the immunoreactivity merged structures CEA-LT in the context of the haplotypes as MHC class I and MHC class II human.

CEA-specific immune response in BALB/c mice were first evaluated using ELISPOT analysis. Enhancing antigen-specific immune response after immunization with plasmid pV1J/hCEA-LT were detected with pools a, b, C, D peptides (pV1J/hCEA-LT: 166, 1353, 796, 899 SFC/106the splenocytes, respectively; pV1J/hCEA: 57, 312, 327, 318 SFC/106the splenocytes, respectively; figure 12). As was observed in mice 57BL/6, N-terminal protein CEA, apparently, is the least immunogenic in comparison with other sections of this tumor antigen. Immunization pV1J/hCEA-LTA was also given the increase in antigen-specific immune response in comparison with pV1J/hCEA. This increase in immune response were detected with pools of peptides b, C and D (936, 727, 650 SFC/106the splenocytes, respectively). In addition, co-injection of these two plasmids pV1J/hCEA-LTA and pV1J/hCEA-LT gave significant additive effect was detected mainly with pools of peptides C and D (1783 and 2141 SFC/106the splenocytes, respectively).

CEA-specific immunodeficiency, the significant response in mice C/DR4 significantly increased immunization pV1J/hCEA-LTB and detected with only one pool of peptides D (figure 13). Staining of intracellular IFNγ performed in the United PBMC from injected mice showed that CD8+reaction to CEA was higher in mice immunized with pV1J/hCEA-LTB (15,32%), whereas was very weak in the treated pV1J/hCEA group (0,5%). Immunization pV1J/hCEA-LTA increased antigen-specific immune response only moderately (0,43%) and non-amplified advanced immunogenicity of CEA co-injection with the design, coding merged design CEA-LTB (13,44%). Interestingly, in these immunized mice are not detected significant CD4+T-cell response (data not shown).

The immune response induced by different CEA-encoding plasmids, were evaluated in mice HHD performing intracellular staining of IFNγ in the United PBMC. Immune responses were detected only with pools of peptides b and D, and, as shown in figure 14, immunization pV1J/hCEA-LT led to a more than 10-fold increase in CD8+response to antigens of the target. In contrast, the increase of the immune response was not detected using pV1J/hCEA-LTA either separately or after joint injection with pV1J/hCEA-LTB. CD4+T-cell response was not detected in these immunized mice (data not shown).

Taken together, these results confirm that the slit design coding LTB sequence with CEA leads to the mean is linoma increase in antigen-specific immune response. Interestingly, this response is predominantly CD8+-specific and can be observed in different strains of mice, indicating, therefore, that a synergistic effect that is manifested by a subunit of LT, is not genotype-restrictionism.

EXAMPLE 13

Tolerance to CEA person in transgenic mice

To determine whether enhanced immunogenic properties of the merged design hCEA-LTB to break tolerance to CEA person more effectively, hCA-transgenic mice were immunized with vectors carrying either fully optimized in terms of codons cDNA hCEA or CEA-LT. These transgenic mice are fully CEA gene of the person and flanking sequences of the gene and Express the protein hCEA in the intestine, mainly in the caecum and colon. Thus, this mouse line is a useful model to study the safety and efficacy of immunotherapy strategies aimed against this tumor autoantigen (Clarke et al., Cancer Research 58: 1469-1477 (1998)).

Immunization pV1J/hCEA-LTBopt led to the significant increase in CEA-specific immune response, as measured by intracellular IFNγ staining on PBMC injected mice (figure 15A). Enhancing T-cell responses were detected with a pool of peptides D, and it was mainly increased CD8+-reaction. The AOC is e, CEA-specific humoral response was increased in the CEA-LT-treated mice, as shown 47-fold increase in the values of the geometric mean Ab titer in comparison with pV1J/hCEAopt-treated group (figure 15C).

To determine whether there synergistic effect shown LT on CEA-specific immune response after immunization with vectors other than plasmid DNA, a group of 12 CEA-transgenic mice were immunized Ad5/hCEAopt-LTB and Ad/hCEAopt at a dose of 1×107, 1×108and 1×109RF. Mice were subjected to two injections at two-week intervals and the immune response was measured by intracellular IFNγ staining on PBMC two weeks after the last injection. The immune response was evaluated using pooled peptides D. Ad/hCEAopt-LTB was more immunogenic than Ad/hCEAopt as significant immune responses to CEA can be detected with a dose of 1×108RF, whereas a dose of 1×109HF Ad/hCEAopt were required to fracture a tolerance to this antigen target (figure 16). CD4+the reaction could not be detected in any of the immunized mice (data not shown).

These data suggest that tolerance to this autoantigen can be destroyed more effectively due to increased immunogenic properties of the merged design CEA-LT. In addition, a synergistic effect on LTB immunogenic its the STV CEA may also be observed after the injection of the plasmid, carrier is fully optimized in terms of codons cDNA fused design CEA-LTB. Finally, these results show that the enhanced immunogenicity of CEA-LTB is not limited to immunization with plasmid DNA.

EXAMPLE 14

The kinetics of tumor growth in CEA-transgenic mice immunized with the slit structures CEA-LT

The authors considered useful to determine whether increased immunogenicity merged design CEA-LT to enhanced therapeutic effect, can prevent progression of the tumor. For this purpose a group of 10 CEA-transgenic (tg) mice were subjected to 5 injections once a week plasmids pV1J/hCEAopt or pV1J/hCEAopt-LTB followed the ultimate stimulation of 1×1010RF corresponding Ad-vector. In connection with the recent messages that indicate that high levels of cellular immunity can be induced against viral and bacterial antigens using schema priming with plasmid DNA stimulation of Ad in this study used the same Protocol of immunization. Two weeks after the last immunization CEA-transgenic mice provoked by subcutaneous injection of 5 x 105tumor cells MS-CEA. This syngeneic cell line was obtained from chemically induced colon cancer, and she expresses CEA. Development of tumors in odnoobraznyh mice were detected on day 22 after stimulation (injection of tumor antigen), when all treated mice were not without tumors (figure 17A). In addition, there was a concomitant increase in the average size of the tumor mass, which reached a significant volume on day 34 after stimulation. Mice vaccinated with vectors encoding pV1J/hCEAopt showed partial resistance to tumor development, as 3 out of 10 treated mice remained without tumor at day 34 after stimulation. The average size of the tumors in this group was less than the volume of the tumors observed in ionoobmennyh mice. Immunization with vectors encoding the merged design opt-LTB, led to the significant protective effect against the development of tumors. Five of the 10 treated mice remained not containing tumors in day 34 after stimulation, and the average size of a tumor mass in this group was significantly smaller than the average size of a tumor mass in ionoobmennyh or processed pV1J/hCEAopt mice. Thus, these results indicate that enhanced CEA-specific immune response associated with the vectors encoding the merged design CEA-LTB, correlates with a significant antitumor effect, leading to partial protection against tumor growth and reduced the growth kinetics of the tumor mass.

EXAMPLE 15

Fused design CEA-DOM and CEA-FcIgG enhancing the immunogenicity of CEA protein

To research the Finance immune responses, induced by a plasmid encoding the merged design CEA-FrC, CEA-DOM, CEA-VSV-G, CEA-FcIgG, CEA-HSP70 and CEA-LAMP, a group of 9 mice 57BL/6 were immunized two i.m. injections of 50 or 5 μg of each plasmid. Immunization was performed with a three-week intervals. Due to the enhanced transduction and immunogenicity reported for electroporation (Zucchelli et al. J. Virology 74: 11598 (2000), Widera et al., J. Immunol. 164: 4635 (2000)), plasmid DNA routinely have electroporative (DNA-EP) in the skeletal muscle of the mouse.

The immune response induced by different plasmids was measured IFNγELISPOT-analysis 2 weeks after the last injection. Antigen-specific secretion of IFNγ stimulated of splenocytes was measured using a pool of 15-dimensional peptides, overlapping by 11 amino acids and includes With the end of CEA (pool D, amino acids 497-703) (Zucchelli et al., supra). Analysis of the immune response to CEA was performed with a pool of peptides D, as the cellular immune response to CEA in mice 57BL/6 initially shifted With the end of this protein (Zucchelli et al., supra). As negative control the production of cytokines was measured after stimulation of splenocytes DMSO at the same concentration, which was used to solubilize the peptides CEA.

Injection pV1J/CEA-DOM or pV1J/CEA-Fc induced a higher immune response to CEA in comparison with pV1J/CEA. Higher immunogenicity of these two fused proteins Pref is present to higher values of geometric mean values forming stains cells (SFC) of 10 6the splenocytes (figure 20A). Plasmids pV1J/CEA-DOM and pV1J/CEA-FcIgG had similar immunogenic properties and showed a 3-4-fold increase in CEA-specific immune responses after injection of 5 or 50 μg of plasmid DNA (pV1J/CEA-DOM: 590 and 1098 SFC/106the splenocytes, pV1J/CEA-FcIgG: 510 and 1160, pV1J/hCEA: 264 146 and SFC/106the splenocytes, respectively). Not noted significant differences between the values of SFC, induced pV1J/CEA-FrC, pV1J/CEA-LAMP, pV1J/CEA-HSP70 and pV1J/CEA. Not detected CEA-specific immune responses in negative control samples.

To determine the effect of the slit structures CEA on the humoral response to CEA, serum from immunized mice was tested using ELISA using purified CEA protein as a substrate (figure 20B). The increase in titer of CEA-specific antibodies was observed after injection of 50 μg of plasmids pV1J/CEA-DOM, pV1J/CEA-FcIgG, pV1J/CEA-FrC and pV1J/CEA-HSP70. In contrast, injection of pV1J/CEA-LAMP and pV1J/CEA-VSV-G resulted in CEA-specific antibody response similar to the response observed after immunization pV1J/CEA. Taken together, these data demonstrate that the merged design coding CEA sequence with cDNA DOM or FcIgG leads to an increase in CEA-specific cell-mediated and humoral immune response.

EXAMPLE 16

Fused design CEA-DOM and CEA-FcIgG, breaking tolerance to the target antigen at CEA-transgen the x mice

Tolerance to the target antigen is one of the barriers that anticancer vaccine must overcome for the induction of immune responses and for the manifestation of effective anti-cancer actions. Thus, the authors considered it important to determine whether enhanced immunogenic properties of the merged structures CEA-DOM and CEA-FcIgG to break tolerance to CEA is more effective than protein CEA. For this purpose, CEA-transgenic mice were used to conduct comparative studies of immunization. These transgenic mice are fully CEA gene of the person and flanking sequences of the gene CEA person and Express CEA protein in the intestine, mainly in the caecum and colon. Thus, this mouse line is a useful model to study the safety and efficacy of immunotherapy strategies aimed against this tumor autoantigen (Clarke et al., supra).

Due to the enhanced immunogenic properties of vectors carrying optimized in terms of codons cDNA (cDNAopt) CEA, designed as a plasmid and adenoviral vectors that are optimized in terms of codons cDNA (cDNAopt) CEA-DOM (CEA-DOMopt) or CEA-FcIgG (CEA-FcIgGopt) merged structures. As observed for CEA, CEA cDNA-DOMopt and CEA-FcIgGopt been shown to be expressed with greater efficiency than the corresponding cDNA is IR type, leading to an increased immune response to CEA (data not shown).

The immunogenicity of these two fused proteins was compared with the immunogenicity of CEA through a series of studies of immunization, based on the use of plasmid DNA and Ad-vectors introduced separately or in combination. Group CEA-transgenic mice were immunized with the following varying schemes introduction: i) 5 injections at one-week intervals with 50 μg of plasmid DNA (DNA/DNA), (ii) 2 injections, one every two weeks plasmid DNA with subsequent final injection of adenovirus in doses in the range of 1×107-1×109viral particles (VLPs) adenovirus (Ad/Ad) or (iii) 5 injections once a week plasmid DNA with subsequent final injection of 1×109RF adenovirus (DNA/Ad). Immune responses were analyzed by intracellular IFNγ staining on PBMC or splenocytes of each immunized mouse using peptide pool D. in Addition, the induction of CEA-specific antibodies were monitored using ELISA.

DNA/DNA immunization CEA-transgenic mice revealed that the vectors CEA-DOMopt and CEA-FcIgGopt showed measurable CD8+T-cell response to the antigen target (figure 21A). Thus, both structures were able to break tolerance to CEA in these mice. Antigen-specific response induced fused proteins DOM and CEA-FcIgG, was comparable, as shown by average values of intracellular staining of IFNγ (0.22 and 0.34 percent, respectively). However, the immune response induced by these two constructs was greater than the immune response observed after vaccination pV1J/hCEAopt (0,07%). Similarly, the humoral response against CEA was also greater after vaccination these fused proteins. The titer of CEA-specific antibodies were detected in all mice immunized with pV1J/CEA-DOMopt and pV1J/CEA-FcIgGopt, and the average antibody titer was equal 56136 and 24725, respectively. In contrast, immunized pV1J/CEAopt group showed at least 77 times lower titer of CEA-specific antibodies (318) (figure 21B).

CEA-transgenic mice treated according to the scheme of vaccination Ad/Ad also showed the best efficiency in breaking tolerance to CEA after vaccination with Ad-vectors CEA-DOMopt and CEA-FcIgGopt than Ad-Copt. CEA-specific CD8+T-cell response could be observed in vaccinated mice after injection of only 107HF Ad-CEA-DOMor Ad-CEA-FcIgG, this CEA-specific response was comparable between the two antigens, and increased after the injection of 109RF (1,55% and 1.15%, respectively). In contrast, the 109HF Ad-opt was required for the induction of significant frequencies of precursor CD8+T cells (2,1%) (figure C). CEA-Spa is specific antibodies detected in all mice, immunized Ad-CEA-DOMopt and Ad-CEA-FcIgGopt. The average antibody titer was 19600, 33,000, respectively. Injection of Ad-opt led to measurable CEA-specific reaction only 2 of the treated mice, and antibody titer was significantly lower (Zucchelli et al., supra) (figure 21D). Interestingly, immunization with DNA/Ad showed reduced differences in the frequencies of precursor CD8+T cells induced by vectors CEA, CEA-DOM and CEA-FcIgG (figure 22A). However, the average titers of CEA-specific antibodies were higher after vaccination vectors expressing CEA-DOM and CEA-FcIgG than CEA (31200, 26120, and 412, respectively) (figure 22B).

Interestingly, regardless of the antigen was not detected visible CD4+cellular Th1 response to CEA in none of the three schemes of vaccination (data not shown). However, significant CD4+cellular Th1 response against helper epitope, P30, present in the sequence of DOM (Rice et al., J. Immunol. 167: 1558-65 (2001)), was discovered after DNA/DNA vaccination (0,4%) (figure 23).

Thus, these results demonstrate that slit proteins CEA-DOM and CEA-FcIgG can break tolerance to CEA transgenic mice more efficiently than protein CEA. Enhanced immunogenic properties of these fused proteins can be observed after immunization with DNA vectors or Ad-vectors. However, the higher the ability of these two merged Belko is in the induction of CD8 +T cells in CEA can be overcome, at least partially, the scheme vaccination DNA/Ad.

EXAMPLE 17

Studies with depletion of T cells

Immunized animals overburden in relation to CD4+T cells, CD8+T cells, NK cells by intraperitoneal injection (I.P. Pavlova.) anti-CD4 (hybridoma GK1.5), anti-CD8 (hybridoma Lyt2.2) or anti-Asialo GM1 (Wako Chemicals, Richmobd, VA)as described (Perricone et al., J. Immunother. 27(4): 273-81 (2004); Yoon et al., J. Ethnopharmacol. 93 (2-3): 247-53 (2004)). Antibodies (100 μl of diluted ascitic fluid per dose) were injected with a day -7 relative to stimulation with tumor antigen and then were injected with each week for 3 weeks after injection of 5×105cells MS-CEA. Conditions of exhaustion was validational analysis flow cytometry peripheral blood using conjugated with phycoerythrin mAb anti-CD4, anti-CD8 and anti-NK (PharMimgen, San Diego, CA); 99% of the relevant subpopulations of cells were depleted, whereas all other subpopulations remained within normal levels.

EXAMPLE 18

CEA-DOM-immunization exhibiting antitumor effects in CEA-transgenic mice

Then the authors present invention was determined, does the increased immunogenicity of the slit structures CEA-DOM or CEA-FcIgG also to increased therapeutic effect, can prevent progression of the tumor. For this Caligraphy from 10 CEA-transgenic mice were subjected to vaccination schedules DNA/DNA, Ad/Ad or DNA/Ad using vectors CEA-DOM, CEA-FcIgG or CEA. Two weeks after the last immunization CEA-transgenic mice stimulated by subcutaneous injection of 5×105MS-CEA cells, syngeneic lines of tumor cells, which expresses CEA (larke et al., supra). Immunization schemes DNA/DNA or Ad/Ad did not lead to any significant antitumor effect, regardless of the protein expressed by the injected vectors (figure 24). In contrast, immunization with DNA-EP/Ad vectors encoding the protein CEA/DOM, resulted in significant antitumor effect, and 7 out of 10 treated mice remained without tumor at day 34 after stimulation with tumor antigen. Thus, these results show that enhanced CEA-specific immune response associated with CEA cDNA-DOMopt, and the scheme of vaccination DNA/Ad correlate with a significant antitumor effect in CEA-transgenic mice.

EXAMPLE 19

Antitumor activity of CEA-DOM-dependent CD4+T cells, CD8+T cells and NK cells

Were characterized effector cells involved in the antitumor action after electroporation of DNA (DNA-EP) and immunization of Ad vectors encoding the merged design CEA-DOM. After DNA/Ad-immunization, but before tumor stimulation, mice istos is whether in respect of CD4 +, CD8+T cells or NK cells using MAb. Antibodies were injected during tumor stimulation to guarantee continuous depletion relevant NK - and T-cell subpopulations. Depletion of all three types of cells were monitored using flow cytometry analysis using antibodies specific against human cell surface markers (data not shown). Depletion of CD4+, CD8+T cells or NK cells had a negative effect on the survival of immunized mice, leading to a dramatic reduction not having tumors in mice compared to the vaccinated group (figure 25). Thus, these results indicate that NK, CD4+and CD8+T-cells play an important role in protivoopujolevam the action expressed by vaccination CEA-DOM.

EXAMPLE 20

Statistical analysis

As indicated, the results were analyzed using logarithmic rank or two-sided t-student criterion. The value of p<0.05 is considered significant.

1. The nucleic acid molecule, inducing an immune response against carcinoembryonic antigen (CEA), containing a nucleotide sequence that encodes a protein CEA, where protein CEA contains protein CEA or functioning of the social option merged with the subunit In thermolabile of enterotoxin E. coli (LTB) or a functional variant; where specified CEA protein contains an amino acid sequence essentially corresponding to SEQ ID NO: 20, and where the protein is able to induce an immune response in a mammal.

2. The nucleic acid molecule according to claim 1, where CEA protein is truncated at the C-end and C-terminal shortening consists of amino acids 679-702 sequence SEQ ID NO: 20.

3. The nucleic acid molecule according to claim 1, where LTB shorter signal sequence.

4. The nucleic acid molecule according to claim 1, where the nucleotide sequence contains the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 12.

5. The nucleic acid molecule, inducing an immune response against CEA, containing a nucleotide sequence which encodes a protein CEA, where the nucleotide sequence presented in SEQ ID NO: 12.

6. The nucleic acid molecule according to claim 3, where the end of CEA protein is fused with N-end of subunit B LT.

7. The expression vector containing the nucleic acid molecule according to claim 1, which is functionally linked to the promoter, inducing an immune response against CEA in mammals.

8. The expression vector according to claim 7, where the vector is an adenoviral vector or a plasmid vector.

9. The expression vector of claim 8, where the vector is the tsya the Ad5 vector.

10. The expression vector of claim 8, where the vector is a vector or Ad6 vector RAD24.

11. The expression vector of claim 8, where the vector is the vector Ad chimpanzees.

12. The expression vector of claim 8, where the vector is pV1JnsB.

13. A host cell containing the expression vector of claim 8, expressing the fused protein CEA-LTB.

14. The way of expression of the slit CEA protein in the recombinant cell host, providing
(a) introducing a vector containing a nucleic acid molecule according to claim 1, in a suitable cell host; and
(b) culturing the host cell under conditions which allow expression of the specified fused protein CEA person.

15. Purified protein CEA encoded by the nucleic acid molecule according to claim 1, for the induction of an immune response against CEA.

16. Purified protein CEA indicated in paragraph 15, where the protein contains an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 13 and 15.

17. Adenoviral vaccine vector to induce an immune response against CEA in mammals, containing adenoviral genome with a deletion in the region E1 and the insertion in area E1, where the insertion contains an expression cassette containing
(a) polynucleotide containing a nucleotide sequence which encodes a protein CEA, where protein CEA contains a CEA protein or variant, fusion with the protein LTB; and RG is a protein able to induce an immune response in a mammal; and
(b) a promoter functionally linked with polynucleotides.

18. An adenoviral vector according to 17, which is the Ad5 vector.

19. An adenoviral vector according to 17, which is the Ad6 vector or vector RAD24.

20. Vaccine plasmid for the induction of an immune response against CEA, plasmid containing part and the expression cassette, and the part of the expression cassette contains
(a) polynucleotide containing a nucleotide sequence which encodes a protein CEA, where protein CEA contains a CEA protein or variant, fusion with the protein LTB; and where a protein is able to induce an immune response in a mammal; and
(b) a promoter functionally linked with polynucleotides.



 

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