Cdca1 epitope peptides and vaccines containing same

FIELD: chemistry.

SUBSTANCE: isolated peptide having cytotoxic T lymphocyte (CTL) inducing capacity in the presence of an antigen-presenting cell bearing HLA-A*2402, is used to obtain antigen-presenting cells and therefore CTL. The obtained CTL are used for targeted action against CDCA1-expressing cancer cells.

EFFECT: invention provides an effective vaccine for inducing anti-tumour immunity in a subject.

16 cl, 4 dwg, 1 tbl, 1 ex

 

The technical field to which the invention relates.

The present application claims the priority of provisional patent application U.S. 61/074,062, filed on June 19, 2008, and 61/197,599, filed October 28, 2008, the entire contents of which is incorporated into the present application by reference.

The present invention relates to the field of biology, more specifically, to the field of cancer therapy. In particular, the present invention relates to new peptides, which are extremely effective as anti-cancer vaccines and medicines for the treatment and prevention of tumors.

The level of technology

It has been demonstrated that CD8-positive cytotoxic T-lymphocytes (CTLs) recognize epitope peptides derived from tumor-specific antigens (OCA), found on the molecule major histocompatibility complex (MHC) class I, and then kill the tumor cells. After the opening of the collection melanoma antigens (MAGE), as a first example, the WASP were found many other WASP, primarily using immunological methods (Boon T, Int J Cancer 1993 May 8, 54(2): 177-80; Boon T&van der Bruggen P, J Exp Med 1996 Mar 1, 183(3): 725-9). Some of these WASP are currently undergoing clinical trials as an immunotherapy target.

Identification of new WASP, can stimulate potent and specific antitumor immune is e answers, provides further development and clinical application of strategies of peptide vaccination for different types of cancers (Harris CC, J Natl Cancer Inst 1996 Oct 16, 88(20): 1442-55; Butterfield LH, et al., Cancer Res 1999 Jul 1, 59(13): 3134-42; Vissers JL et al., Cancer Res 1999 Nov 1, 59(21): 5554-9; van der Burg SH, et al., J Immunol 1996 May 1, 156(9): 3308-14; Tanaka F et al., Cancer Res 1997 Oct 15, 57(20): 4465-8; Fujie T, et al., Int J Cancer 1999 Jan 18, 80(2): 169-72; Kikuchi M et al., Int J Cancer 1999 May 5, 81(3): 459-66; Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94). Today there are a few reports of clinical trials using these peptides derived from tumor-specific antigens. Unfortunately, in testing these cancer vaccines while there was only a low frequency of objective responses (Belli F, et al., J Clin Oncol 2002 Oct 15, 20(20): 4169-80; Coulie PG, et al., Immunol Rev 2002 Oct, 188: 33-42; Rosenberg SA et al., Nat Med 2004 Sep, 10(9): 909-15).

WASP required for proliferation and survival of cancer cells, important as targets for immunotherapy, because the use of such WASP can minimize a well-described risk of escape of cancer cells from the immune response, which refers to deletions, mutations or negative modulation of the OCA as a consequence therapeutically managed immune selection.

CDCA1 - gene associated with the cycle of cell division 1, was identified as representative of a class of genes that coexpressed with the genes of the cell cycle, such as CDC2, cyclin, topoisomerases is II and others (Walker et al., Curr Cancer Drug Targets 2001 May;1(1):73-83). CDCA1, in particular, as found, was associated with centromere mitotic HeLa cells and therefore was considered to be the functional homologue of yeast Nuf2 (J Cell Biol 2001 Jan 22; 152(2):349-60).

In addition, by analyzing the expression profile of genes using genome-wide set of cDNA microarray containing 23040 genes (Cancer Res 2006 Oct 1; 66(21):10339-48), CDCA1 was also identified as a new molecule, positively regulated in breast cancer (WO2005/028676), bladder cancer (WO 2006/085684), cancer of the esophagus (WO2007/013671), small cell lung cancer (SCLC) (WO2007/013665) and non-small cell lung cancer (NSCLC) (WO2005/089735), the content of the above descriptions are hereby incorporated by reference in the present application. Expression of CDCA1 particularly positively regulated in cell lines tumors SCLC and NSCLC, although in 23 normal tissues, except testis, expression was not detected. In addition, negative regulation of expression of CDCA1 by miRNAs caused suppression of cell growth in cell lines of lung cancer expressing CDCA1 (WO 2005/089735).

Taken together, these data indicate that CDCA1 is new, potentially universal encountering. Thus, the epitope-peptides derived from CDCA1, can be used as anticancer immunotherapy for the treatment of a wide variety of cancer tumor is th.

The invention

The present invention is partially based on the discovery of suitable epitope-peptides that can serve as targets for immunotherapy. Considering the fact that CDCA1 positively regulated in many types of cancer, including breast cancer, bladder cancer, non-small cell lung cancer, small cell lung cancer and esophageal cancer, the present invention is directed to a specified product of a gene associated with the cycle of cell division 1 (CDCA1) (SEQ ID NO: 35, encoded by the genome GenBank NM_145697 (SEQ ID NO: 34)) for further analysis. In particular were selected gene products CDCA1 containing the epitope-peptides, which induce CTLs that are specific to the respective molecules. Mononuclear cells from peripheral blood (MCPC)obtained from a healthy donor were stimulated using HLA-A*2402 binding candidate peptides derived from CDCA1. Received line CTLs that are specific recognize HLA-A24-positive target cells sensitized with relevant candidate peptides were identified HLA-A24 restrictively epitope peptides that can induce potent and specific immune responses against CDCA1. The presented results demonstrate that CDCA1 is strongly immunogenic, and its epitopes are effective targets for antitumor immunotherapy.

So about the time, the purpose of the present invention is to provide peptides having the ability to induce CTLs, as well as having an amino acid sequence selected from SEQ ID NO: 3, 4, 11, 14, 22 and 23. The present invention considers the modified peptides having amino acid sequence SEQ ID NO: 3, 4, 11, 14, 22 or 23, in which one, two or more amino acids replaced, entered, deleted or added, provided that the modified peptides retain the original ability to induce CTLs.

With the introduction of the subject, present the peptides on the surface of antigen presenting cells or actsoma and induce CTLs directed against the respective peptides. Therefore, the aim of the present invention is to provide antigen-presenting cells and actcom, presenting any of these peptides, and methods induction of antigen presenting cells.

Antitumor immune response induced by the introduction of true CDCA1 polypeptide or polynucleotide encoding the polypeptides, as well as actcom and antigen-presenting cells, which presenterat CDCA1 polypeptide. Thus, the purpose of the present invention is to provide pharmaceutical products containing the polypeptides of the present invention or encoding their polynucleotide and actsoma antigenpresenting cells, containing them as active ingredients. The pharmaceutical agents of the present invention find particular use as vaccines.

Another objective of the present invention is to provide methods of treatment and/or prevention (i.e., prevention) cancer (tumors), and/or prevent post-operative recurrence, as well as methods of inducing CTLs, methods of induction of antitumor immunity, where these methods include the stage of introduction of CDCA1 polypeptide, polynucleotide coding CDCA1 polypeptide, eksasol or antigen presenting cells presenting CDCA1 polypeptides, or pharmaceutical products according to the invention. In addition, CTLs according to the invention also find use as vaccines against cancer. Examples of cancerous tumors dealt with in accordance with the present invention, include, among others, breast cancer, bladder cancer, esophageal cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).

In addition, other objectives and features of the invention will be understood more fully when reading the subsequent detailed description in conjunction with the accompanying figures and examples. However, it should be understood that the previous entity and further detail is e description of the invention relate to the embodiments and should not limit the invention or other additional embodiments of the invention. In particular, although the invention described in this application in respect of a number of specific embodiments, it will be obvious that the description serves to explain the invention and should not be construed as limiting the invention. Professionals, skilled in the art, may have ideas regarding various modifications and applications that do not depart from the essence and scope of the invention as described in accordance with the attached formula. Similarly, other objects, characteristics, effects and advantages of the present invention will be apparent from the entity and specific embodiments described below, and will easily clear professionals, skilled in the art. Such objects, features, effects and advantages will be apparent from the foregoing in conjunction with the accompanying examples, data, figures and all the ensuing rational conclusions, alone or with consideration of the references included in this application.

Brief description of figures

Various aspects and applications of the present invention will become clear qualified after consideration brief description of the figures and detailed description of the present invention and its preferred embodiments, the following.

[Fig. 1] Phi is ur 1 is formed from a series of photos (a)-(g), which depicts the results of IFN-gamma ELISPOT analysis of CTLs induced by peptides derived from CDCA1. TLCs in hole #8 stimulated CDCA1-A24-10-119 (SEQ ID NO: 3) (a), #1, stimulated CDCA1-A24-10-335 (SEQ ID NO: 4) (b), #1, stimulated CDCA1-A24-10-48 (SEQ ID NO: 11) (c), #4 stimulated CDCA1-A24-10-5 (SEQ ID NO: 14) (d), #2, stimulated CDCA1-A24-9-8 (SEQ ID NO: 22) (e), and #2, stimulated CDCA1-A24-9-56 (SEQ ID NO: 23) (f), showed a powerful production of IFN-gamma compared with control, respectively. On the contrary, any specific secretion of IFN-gamma against peptide sensitized target cells in CTL stimulated CDCA1-A24-10-74 (SEQ ID NO: 2) (g)was not found. Cells in the wells indicated by a rectangular frame, used to get lines CTLs. On the figures, " + " indicates the production of IFN-gamma against target cells sensitized with the appropriate peptide, and "-" indicates the production of IFN-gamma against target cells not sensitized to any peptides.

[Fig. 2] Figure 2 is composed of a series of line graphs (a)-(g), which presents the results of the analysis of IFN-gamma ELISA on the CTL lines derived from CDCA1-A24-10-119 (SEQ ID NO: 3) (a), CDCA1-A24-10-335 (SEQ ID NO: 4) (b), CDCA1-A24-10-48 (SEQ ID NO: 11) (c), CDCA1-A24-10-5 (SEQ ID NO: 14) (d), CDCA1-A24-9-8 (SEQ ID NO: 22) (e) and CDCA1-A24-9-56 (SEQ ID NO: 23) (f) in the above analysis of IFN-gamma ELISA. The results show that the lines CTLs obtained upon stimulation with each peptide showed potent IFN-gamma compared with the control. On the contrary, in line CTLs derived from CDCA1-A24-10-74 (SEQ ID NO: 2) (g), any specific secretion of IFN-gamma against peptide sensitized target cells were not observed. On the figures, " + " indicates the production of IFN-gamma against target cells sensitized with the appropriate peptide, and "-" indicates the production of IFN-gamma against target cells not sensitized to any peptides.

[Fig. 3] Figure 3 is a line graph depicting the production of IFN-gamma CTL clone, obtained by serial dilution of CTLs stimulated using SEQ ID NO: 23. The results demonstrate that the CTL clone, obtained by stimulation using SEQ ID NO: 23, showed a powerful production of IFN-gamma compared with the control. In the figure, " + " indicates the production of IFN-gamma against target cells sensitized with SEQ ID NO: 23, and "-" indicates the production of IFN-gamma against target cells not sensitized to any peptides.

[Fig. 4] Figure 4 is a line graph that shows the specific activity of CTLs against target cells that exogenously Express CDCA1 and HLA-A*2402. The CTL clone obtained using CDCA1-A24-9-56 (SEQ ID NO: 23), showed high specific activity of CTLs against COS7 cells, transfected with CDCA1, and with HLA-A*2402 (diamond). On the contrary, against target cells expressing HLA-A*2402 (t is ugolnik) or CDCA1 (circle), any significant specific activity of CTLs was not found.

Description of embodiments

Although any methods and materials similar or equivalent to those described in this application can be used in practice or testing of embodiments of the present invention, the following describes the preferred methods, devices, and materials. However, before will be described in these materials and methods, it should be understood that this invention is not limited to specific sizes, shapes, dimensions, materials, methodologies, methods, etc. described in this application, as they may change in accordance with the standard experimentation and optimization. It should also be understood that the terminology used in the description is solely for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the present invention, limited only in accordance with the attached formula.

The content of each publication, patent or patent application mentioned in the present description, is expressly incorporated in this application by reference in full. However, in this application, nothing should be seen as the assumption that the invention does not have the authority preds is to act like the description of the effect of prior inventions.

I. Definitions

If not stated otherwise, all technical and scientific terms used in this application have the same meaning, by which they usually mean the average specialist in the field of engineering that applies the present invention. However, in case of conflict, the present description, including definitions, will prevail.

Articles are the only numbers used in the original text of the present application, to mean "at least one", unless expressly specified otherwise.

The terms "polypeptide", "peptide" and "protein" in the present application are used interchangeably and refer to a polymer of amino acid residues. The terms refer to polymers of amino acids, in which one or more amino acid residues are modified residues, or non-natural residues, such as artificial chemical mimetic of a corresponding natural amino acids, as well as natural polymers of amino acids.

The term "amino acid", as used in this application refers to natural and synthetic amino acids, and analogs of amino acids and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are the amino acids encoded by the genetic code and amino acids, modified after launched the cells (for example, hydroxyproline, gamma-carboxyglutamate and O-phosphoserine). The phrase "similar amino acid" refers to compounds that have the same basic chemical structure (alpha-carbon atom linked to a hydrogen, carboxy group, amino group and R group), as well as natural amino acids, but contain a modified group R or modified skeletons (for example, homoserine, norleucine, methanesulfonic, metonymically). The phrase "mimetic amino acid" refers to chemical compounds that have different patterns, but have functions similar to the functions of the conventional amino acids.

Amino acids can be identified in the present application for its well-known three letter symbols or one-letter symbols recommended by the Commission on biochemical nomenclature of the IUPAC-IUB.

The terms "gene", "polynucleotide", "nucleotide" and "nucleic acid" in the present application are used interchangeably and, unless expressly stated otherwise, are referred to by their commonly accepted single-letter codes.

Unless otherwise indicated, the term "cancer" refers to cancer, sverkhekspressiya gene CDCA1, including, for example, breast cancer, bladder cancer, esophageal cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).

Unless otherwise indicated, the terms "cytotoxic T-lymphocyte", "cytotoxics the I am T-cell" and "CTL" in this application are used interchangeably and, unless explicitly stated otherwise, belong to the subgroup of T-lymphocytes, which are able to recognize "foreign" cells (e.g. tumor cells, cells infected with the virus) and to cause the death of such cells.

II. Peptides

To demonstrate that peptides derived from CDCA1, function as antigen recognizable cytotoxic T-lymphocytes (CTL), peptides derived from CDCA1 (SEQ ID NO: 35), were analyzed to determine was whether they were antigen epitopes, restrictionenzyme HLA-A24, which are commonly encountered HLA alleles (Date Y et al., Tissue Antigens 47: 93-101, 1996; Kondo A et al., J Immunol 155: 4307-12, 1995; Kubo RT, et al., J Immunol 152: 3913-24, 1994). Candidate HLA-A24-binding peptides derived from CDCA1, identified on the basis of their affinely binding to HLA-A24. After in vitro stimulation of T-cells by dendritic cells (DC)loaded with these peptides was successfully received line CTLs using each of the following peptides:

CDCA1-A24-10-119 (SEQ ID NO: 3),

CDCA1-A24-10-335 (SEQ ID NO: 4),

CDCA1-A24-10-48 (SEQ ID NO: 11),

CDCA1-A24-10-5 (SEQ ID NO: 14),

CDCA1-A24-9-8 (SEQ ID NO: 22), and

CDCA1-A24-9-56 (SEQ ID NO: 23).

These lines CTLs showed a powerful specific activity of CTLs against target cells sensitized with the corresponding peptides. These results show that CDCA1 is the antigen recognized CTLs, and that the peptides are epitope-peptides CDCA1, restrictionenzyme HA-A24.

Since the gene CDCA1 sverkhekspressiya in most cancer tissues, including, for example, breast cancer, bladder cancer, esophageal cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), it represents a good target for immunotherapy. Thus, in the present invention proposed nonapeptide (peptides consisting of nine amino acid residues) and the Decapeptide (peptides consisting of ten amino acid residues)corresponding to CTL-recognized epitopes CDCA1. The most preferred examples of nonapeptide and Decapeptide of the present invention include peptides comprising amino acid sequences selected from SEQ ID NO: 3, 4, 11, 14, 22 and 23.

In most cases, programs that are currently available on the Internet, such as the programs described in KC Parker et al., J Immunol 1994 Jan 1, 152(1): 163-75, can be used to calculate affinely linking between different peptides and HLA antigens in silico. The affinity of binding to HLA antigens can be measured as described, for example, Parker KC, et al., J Immunol 1994 Jan 1, 152(1): 163-75; and Kuzushima K, et al., Blood 2001, 98(6): 1872-81. Methods for determining the affinity of binding is described, for example, in the Journal of Immunological Methods, 1995, 185: 181-190 and Protein Science, 2000, 9: 1838-1846. Thus, the present invention encompasses certain CDCA1 peptide that bind to antigen and HLA, identified with the use of such well-known programs.

Nonapeptide and the Decapeptide of the present invention may not necessarily be flanked by additional amino acid residues, with the proviso that the peptide retains its ability to induce CTLs. Such peptides having the ability to induce CTLs, in most cases have a length of less than about 40 amino acids, often less than about 20 amino acids, usually less than about 15 amino acids. Specific amino acid sequence (sequence), flanking peptides comprising amino acid sequences selected from SEQ ID NO: 3, 4, 11, 14, 22 and 23 is not limited, and may consist of amino acids of any type, provided that it does not impair the ability of the original peptide to induce CTLs. Thus, the present invention also provides peptides with the ability to induce CTLs and having an amino acid sequence selected from SEQ ID NO: 3, 4, 11, 14, 22 and 23.

In most cases, the modification of one, two or more amino acids in the protein will not affect the function of the protein, and in some cases will even amplify the desired function of the original protein. In fact, the modified peptides (i.e. peptides consisting of amino acid sequence in which biomodification two or more amino acid residues (i.e., substituted, added, deleted or inserted) compared to the original reference sequence)are known to retain biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Thus, in one embodiment, the peptides of the present invention may have the ability to induce CTLs and to have an amino acid sequence selected from SEQ ID NO: 3, 4, 11, 14, 22 and 23, in which one, two or even more amino acids are added, inserted, deleted and/or replaced.

Professionals qualified in this field, it is known that usually when an individual additions or substitutions in the amino acid sequence in which changes one amino acid or a small percentage of amino acids, the properties of the original side chain amino acids are preserved. However, they are often referred to as "conservative substitution" or "conservative modifications", in which the change of the protein results in a modified protein having a similar source protein function. Table conservative substitutions which are functionally similar amino acids are known in the prior art. Examples of the properties of the side chains of the amino acids are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilinae the amino acid is s (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics simultaneously: aliphatic side chain (G, A, V, L, I, P); a side chain containing a hydroxyl group (S, T, Y); a side chain containing a sulfur atom (C, M); a side chain containing a carboxyl and amide group (D, N, E, Q); a side chain containing base (R, K, H); and a side chain containing an aromatic group (H, F, Y, W). In addition, each of the following eight groups contain amino acids that are conservative substitutions for one another:

1) Alanine (A), glycine (G);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), lysine (K);

5) Isoleucine (I), leucine (L), methionine (M), valine (V);

6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); and

8) Cysteine (C), methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified peptides are also considered as the peptides of the present invention. However, the peptides of the present invention is not limited to this and may include non-conservative modifications, provided that the modified polypeptide retains the ability of the original peptide to induce CTLs. In addition, the modified peptides should not exclude CTL-inducing peptides of polymorphic variants, interspecies homologues, and all the lei CDCA1.

In order to maintain the ability to induce CTLs, it is possible to modify (insert, add, delete and/or replace) a small number (e.g. 1, 2 or more) or a small percentage of amino acids. In this case, the term "several" means 5 or fewer amino acids, for example, of 3 or less. The percentage of modified amino acids is preferably 20% or less, more preferably 15% or less, more preferably 10% or less, or 1-5%.

Analysis of homology preferred peptides of the present invention CDCA1-A24-10-119 (SEQ ID NO: 3), CDCA1-A24-10-335 (SEQ ID NO: 4), CDCA1-A24-10-48 (SEQ ID NO: 11), CDCA1-A24-10-5 (SEQ ID NO: 14), CDCA1-A24-9-8 (SEQ ID NO: 22), CDCA1-A24-9-56 (SEQ ID NO: 23) confirmed that these peptides do not have significant homology with peptides derived from any other well-known products of human genes. Thus, the ability of these peptides to induce unknown or unwanted immune responses when used for immunotherapy significantly reduced. Accordingly, these peptides are expected to be extremely useful to stimulate immunity against CDCA1 cancer patients.

When applied in the context of immunotherapy, the peptides of the present invention should be presented on the cell surface or actsoma, preferably in the form of a complex with an HLA antigen. Thus, preferably the select peptides, not only induce CTLs, but also possess high affinity binding to HLA antigen. With this purpose, the peptides can be modified by substitution, insertion, deletion and/or addition of amino acid residues, with the receipt of the modified peptide having high affinity binding. In addition to peptides that are naturally the, because the regularity of the sequences of the peptides, prezentirani when binding to HLA antigens are already known (J Immunol 1994, 152: 3913; Immunogenetics 1995, 41: 178; J Immunol 1994, 155: 4307), modification, based on this regularity, can be introduced into immunogenic peptides of the invention. For example, you may need to replace the second amino acid from the N-Terminus of the phenylalanine, tyrosine, methionine, or tryptophan, and/or the amino acid at the C-end of phenylalanine, leucine, isoleucine, tryptophan or methionine, to increase the affinity of binding HLA-A24. Thus, peptides that have an amino acid sequence selected from SEQ ID NO: 3, 4, 11, 14, 22 and 23, in which the second amino acid from N-Terminus amino acid sequence of SEQ ID NO replaced by phenylalanine, tyrosine, methionine, or tryptophan, and/or in which the amino acid at the C-end of the sequence SEQ ID NO replaced by phenylalanine, leucine, isoleucine, tryptophan or methionine, included in the present of Britanie. Substitutions can be introduced not only into the end amino acids, but in a position of potential TCR recognition of peptide. In several studies it has been demonstrated that amino acid substitutions in the peptide can meet or exceed original, for example, CAP1, p53(264-272), Her-2/neu(369-377)or gp100(209-217)(Zaremba et al. Cancer Res. 57, 4570-4577, 1997, T. K. Hoffmann et al. J Immunol. (2002) Feb 1;168(3):1338-47., S. O. Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206, and S. O. Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-314).

The present invention also considers the addition of one or two amino acids to the N - and/or C-Termini of the described peptides. Such modified peptides with high affinity binding to HLA-antigen and retaining the ability to induce CTLs, are also included in the present invention.

However, when the peptide sequence identical to part of the amino acid sequence of an endogenous or exogenous protein with a different function may develop side effects such as autoimmune disorders and/or symptoms of allergic against certain substances. Thus, it is preferable to first perform a homology search using the available databases, in order to avoid situations in which the sequence of the peptide corresponds to the amino acid sequence of another protein. When search homolo the AI becomes clear there is no peptide even with 1 or 2 amino acid differences compared with the target peptide, the target peptide can be modified to increase the affinity of binding to HLA antigens, and/or improve its ability to induce CTLs without any risk of such side effects.

Although the peptides having the above-described high affinity binding to HLA antigens are expected to be very effective, candidate peptides, which are selected according to the presence of high affinity binding as an indicator that further research on the subject in the presence of the ability to induce CTLs. In this case, the phrase "the ability to induce CTLs" indicates the ability of the peptide to induce cytotoxic T-lymphocytes (CTLs) when prezentowanie on antigen presenting cells. In addition, the ability to induce CTLs " includes the ability of the peptide to induce the activation of CTLs, the proliferation of CTLs, activate CTL lysis of target cells and increase the production of IFN-gamma in CTLs.

Demonstrate an ability to induce CTLs is carried out by inducing antigen-presenting cells bearing MHC-antigens person (for example, B-lymphocytes, macrophages and dendritic cells (DC)), or, more specifically, DK, obtained from mononuclear leukocytes in human peripheral blood, and, after stimulat and peptides, mixing with CD8-positive cells, with subsequent measurement of the level of production of IFN-gamma, which secrete CTLs against target cells. As the reaction system can be used transgenic animals expressing human HLA-antigen (for example, described in BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000 Aug, 61(8): 764-79 related articles, books, Linkout Induction of CTL response by a minimal an epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response). For example, target cells can be labeled with radioactive isotope51Cr etc., while the cytotoxic activity can be calculated from the radioactivity emitted by the target cells. In the alternative, the ability to induce CTLs can be assessed by measuring the level of IFN-gamma produced and secreted CTLs in the presence of antigen presenting cells (APCS), which are immobilized peptides, and visualization of zones of inhibition on the environment with the use of monoclonal antibodies against IFN-gamma.

The study of the ability of peptides to induce CTLs, as described above, it was found that peptides that have high affinity binding to HLA-antigen does not necessarily have a high ability to induce CTLs. However, of all the identified and analyzed peptides, nonapeptide or Decapeptide, to the which have the amino acid sequence, selected from SEQ ID NO: 3, 4, 11, 14, 22 and 23, as found, showed the highest ability to induce CTLs, as well as high affinity binding to HLA-antigen. Thus, these peptides are presented as preferred embodiments of the present invention.

In addition to the above modifications, the peptides of the present invention can also be associated with other substances, provided that the resulting linked peptide retains the ability of the original peptide to induce CTLs. Examples of suitable substances include, for example, peptides, lipids, sugars and sacharine chain, acetyl groups, natural and synthetic polymers, etc. Peptides may contain modifications, such as glycosylation, oxidation of the side chain or phosphorylation, etc. if the modification does not lead to the disappearance of the biological activity of the original peptide. These types of modifications can be made in order to give additional functions (e.g., directional and shipping functions) or stabilizirovannye polypeptide.

For example, to enhance in vivo stability of the polypeptide, as is known from the prior art, introducing D-amino acids, amino acid mimetics or unnatural amino acids; this concept can also be adapted to the present polypeptide is m The stability of the polypeptide can be assessed in a variety of ways. For example, peptidases and various biological environment, such as human plasma and serum can be used for stability analysis (see, e.g., Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).

The peptides of the present invention on the cell surface (for example, antigen presenting cells) or actsoma in the form of complexes in combination with HLA-antigens, and then induce CTLs. Therefore, the peptides that form complexes with HLA-antigens on the cell surface, or actsoma also included in the present invention. Such actsoma can be obtained, for example, using methods described in the published patent applications of Japanese Kohyo. Hei 11-510507 and WO99/03499, and can be obtained using the AIC obtained from patients undergoing treatment and/or prevention. Actsoma or cells presenting the peptides of the present invention, can be introduced in the form of vaccines.

The type of HLA antigens contained in the above complexes, must match the type of HLA antigens of the subject in need of treatment and/or prevention. For example, the population of Japan, HLA-A24, particularly HLA-A2402, is the most common, and therefore suitable for the treatment of a patient in Japan. Use type A24, which is expressed in the juice level in the Japanese and Europeans, favorable for obtaining effective results, these subtypes, as A2402, are also used. As a rule, in the clinic, the type of HLA antigen of the patient in need of treatment, research in advance to allow proper selection of peptides with high affinity binding to a specific antigen, or possessing the ability to induce CTLs by prezentowania antigen.

When using type A24 HLA-antigen for actsoma or cells, preferably used peptides that have amino acid sequence, vybranou of SEQ ID NO: 3, 4, 11, 14, 22 and 23.

In this case, the peptides of the present invention can also be described as "CDCA1 peptide(s)" or "CDCA1 polypeptide(s)".

III. Getting CDCA1 peptide

The peptides of the invention can be obtained using known methods. For example, peptides can be obtained artificially, using genetic engineering techniques or chemical synthesis. The peptide of the invention can be synthesized individually or as longer polypeptides comprising two or more peptides. Then, the peptides can be selected, that is, cleansed so that they practically do not contain any other natural proteins of the host cell and their fragments, or any other chemical substances.

The peptide of the present invention can b is to be obtained through chemical synthesis, based on the selected amino acid sequence. Examples of standard methods for the synthesis of peptides, which can be used in the synthesis include:

(i) Peptide Synthesis, Interscience, New York, 1966;

(ii) The Proteins, Vol. 2, Academic Press, New York, 1976;

(iii) Peptide Synthesis (in Japanese), Maruzen Co., 1975;

(iv) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;

(v) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;

(vi) WO99/67288; and

(vii) Barany, G. & R.B. Merrifield, the Peptides, Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

In the alternative, the present peptides can be obtained using any known method of genetic engineering to obtain peptides (e.g., Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss &Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first get a suitable vector carrying polynucleotide encoding the target peptide expressed in the form (for example, located after regularatory sequence corresponding to the sequence of the promoter), which then transform a suitable cell host. Then the cell host is cultivated, getting the desired peptide. The peptide can also be obtained in vitro with the use of system broadcast in vitro.

IV. Polynucleotide

The present invention also provides polynucleotide that encodes any of the above peptides of nastoyascheevremya. They include polynucleotide derived from the natural gene CDCA1 (GenBank NM_145697 (SEQ ID NO: 34)), and polynucleotide that have conservatively modified nucleotide sequence. In this case, the phrase "conservatively modified nucleotide sequence" refers to sequences that encode identical or essentially identical amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode a particular protein. For example, the codons GCA, GCC, GCG and GCU encode the amino acid alanine. Thus, at each position, in which alanine is specified by a codon, the codon can be changed to any of the described respective codons without changing the encoded polypeptide. Such changes nucleic acids are "silent variations" are one of the options conservatively modified variations. Each sequence of nucleic acid in the present application, which encodes a peptide, also describes every possible silent variation of the nucleic acid. The average person skilled in the art it is known that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon on what I tryptophan) can be modified to obtain a functionally identical molecule. Thus, each of Malcha variation of a nucleic acid which encodes a peptide, implicitly described in each of the disclosed sequence.

Polynucleotide of the present invention may comprise DNA, RNA, and derivatives thereof. DNA preferably comprises such basis as A, T, C and G, while in RNA, T is replaced by U.

Polynucleotide of the present invention can encode several peptides of the present invention, with or without intermediate amino acid sequence between them. For example, the intermediate amino acid sequence can provide a cleavage site (e.g., sequence recognition enzyme) polynucleotide or translated peptides. In addition, polynucleotide may include any additional sequence in the coding sequence that encodes the peptide of the present invention. For example, polynucleotide may be a recombinant polynucleotide, which includes regulatory sequences required for expression of the peptide, or may be an expression vector (plasmid) with marker genes, etc. In most cases, such recombinant polynucleotide can be obtained through the manipulation of polynucleotide using standard genetic engineering methods, for example using the polymerase and e is gnucleus.

To obtain polynucleotides of the present invention can be used recombinant and chemical synthesis methods. For example, polynucleotide can be obtained by embedding into an appropriate vector, which can be expressed by transfection into competent cell. In the alternative, polynucleotide can be amplified using PCR or expression in suitable organisms hosts (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989). In the alternative, polynucleotide can be synthesized using the methods of solid-phase synthesis, as described in Beaucage SL&Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.

V. Antigenpresenting cells (APCS)

The present invention also provides antigenpresenting cells (APCS), which presenterat complexes formed between HLA antigens and peptides of the present invention on its surface. AIC, which is obtained by contact with the peptides of the present invention or by introduction of nucleotide encoding the peptides of the present invention expressed in the form, can be obtained from patients who underwent treatment and/or prevention, and can be introduced in the form of vaccines or in combination with other drugs including the peptides of the present invention, actso is s or cytotoxic T cells.

AIC is not limited to specific type of cells include dendritic cells (DC), Langerhans cells, macrophages, B-cells and activated T-cells, which are known to presentyou on its surface protein antigens recognizable by lymphocytes. Since DK is a typical AIC, with the strongest inducing activity against CTLs from all APK, DC find application as an APK of the present invention.

For example, the AIC can be obtained through the induction of DC from peripheral blood monocytes and their subsequent contact (stimulation) with peptides of the present invention in vitro, ex vivo or in vivo. With the introduction of the peptides of the present invention to subjects, APK that presenterat peptides of the present invention, are induced in the body of the subject. The phrase "induction of agriculture" includes contact (stimulation) of the cells with the peptides of the present invention, or a nucleotide encoding the peptides of the present invention, with prezentowanie complexes formed between HLA antigens and peptides of the present invention on the cell surface. In the alternative, after the introduction of the peptides of the present invention in agriculture to agribusiness presented peptides, APC can be administered to the subject in the form of a vaccine. For example, the introduction of ex vivo may include stages:

a) get the APK from the first entity,

c) introduction of the loaded peptide APK the second entity.

The first subject and the second subject may be one and the same person or may be different persons. In the alternative, according to the present invention, the application of the peptides of the present invention for the preparation of pharmaceutical compositions that induce antigenpresenting cells. In addition, the present invention provides a method of preparation of pharmaceutical compositions that induce antigenpresenting cells, comprising a stage of mixing or incorporation of the peptide of the present invention in a formulation with a pharmaceutically acceptable carrier. In addition, the present invention also provides peptides of the present invention for the induction of antigen presenting cells. APK obtained in stage (b), can be administered to the subject in the form of a vaccine.

According to the aspect of the present invention, APK have a high level of ability to induce CTLs. The term "high level of ability to induce CTLs", high level refers to the corresponding level in the absence of contact APK with peptide or in contact with peptides, which are unable to induce CTLs. Such agriculture, with a high level of ability to induce CTLs can be obtained by a method that includes a step of transferring Geno is, containing polynucleotide that encode the peptides of the present invention, in agro-industrial complex in vitro. Introduced genes may be in the form of DNA or RNA. Examples of methods of introduction include, without particular limitations, various methods traditionally used in this field, such as lipofection, electroporation, can also be used a method using calcium phosphate. More specifically, it can be performed as described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; published Japanese translation of International publication 2000-509281. When gene transfer in agriculture, gene undergoes transcription, translation, etc. in the cell, after which the protein is processed MHC class I or class II and passes through the path prezentowania with subsequent prezentowanie peptides.

VI. Cytotoxic T-cells

Cytotoxic T-cells induced against any of the peptides of the present invention, enhances the immune response directed against the cancer cells in vivo, and thus, can be used as a vaccine, essentially similar to the peptides. Thus, the present invention also provides a selected cytotoxic T cells that are specific induced or activated by any of these peptides.

Such cytotoxic T cells can be obtained by: (1) the introduction of Pat the species of the present invention to a subject, collection of cytotoxic T-cells in the subject, or (2) contact (promotion) obtained from the subject APC and CD8-positive cells or mononuclear peripheral blood leukocytes in vitro with the peptides of the present invention with subsequent isolation of cytotoxic T-cells.

Cytotoxic T-cells, which were induced by stimulation with the use of AIC, which presenterat peptides of the present invention, can be obtained from patients who undergo treatment and/or prevention, and can be introduced alone or in combination with other drugs including the peptides of the present invention or actsoma, in order regulating effects. Received cytotoxic T-cells specific act against target cells presenting the peptides of the present invention, for example, the peptides that are used for induction. Target cells can be cells that endogenously Express a CDCA1 or cells that transliterowany genome CDCA1, and cells that presenterat peptide of the present invention on the surface due to stimulation by the peptide, can also serve as targets for attack of activated CTLs.

VII. T-cell receptor (TCR)

The present invention also provides a composition comprising a nucleic acid coding for the interacting polypeptides, which are capable of forming subunit of T-cell receptor (TCR)and its applications. Subunit of the TCR have the ability to form TCR, which give T-cell specificity against tumor cells, presenting CDCA1. Using methods known from the prior art (WO2007/032255 and Morgan et al., J Immunol, 171, 3288 (2003)), can be identified nucleic acid alpha - and beta-chains as a subunit of the TCR on the CTLs induced by one or more peptides of the present invention. Derived TCR can bind target cells presenting CDCA1 peptide with high avidity, and is not necessary to mediate efficient cytolysis of target cells presenting CDCA1 peptide in vivo and in vitro.

Nucleic acid encoding the subunit of the TCR may be incorporated into suitable vectors, such as retroviral vectors. These vectors are known in the prior art. Nucleic acids or vectors containing them, preferably can be transferred to T-cell, for example, T-cell obtained from the patient. Preferably, the invention provides ready-to-use composition, which allows quick modification of your own T-cells (or T-cells other mammal) with quick and easy obtaining of modified T-cells, which are excellent biocidal properties against R the same cell.

In addition, the present invention provides CTLs which are obtained by transduction with the nucleic acids encoding polypeptides of the TCR subunits that are associated with CDCA1 peptide, for example, SEQ ID NO: 3, 4, 11, 14, 22 and 23 in relation to HLA-A24. Transfection CTLs are able to migrate to tumor cells in vivo, and can be obtained culture using well-known methods of in vitro cultivation (e.g., Kawakami et al., J Immunol., 142, 3452-3461 (1989)). T cells of the invention can be applied to obtain a composition that can be used in the treatment or prophylaxis of cancer in a patient in need of treatment or protection (WO2006/031221).

Prevention and prophylaxis include any activity that reduces the percentage of mortality or severity of the disease. Prevention and prevention can occur on primary, secondary and tertiary levels of prevention." While primary prevention and prevention to avoid the disease, secondary and tertiary levels of prevention and prophylaxis cover actions aimed at preventing and prevention of disease progression and the onset of symptoms, as well as reducing the negative impact already developed the disease by restoring function and reducing associated with disease complications. In alternative prevention and prevention includes a wide range of preventive therapies, aimed at facilitating the specific gravity of the violation, for example, reducing the proliferation and metastasis of tumors, reduction of angiogenesis.

Treatment and/or prevention of cancer, and/or prevention of postoperative recurrence of cancer include any of the following stages, such as surgical removal of cancer cells, inhibition of growth of malignant cells, involution or regression of tumors, induction of remission and suppression of cancer, regression of the tumor and the reduction or inhibition of metastasis. In fact, the treatment and/or prevention of cancer reduces mortality and improves the prognosis for people with cancer, reduce the levels of tumor markers in the blood and facilitate detected symptoms that accompany cancer. For example, reduction or improvement of symptoms constitute the treatment and/or prevention, including 10%, 20%, 30% or greater percentage reduction or stabilization of the disease.

VIII. The pharmaceutical agents or compositions

Because the expression of CDCA1 specifically increased in several types of cancer, including breast cancer, bladder cancer, esophageal cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)compared with normal tissue (Cancer Res 2006 Oct 1; 66(21):10339-48, WO2005/028676, WO2005/089735, WO2006/085684, WO2007/013665, WO2007/013671), the peptides of the present invention or polynucleotide is s, encoding the peptides can be used for the treatment and/or prevention of cancer or tumor, and/or to prevent post-operative recurrence. Thus, the present invention provides a pharmaceutical agent or composition for the treatment and/or prevention of cancer or tumor, and/or prevent post-operative recurrence, which include one or more of the peptides of the present invention, or polynucleotides encoding the peptides as an active ingredient. In the alternative, the present peptides can be expressed on the surface of any of the above actcom or cells, such as AIC, for use as pharmaceutical agents or compositions. In addition, the above cytotoxic T-cells, which are directed to any of the peptides of the invention can also be used as an active ingredient of the present pharmaceutical agents or compositions.

In another embodiment, the present invention also provides the use of an active component selected from the following:

(a) the peptide of the present invention,

(b) a nucleic acid encoding such a peptide disclosed in the present application, expressed in the form

(c) agricultural or actsoma, presenting the peptide of the present invention on its surface, and

(d) cytotoxic T-Claix the ka of the present invention,

in the manufacture of pharmaceutical compositions or tools for the treatment of cancer or tumor.

In the alternative, the present invention further provides an active component, selected from the following:

(a) the peptide of the present invention,

(b) a nucleic acid encoding such a peptide disclosed in the present application, expressed in the form

(c) agricultural or actsoma, presenting the peptide of the present invention on its surface, and

(d) cytotoxic T cells of the present invention,

for use in the treatment of cancer or tumor.

In the alternative, the present invention additionally provides a method for preparing a pharmaceutical composition or means for the treatment of cancer or tumor, including the stage of inclusion in the dosage form of pharmaceutically or physiologically acceptable carrier with active component, selected from the following:

(a) the peptide of the present invention,

(b) a nucleic acid encoding such a peptide disclosed in the present application, expressed in the form

(c) agricultural or actsoma, presenting the peptide of the present invention on its surface, and

(d) cytotoxic T cells of the present invention,

as the active component.

In another embodiment, the present invention also provides a method of preparation of farmacevticheskoi composition or means for the treatment of cancer or tumor, including the stage of introduction of the active component in a pharmaceutical form with a pharmaceutically or physiologically acceptable carrier, where the active component is selected from the following:

(a) the peptide of the present invention,

(b) a nucleic acid encoding such a peptide disclosed in the present application, expressed in the form

(c) agricultural or actsoma, presenting the peptide of the present invention on its surface, and

(d) cytotoxic T cells of the present invention.

In the alternative, the pharmaceutical composition or means of the present invention can be applied for prevention of cancer or tumor and/or to prevent post-operative recurrence.

The present pharmaceutical agents or compositions find use as vaccines. In the framework of the present invention, the phrase "vaccine" (also called immunogenic composition) refers to the substance that has the function to induce anti-tumor immunity with the introduction of the animal.

The pharmaceutical agents or compositions of the present invention can be used for the treatment and/or prevention of cancer or tumor, and/or prevent post-operative recurrence in subjects or patients, including humans and any other mammal, including, among others, mouse, rat, Guinea pig, Krol is ka, cat, dog, sheep, goat, pig, cattle, horse, monkey, baboon, chimpanzee, particularly a commercially important animal or a domesticated animal.

According to the present invention, polypeptides that have an amino acid sequence selected from SEQ ID NO: 3, 4, 11, 14, 22 and 23, as found, was HLA-A24-restrictionenzyme epitope-peptides or candidates that can induce potent and specific immune response. Therefore, the present pharmaceutical agents, which include any of the above polypeptides having the amino acid sequence of SEQ ID NO: 3, 4, 11, 14, 22 and 23, especially suited for administration to subjects with HLA antigen is HLA-A24. The same applies to pharmaceutical means or compositions that contain polynucleotide encoding any of these polypeptides.

Cancer tumors or tumor being treated with the use of pharmaceutical agents or compositions of the present invention is not limited, and include all types of cancerous growths or tumors associated with CDCA1, including for example, breast cancer, bladder cancer, esophageal cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).

The present pharmaceutical agents or compositions, in addition to the above active ingredients, can is to contain other peptides, which have the ability to induce CTLs against malignant cells, other polynucleotide encoding other peptides, other cells, which presenterat other peptides, etc. In the present application is a non-limiting examples of other peptides, which have the ability to induce CTLs against tumor cells are tumor-specific antigens (e.g., identified OSA).

If necessary, the pharmaceutical agents or compositions of the present invention as an active component optionally can include other therapeutic substances, provided that the substance did not inhibit anti-tumor effect of the active component, for example, any of these peptides. For example, the dosage form may include anti-inflammatory agents, analgesic tools, chemotherapeutic agents, etc. In addition to the content of other therapeutic substances in the drug directly, drugs of the present invention can also be introduced sequentially or simultaneously with one or more other pharmacological means. The number of drug and pharmacological tools depend, for example, from what pharmacological means (means) is applied from the disease is, which heal, as well as from the scheme and pathways of introduction.

You must understand that in addition to the components specified in this application, the pharmaceutical agents or compositions of the present invention may include other means, standard in the art, which are considered medicinal forms.

In one embodiment of the present invention the present pharmaceutical agents or compositions can be incorporated into products and kits containing materials, used for the treatment of pathological conditions of the disease, which is subjected to treatment, such as cancer. The product may include a container with any of these pharmaceutical agents or compositions with a label. Suitable containers include bottles, vials and tubes. Containers can be made of various materials, such as glass or plastic. The label on the container should state that the agent is used for treatment or prevention of one or more disease States. The label may also indicate the route of administration, etc.

In addition to the container described above, the kit including a pharmaceutical agent or composition of the present invention optionally may further include a second container containing a pharmaceutically acceptable the first diluent. It may additionally include other materials necessary from a commercial and user positions, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The pharmaceutical agents or compositions optionally may be provided on the packaging or the metering device, which may contain one or more unit dosage forms containing the active ingredient. The package may, for example, include a metal foil or plastic film, such as a blister pack. Packing or dosing device can be accompanied by instructions for administration.

(1) the Pharmaceutical agents or compositions containing the peptides as an active ingredient

The peptide of the present invention can be introduced directly in the form of the pharmaceutical agents or compositions, or, if necessary, included in the dosage form using standard pharmaceutical techniques. In the latter case, in addition to the peptides of the present invention, carriers, fillers, etc. which are normally used in the manufacture of medicines, if necessary, can be included without certain restrictions. Examples of such media is sterilized water, saline, phosphate buffer, culture is idcast etc. In addition, the pharmaceutical agents or compositions may contain, if necessary, stabilizers, suspension, preservatives, surfactants and other Pharmaceutical agents or compositions of the present invention can be applied against cancer.

The peptides of the present invention can be prepared in combination of two or more peptides of the present invention, for the induction of CTLs in vivo. The combination of the peptides may be in the form of a mixture or conjugate obtained using standard methods. For example, the peptides can be chemically linked or expressed in the form of a single fused polypeptide sequence. Peptides in combination may be the same or different.

With the introduction of the peptides of the present invention, the peptides in the high density of HLA antigens on agriculture, while the induced CTLs that react with specific complex formed between the presented peptide and HLA-antigen. In the alternative, APK that presenterat any of the peptides of the present invention on its surface and can be obtained by stimulation of the APK (for example, DK)obtained from the subject, the peptides of the present invention, can be administered to the subject, resulting CTLs induced in the subject, and may be increased aggressiveness in related and cancer cells.

The pharmaceutical agents or compositions for the treatment and/or prevention of cancer or tumor, comprising the peptide of the present invention as an active ingredient, may also include adjuvant, which is known to effectively activates cellular immunity. In the alternative, the pharmaceutical agents or compositions may be introduced with other active components or may be in the form of a pharmaceutical form granules. Adjuvant refers to a compound that enhances an immune response against a protein with a simultaneous (or sequential) introduction to protein having immunological activity. Adjuvants discussed in the present application is described in the literature (Cl Clin Rev Environ 1994, 7: 277-89). Examples of suitable adjuvants include, without limitation, aluminum phosphate, aluminum hydroxide, potassium alum, cholera toxin, the toxin Salmonella, etc.

In addition, can be applied liposomal dosage forms, granular dosage form in which the peptide is sewed to the pellets with a diameter of several microns, and dosage forms, in which the peptide is associated with a lipid.

In some embodiments, the implementation of the pharmaceutical agents or compositions of the present invention may additionally include a component that will primire CTLs. Lipids were identified to the to the agents, can premirovat CTLs in vivo against viral antigens. For example, palmitic acid residues can be attached to the Epsilon - and alpha-amino groups of lysine residue and then linked to the peptide of the present invention. Then libidinously peptide can be entered either directly in the micelle, or in the form of particles included in the liposome, or emulsified in Freund. As another example of lipid primaryusage CTL responses, E. coli lipoproteins, such as dipalmitoyl-S-glyceryltrinitrate (P3CSS), can be used to premirovany CTLs when they are covalently linked with the appropriate peptide (see, e.g., Deres et al., Nature 1989, 342: 561-4).

The route of administration may be oral, intradermal, subcutaneous, intravenous injection and the like, as well as system introduction or local introduction near target areas. The introduction can be performed using a single administration or reinforced by repeated introduction. The dose of the peptides of the present invention can be adjusted depending on the disease being treated, the patient's age, weight, method of administration, etc. and is usually 0.001 to 1000 mg, for example, 0.001 to 1000 mg, for example 0.1 to 10 mg, and may be given once a few days or a few months. Specialist knowledgeable in this area can properly find the suitable dose.

(2) the Pharmaceutical agents or compositions containing polynucleotide as an active ingredient

The pharmaceutical agents or compositions of the present invention may also contain nucleic acids encoding the peptides disclosed in the present application, expressed in the form. In this application the phrase "expressed in the form of" means that polynucleotide when introduced into the cell is expressed in vivo in the form of the polypeptide that induces antitumor immunity. In the above embodiment, the sequence of the nucleic acid target polynucleotide comprises the regulatory elements necessary for expression of polynucleotide. Polynucleotide(s) can be constructed in such a manner that a stable integration into the genome of target cells (see, for example, Thomas KR & Capecchi MR, Cell 1987, 51: 503-12, regarding description cassette vectors for homologous recombination). See, for example, Wolff et al., Science 1990, 247: 1465-8; U.S. patent 5580859; 5589466; 5804566; 5739118; 5736524; 5679647; and WO 98/04720. Examples of delivery technologies based on DNA include "naked DNA", lightweight (bupivacaine, polymers, peptide-mediated) delivery, cationic complexes of lipids and delivery on the particles ("gene gun") or delivery-mediated pressure (see, for example, U.S. patent 5922687).

The peptides of the present invented who I can also be expressed using a viral or bacterial vectors. Examples of expression vectors include attenuated viruses, such as viruses ospowiki or smallpox-diphtheria birds. This approach involves the use of vaccinia virus, for example, as a vector to Express nucleotide sequences that encode the peptide. When introduced into the host organism, the recombinant vaccinia virus expresses the immunogenic peptide, and thus triggers the immune response. Vectors based on vaccinia virus and methods to be used in the methods of immunization are described, for example, in U.S. patent 4722848. Another vector is BCG (Bacillus Calmette-guérin (BCG). Vectors based on BCG described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors used for therapeutic injection or immunization, for example, adeno and adenoassociated viral vectors, retroviral vectors, Salmonella typhi vectors, vectors based on detoxificating anthrax toxin, etc. are also known. See, for example, Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo 2000, 14: 571-85.

Shipping polynucleotide in the body of the subject can be direct, when the subject is directly exposed to contact with the vector carrying polynucleotide, or indirect, when the first target polynucleotide in vitro transformed cells, and the cells are then transferred into organizmebit. Two of the above approach is known as gene therapy in vivo and ex vivo, respectively.

About General surveys on methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3: 87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; Mulligan, Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62: 191-217; Trends in Biotechnology, 1993, 11(5): 155-215. Standard techniques known in the field of genetic engineering, which can also be used in the present invention, described in the manuals Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1993; and Krieger, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY, 1990.

The route of administration may be oral, intradermal, subcutaneous, intravenous injection and the like, is used, systemic injection or local injection near the target areas. The introduction can be performed using a single administration or reinforced by repeated introduction. Dose polynucleotide in a suitable carrier or cells transformed by polynucleotides coding for the peptides of the present invention, can be adjusted depending on the disease being treated, the patient's age, weight, method of administration, etc. and is usually 0.001 to 1000 mg, for example, 0.001 to 1000 mg, for example 0.1 to 10 mg, and may be given once a few days or a few months. Specialist, skilled in this field can correctly retrieve the ü appropriate dose.

IX. Methods of using peptides, actcom, APC and CTL

The peptides of the present invention and polynucleotides encoding such peptides can be used for induction of APCS and CTLs. Actsoma and AIC of the present invention can also be used for the induction of CTLs. Peptides, polynucleotides, actsoma and AIC can be used in combination with any other compounds, provided that these compounds do not inhibit their ability to induce CTLs. Thus, any of the above pharmaceutical agents or compositions of the present invention can be used for the induction of CTLs, and, in addition, the pharmaceutical agents or compositions of the present invention, which include peptides and polynucleotides, can also be used for the induction of APC, as described below.

(1) a Method of inducing antigen-presenting cells (APC)

The present invention provides methods of induction of agriculture, with the use of the peptides of the present invention or polynucleotides encoding the peptides. Induction of APC may be performed as described above in section "VI. Antigenpresenting cells". The present invention also provides a method of induction of agriculture, which have a high level of ability to induce CTLs, induction which was also mentioned in the section "VI. Antigenpresenting cells"above.

(2) Method of indukti the CTLs

In addition, the present invention provides methods of induction of CTLs with the use of the peptides of the present invention, polynucleotides encoding the peptides, eksasol or AIC, presenting peptides. The present invention also provides methods of inducing CTLs using polynucleotide encoding a polypeptide, which is capable of forming subunit of T-cell receptor (TCR)that recognizes a complex of the peptide of the present invention and HLA antigens. Preferably, the methods of induction of CTLs include at least one phase selected from the group consisting of:

a: contact CD8-positive T cells with antigen presenting cell and/or ectosomal, which presents on its surface a complex of an HLA antigen and the peptide of the present invention, and

b: introduction of polynucleotide encoding a polypeptide, which is capable of forming subunit of TCR that recognizes a complex of the peptide of the present invention and HLA-antigen, CD8-positive T-cell.

With the introduction of the peptides of the present invention to a subject, in the body of the subject induced by CTLs, and increases the strength of the immune response against cancer cells. In alternative peptides and polynucleotides, encoding the peptides can be used in ex vivo therapeutic method, which is obtained from the subject APC and CD8-positive cells Il the mononuclear leukocytes of peripheral blood are contacting (stimulating) with peptides of the present invention in vitro, and after the induction of CTLs, activated cells, CTLs are transferred back into the body of the subject. For example, the method may include the following stages:

a) the fence APK from the subject

(b) contacting the AIC stage a) with a peptide

c) mixing APK stage b) with CD8+T-cells and joint cultivation with the aim of inducing CTLs, and

d) collecting CD8+T-cells from a mixed culture stage c).

In the alternative, according to the present invention, the application of the peptides of the present invention for the preparation of the pharmaceutical agents or compositions that induce CTLs. In addition, the present invention provides a method for preparing the pharmaceutical agents or compositions that induce CTLs, comprising a stage of mixing or inclusion in the dosage form of the peptide of the present invention with a pharmaceutically acceptable carrier. In addition, the present invention also provides a peptide of the present invention for the induction of CTLs.

CD8+T cells with cytotoxic activity obtained in stage d)can be administered to the subject as a vaccine. APK, mix with CD8+T-cells in the above stage (c)can also be obtained by transferring the gene encoding these peptides, in agriculture, as described in detail above in the section "VI. Antigenpresenting cells, however, this is e should be considered as a limitation. Thus, any agricultural or actsoma, which effectively presenterat present peptides to T cells, can be used in the present method.

The following examples are provided to illustrate the present invention and promotion of the average person skilled in the art to implement and use. The examples should not be construed as any limitation of the scope of the invention.

EXAMPLES

Materials and methods

Cell line

Lymphoblastic cell line A24 (A24LCL) was obtained through the transformation by the Epstein-Barr HLA-A24-positive B-lymphocyte person.

Selection of candidate peptides derived from CDCA1

9-d and 10-d peptides derived from CDCA1 that are associated with HLA-A*2402 were predicted using the software "BIMAS" to predict potential peptide fragments that bind MHC-I human (http://www-bimas.cit.nih.gov/molbio/hla_bind), the algorithms described KC Parker et al. (J Immunol 1994, 152(1): 163-75) and Kuzushima K et al. (Blood 2001, 98(6): 1872-81). These peptides were synthesized in American Peptide Company Inc. (Sunnyvale, CA) according to standard methods of solid-phase synthesis and purified using reverse-phase high-performance liquid chromatography (HPLC). Purity (>90%) and identity of the peptides was determined by analytical HPLC and mass spectrometry analysis, respectively. The peptides were dissolved in dimetilan is xide (DMSO) at a concentration of 20 mg/ml and stored at -80°C.

Induction of CTLs in vitro

Monocytic dendritic cells (DC) were used as antigen presenting cells (APCS) to induce responses of cytotoxic T-lymphocytes (CTLs) against peptides presented at the human leukocyte antigen (HLA). DC were obtained in vitro, as described in the literature (Nakahara S, et al., Cancer Res 2003 Jul 15, 63(14): 4112-8). In particular, mononuclear cells from peripheral blood (MCPC)isolated from normal volunteers (HLA-A*2402-positive) using a solution of Ficoll-Plaque (Pharmacia), was divided by adhesion to a plastic Cup for tissue culture (Becton Dickinson), enriching them by the fraction of monocytes. Enriched monocytes cell population cultured in the presence of 1000 u/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D System) and 1000 u/ml interleukin (IL)-4 (R&D System) in AIM-V (Invitrogen)containing 2% thermoinactivation autologous serum (AS). After 7 days of cultivation, the cytokine-indutsirovannye DK was senzibilizirani 20 μg/ml of each of the synthesized peptides in the presence of 3 μg/ml of beta-2-microglobulin for 3 hours at 37°C in medium AIM-V Derived cells, as it turned out, expressed DC-associated molecules, such as CD80, CD83, CD86 and HLA class II molecules on their surface (data not shown). Then these DK sensitized peptides, iactiveaware what mitomicina C (MMC) (30 μg/ml for 30 minutes) and was mixed in a ratio of 1:20 with autologous CD 8+T-cells, obtained by positive selection using a set of CD8 Positive Isolation Kit (Dynal). Data culture was transferred into 48-well plates (Corning); each well contained 1.5 x 104DK sensitized peptides, 3×105CD8+T-cells and 10 ng/ml IL-7 (R&D System) in 0.5 ml medium AIM-V/2% AS. After three days in culture was added IL-2 (CHIRON) to a final concentration of 20 IU/ml At day 7 and 14 T-cells are additionally stimulated with autologous DC sensitized peptides. In each case, DK received in the same way as described above. CTLs were tested against sensitized peptides cells A24LCL after the 3rd round of stimulation with peptides at day 21 (Tanaka H et al., Br J Cancer 2001 Jan 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001 Apr 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).

Method of reproduction CTLs

CTLs were propagated in culture, using a method similar to that described Riddell et al. (Walter EA, et al., N Engl J Med 1995 Oct 19, 333(16): 1038-44; Riddell SR, et al., Nat Med 1996 Feb, 2(2): 216-23). A total of 5×104CTLs suspended in 25 ml of medium AIM-V/5% AS 2 types of lines B-lymphoblastic cells, inactivated MMC, in the presence of 40 ng/ml of monoclonal antibodies to CD3 (Pharmingen). After one day after receipt, the cultures were added to 120 IU/ml IL-2. In culture was added to fresh medium AIM-V/5% AS containing 30 IU/ml IL-2 in DN is 5, 8 and 11 (Tanaka H et al., Br J Cancer 2001 Jan 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001 Apr 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).

Obtaining cultures of clones of CTLs

Prepared cultivation CTLs 0.3, 1, and 3 per well in 96-well round-bottom microplate (Nalge Nunc International). CTLs were cultured with 2 types of lines B-lymphoblastic cells at a density of 1×104cells/well, 30 ng/ml antibodies to CD3 and 125 u/ml IL-2 in the medium AIM-V containing 5% of AS, in a total volume of 150 μl/well. On Wednesday after 10 days were added 50 μl/well of IL-2, reaching a final concentration of IL-2 125 U/ml Activity of CTLs was tested on day 14, and CTL clones were propagated using the same technique as described above (Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).

Specific activity of CTLs

To determine specific activity of CTLs was performed interferon (IFN)-gamma enzyme-spot-immunoassay (ELISPOT) and IFN-gamma enzyme-linked immunosorbent assay (ELISA). In particular, sensitised peptides A24LCL (1×104/well) was obtained as a cell stimulant. Culture cells in 48 holes were used as cell-responders. IFN-gamma ELISPOT analysis and IFN-gamma ELISA was performed according to the method of the manufacturer.

Obtaining cell lines, forcibly expressing the target issue for lighting the th gene and/or HLA-A24

cDNA encoding the open reading frame of the target genes or HLA-A24, amplified by PCR. PCR amplificatory product was cloned in the vector pCAGGS. Plasmids were transfusional in line COS7, which is a "zero" cell line for target genes and HLA-A24, using lipofectamine 2000 (Invitrogen) according to the method recommended by the manufacturer. After 2 days after transfection, transfetsirovannyh cells were harvested using Versene®(Invitrogen) and used as target cells (5×104cells/well) to analyze the activity of CTLs.

Results

Prediction of HLA-A24-binding peptides derived from CDCA1

Table 1 shows the HLA-A*2402 binding peptides CDCA1 in descending order of affinity of binding. In total, selected and researched 40 peptides with potential affinity binding to HLA-A24, to determine the epitope-peptides.

185
Table 1
HLA-A24-binding peptides derived from CDCA1
The starting positionAmino acid sequenceScore bindingSEQ ID NO:
36LYPNPKPEVL 3001
74MYPHLMEGFL3002
119RFLSGIINFI25,23
335KTEENSFKRL17,284
432KYHDGIEKAA16,85
181KQLSDGIQEL15,846
64FYMMPVNSEV11,557
295LYQKKIQDLS10,58
309KLASILKESL9,69
146KSSADKMQQL9,610
48IYMRALQIVY911
DGIQELQQSL8,6412
231VSLKEIQESL8,413
5SFPRYNVAE18,2514
394INQEIQKIKLa 7.9215

322DQIESDESELa 7.9216
87NLVTHLDSFL7,217
368QYKRTVIEDC718
295LYQKKIQDL36019
278IYGDSVDCL24020
74MYPHLMEGF18021
8 RYNVAEIVI15022
56VYGIRLEHF10023
422IFLNLKTAL3624
119RFLSGIINF3025
144QYKSSADKM27,526
418KSQEIFLNL24,19227
197DFHQKTIVL2028
275KYEIYGDSV1529
432KYHDGIEKA13,230
387VYERVTTIN10,531
186GIQELQQSL10,36832
48IYMRALQIV933

Start position indicates the number of amino acid residues from the N-Terminus CDCA1.

The point of binding obtained from "BIMAS".

Induction of CTLs predicted peptides from CDCA1, restrictionenzyme HLA-A*2402, and receiving lines CTLs stimulated by peptides derived from CDCA1

CTLs for these peptides, derived from CDCA1, obtained according to the procedures described in section "Materials and methods". Specific activity of CTLs against peptides were determined using an IFN-gamma ELISPOT analysis (Figures 1a-f). It was shown that CDCA1-A24-10-119 (SEQ ID NO: 3), CDCA1-A24-10-335 (SEQ ID NO: 4), CDCA1-A24-10-48 (SEQ ID NO: 11), CDCA1-A24-10-5 (SEQ ID NO: 14), CDCA1-A24-9-8 (SEQ ID NO: 22), CDCA1-A24-9-56 (SEQ ID NO: 23) demonstrated a powerful production of IFN-gamma compared to the control wells. In addition, the cells in the positive well #8 stimulated CDCA1-A24-10-119 (SEQ ID NO: 3), #1, stimulated CDCA1-A24-10-335 (SEQ ID NO: 4), #1, stimulated CDCA1-A24-10-48 (SEQ ID NO: 11), #4 stimulated CDCA1-A24-10-5 (SEQ ID NO: 14), #2, stimulated CDCA1-A24-9-8 (SEQ ID NO: 22), and #2, stimulated CDCA1-A24-9-56 (SEQ ID NO: 23), printed and received the corresponding lines of the CTLs. The activity of CTLs in the received lines CTLs were determined using an IFN-gamma ELISA (Figure 2a-f). It was shown that all the lines CTLs showed a strong production of IFN-gamma against to ATOC target, sensitized corresponding peptide, compared to the target cells without sensitization peptides. On the other hand, the stimulation of other peptides failed to get lines CTLs, despite the fact that these peptides had a possible binding activity with HLA-A*2402. For example, a typical negative response data CTLs stimulated CDCA1-A24-10-74 (SEQ ID NO: 2), shown in Figure 1g and Figure 2g. The presented results indicate that the six peptides derived from CDCA1, have the ability to induce potent lines CTLs.

Receiving lines, CTL clones against CDCA1-specific peptides

Line CTL clones were obtained by serial dilution from the CTL lines, as described in "Materials and methods", and then using IFN-gamma ELISA was determined by the production of IFN-gamma from clones of CTLs against target cells sensitized with peptide. Powerful production of IFN-gamma was detected in clones of CTLs stimulated using SEQ ID NO: 23, as shown in Figure 3.

Specific activity of CTLs against target cells, expressing exogenous CDCA1 and HLA-A*2402

Stable lines CTLs induced against these peptides were examined for the ability to recognize target cells that Express exogenous molecules CDCA1 and HLA-A*2402. Specific activity of CTLs against COS7 cells, transfected Polner smennymi genes molecules CDCA1 and HLA-A*2402 (specific model of target cells, exogenously expressing the gene CDCA1 and HLA-A*2402), were analyzed using line CTLs induced by the corresponding peptide as cell effectors. The COS7 cells, transfetsirovannyh or a full-length genome of CDCA1 or a full-length gene HLA-A*2402, received as a control. As shown in Figure 4, CTLs stimulated using SEQ ID NO: 23, showed potent activity of CTLs against COS7 cells expressing CDCA1 and HLA-A*2402. On the other hand, any significant specific activity of CTLs against control not found. Thus, the data presented clearly demonstrate that the peptide having the amino acid sequence of SEQ ID NO: 23, naturally prezentuetsya on the target cells with a molecule HLA-A*2402 and recognized by CTLs. The results indicate that this peptide, derived from CDCA1, can be used for immunotherapy of cancer, in particular, as a vaccine against cancer patients with tumors expressing CDCA1.

Analysis of homology antigenic peptides

CTLs stimulated CDCA1-A24-10-119 (SEQ ID NO: 3), CDCA1-A24-10-335 (SEQ ID NO: 4), CDCA1-A24-10-48 (SEQ ID NO: 11), CDCA1-A24-10-5 (SEQ ID NO: 14), CDCA1-A24-9-8 (SEQ ID NO: 22), CDCA1-A24-9-56 (SEQ ID NO: 23), showed significant and specific activity of CTLs. The result may be due to the fact that the sequence of CDCA1-A24-10-119 (SEQ ID NO: 3), CDCA1-A24-10-335 (SEQ ID NO: 4), CDCA1-A24-10-48 (SEQ ID NO: 11), CDCA1-A24-10-5 (SEQ ID NO: 14), CDCA1-A24-9-8 (SEQ ID NO: 2) and CDCA1-A24-9-56 (SEQ ID NO: 23) homologous peptides, derived from other molecules, which are known to sensibiliser immune system. To exclude this possibility, conducted analysis of the homology of these peptide sequences, using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi), which revealed no sequences with significant homology. The results obtained in the analysis of homology indicate that the sequence of CDCA1-A24-10-119 (SEQ ID NO: 3), CDCA1-A24-10-335 (SEQ ID NO: 4), CDCA1-A24-10-48 (SEQ ID NO: 11), CDCA1-A24-10-5 (SEQ ID NO: 14), CDCA1-A24-9-8 (SEQ ID NO: 22), CDCA1-A24-9-56 (SEQ ID NO: 23) is unique and, thus, there is a low probability of these molecules induce immune random answers to some extraneous molecules.

In conclusion, it should be noted that the identified new epitope-peptides HLA-A24 derived from CDCA1, and it was demonstrated that they can be used in immunotherapy of cancer.

Industrial applicability

The present invention describes the new WASP, in particular, derived from CDCA1, which induce potent and specific antitumor immune responses and can be used in a wide variety of cancer types. Such WASP ensure further development as peptide vaccines against diseases associated with CDCA1, for example, cancerous tumors, more specifically, tumors of the testis, cancer of the pancreas, cancer of the bladder, nemelkokletochnogo lung cancer, small-cell lung cancer and esophageal cancer.

Although in this application the invention is described in detail and in relation to specific embodiments, it should be understood that the preceding description is exemplary and explanatory in nature and serves to illustrate the invention and its preferred embodiments. Through standard experimentation, specialist, skilled in the art will easily appreciate that the present invention can be made various changes and modifications without departure from the essence and scope of the invention, the boundaries of which are determined in accordance with the attached formula.

1. The selected peptide having the ability to induce cytotoxic T-lymphocyte (CTL) in the presence of antigen presenting cells bearing HLA-A*2402, where the peptide is a (a) or (b) below:
(a) a peptide characterized by the amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 4, 11, 14, 22 and 23; and
(b) a peptide characterized by the amino acid sequence in which the second amino acid from the N-end amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 11, 14, 22 and 23, modified to be an amino acid selected from the group consisting of phenylalanine, slapshot is in, methionine and tryptophan, and/or C-terminal amino acid amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 11, 14, 22 and 23, modified to be an amino acid selected from the group consisting of phenylalanine, leucine, isoleucine, tryptophan and methionine.

2. Selected polynucleotide encoding the peptide according to claim 1.

3. Method of induction of antigen presenting cells bearing HLA - A*2402, with the ability to induce CTLs with the use of the peptide according to claim 1.

4. The method according to claim 3, where the method involves the step of contacting antigen presenting cells bearing HLA-A*2402, a peptide according to claim 1.

5. Selected antigen presenting cell with the ability to induce CTLs, which presents the complex formed between HLA-A*2402 and the peptide according to claim 1 on its surface.

6. Antigen presenting cell according to claim 5, where the specified cell induced by the method according to claim 3 or 4.

7. Method of induction of CTLs using:
(a) the peptide according to claim 1 and antigen presenting cells bearing HLA-A*2402; or
(b) antigen presenting cell presenting the complex formed between HLA-A*2402 and the peptide according to claim 1 on its surface.

8. The method according to claim 7, where the method involves the step of contacting CDS-positive T cells with antigen presenting cell which presents the complex formed between HLA-A*2402 and the peptide according to claim 1 on its surface.

9. Means for inducing CTLs, where the tool includes an antigen presenting cell that presents the complex formed between HLA-A*2402 and the peptide according to claim 1 on its surface, as the active component.

10. Tool for the induction of CTLs in the presence of antigen presenting cells bearing HLA-A*2402, where the tool includes a peptide according to claim 1 as an active ingredient.

11. Allocated for specific CTLs action against cancer cells expressing CDCA1, which is induced by the application of the method, including the stage of contacting CDS-positive T cells with antigen presenting cell which presents the complex formed between HLA-A*2402 and the peptide according to claim 1 on its surface in vitro.

12. Pharmaceutical agent, comprising as an active ingredient are from 0.001 mg to 1000 mg of at least one peptide according to claim 1 in combination with a pharmacologically acceptable carrier, made for a purpose selected from the group consisting of:
(i) cancer treatment;
(ii) prevention of cancer;
(iii) prevention of postoperative recurrence of cancer; and
(iv) combinations of the above,
where specified pharmaceutical composition for administration to a subject whose HLA antigen is an HLA-A*2402.

13. A method of inducing an immune response against cancer in a subject whose HLA antigen Ave is dstanley a HLA-A*2402, including the stage of introduction of a specified subject peptide according to claim 1 at a dose of from 0.001 mg to 1000 mg

14. A method of inducing an immune response against cancer in a subject whose HLA antigen is an HLA-A*2402, which includes stages:
(a) obtaining antigen presenting cells from a subject;
(b) contacting the antigen presenting cells from step (a) with the peptide according to claim 1; and
(c) the introduction of antigen presenting cells from step (b) to the subject.

15. A method of inducing an immune response against cancer in a subject whose HLA antigen is an HLA-A*2402, which includes stages:
(a) obtaining antigen presenting cell and CDS-positive T cells from the subject;
(b) contacting the antigen presenting cells from step (a) with the peptide according to claim 1;
(c) mixing the antigen presenting cells with a stage with CD8 - positive T-cell from step (a) and joint cultivation for the induction of CTLs; and
(d) the introduction of CTLs induced in stage (C), the entity.

16. The vaccine for the induction of antitumor immunity in a subject whose HLA antigen is an HLA-A*2402, including from about 0.001 mg to 1000 mg of the peptide according to claim 1 as an active ingredient.



 

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4 cl, 5 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: expression plasmid vector is described for heterologous expression of recombinant proteins, high-frequency integration and accelerated amplification of an expression cassette in cells of mammals. The vector comprises functional promotor and terminator of a gene of elongation factor 1 alpha of a Chinese hamster, flanked by 5' and 3' HTO of this gene; a section of cloning of open frames of reading target proteins; an internal ribosome entry site (IRES) of a encephalomyocarditis virus (EMCV); an open frame of reading of dihydrofolate reductase (DHFR) of a mouse, expressing within a bicistronic messenger riziform together the target gene (IRES DHFR); and a section of Epstein-Barr virus terminal repetition (EBVTR). Also the method is described to produce stable lines of recombinant protein producents with high representation of an expression cassette in a genome with usage of the specified expression plasmid vector.

EFFECT: method improvement.

12 cl, 10 dwg, 3 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to peptide derivatives and peptidomimetics as transglutaminase inhibitors, methods for their preparation, pharmaceutical compositions containing said compounds as well as use of said transglutaminase inhibitors in particular for treatmenting coeliac disease and transglutaminase dependent diseases.

EFFECT: high efficiency of using said compounds.

12 cl, 20 dwg, 2 tbl, 7 ex

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