Bcl-2 proteins, fragments thereof, and application thereof in patients with malignant tumour

FIELD: medicine.

SUBSTANCE: invention relates to Bcl-2 proteins, fragments thereof, and to application thereof in patients with a malignant tumour. The declared proteins and peptide fragments particularly are applicable in vaccine compositions for treatment of malignant tumour. Besides, the invention concerns the methods of treatment with application of specified compositions. Also, an aspect of the invention is production of T-cells and receptors thereof which are specifically recognise declared proteins and peptide fragments.

EFFECT: higher clinical effectiveness with respect to tumours.

61 cl, 5 ex, 2 tbl, 12 dwg

 

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention in General relates to the field of prevention and therapy of malignant tumors. In particular, it offers isolated proteins regulating apoptosis, or their peptide fragments that can induce immune responses against malignant tumors. In particular, it is proposed the use of such proteins, belonging to the family of proteins Bcl-2, and immunogenic peptide fragments in the treatment, diagnosis and prognosis of malignant tumors.

The LEVEL of TECHNOLOGY

The development of resistance of malignant cells to a wide variety of chemotherapeutic agents is a major obstacle to the successful treatment of malignant tumors. Drug resistance is observed in a wide range of types of malignant cells. Many mechanisms contribute to drug resistance, including the inactivation of drugs, displacement medicines sediment cell membrane, mutations in drug targets and the inability to initiate apoptosis. Prevention of apoptosis can result from many conditions, including the retention of mitochondrial membrane potential and cytokine stimulation.

The search for proteins responsible for drug resistant environments is Tam phenotypes showed part protivopoloznoj molecule Bcl-2. Overexpression of Bcl-2 plays a role in the development of drug resistance in leukemia and other susceptible to apoptosis of the tumor and, therefore, a poor prognosis in a variety of malignant human tumors. Bcl-2 belongs to the family of proteins, the family of Bcl-2, whose members regulate apoptosis. The collection includes both proapoptotic and protivoponosnye representatives. Although an accurate understanding of how Bcl-2 exerts its protivopolozhnoe impact remains elusive, found that it is expressed in many malignant tumors, including lung cancer, colon and rectum, prostate and breast cancer, and leukemia and lymphoma.

Thus, Bcl-2 is a critical cellular factor, as elevated levels of expression of this protein gives resistance to apoptotic stimuli, thereby contributing to the pathogenesis and progression of malignant tumors.

The method by which the immune system of mammals learns and interacts with alien or foreign materials is difficult. An important aspect of the system is T-cell response. The response requires T-cells recognize and interact with complexes of cell surface molecules, called leukocyte of antig the us human (HLA), components of the major histocompatibility complex of man (MHC), and peptides. Peptides originate from larger molecules, which are processed by the cells, which, in turn, represent a molecule HLA/MHC. The interaction of T-cells and complexes of HLA/peptide limited, this requires T-cell, which is specific to a particular combination of molecules HLA and peptide. If specific T-cell is not present, the T-cell response is missing, even if you have a complex partners. Similarly, there is no answer, if there is no specific complex, and T-cell is present.

The mechanism by which T cells recognize cellular abnormalities, is also involved in the development of malignant tumors. For example, in W092/20356 described family of genes, which are processed into peptides, which, in turn, are expressed on cell surfaces and can lead to lysis of tumor cells by specific CTL. These genes are called MAGE family and encode the so-called "precursor antigens of tumor rejection", or molecules "TRAP", and derived peptides are called "antigens of tumor rejection", or "TRA".

In WO 94/05304 described nonapeptide that are associated with a molecule HLA-A1. In this work found that, given the known specificity of specific peptides in relation to concreteimplementor HLA, it should be assumed that the specific peptide binds one molecule HLA, but does not bind others. This is important, as different people have different HLA phenotypes. As a result, although the identification of specific peptide as partner-specific HLA molecules has diagnostic and therapeutic areas, they are only appropriate for people with this particular HLA phenotype.

Thus, it is well established that peptide epitopes originating from associated with tumor antigens (TAA)can be recognized as antigens to cytotoxic T-lymphocytes (CTL) in the context of MHC molecules. However, although it is generally accepted that most, if not all tumors are antigenic, only a few really are immunogenic in the sense that the progression of the tumor is easily controlled by the immune system.

To overcome this limitation, started several immunotherapy studies, such as vaccination derived from TAA peptides. In the case of myeloma, tumor, which is characterized by the largest number of defined CTL TAA, strong CTL responses against antigens were induced by vaccination, and some patients had complete remission of the disease. However, most of the peptide epitopes used in these tests, vaccination, Aleuts is specific for melanocytes, and these peptides can not be applied in the case of tumors amelanotic origin. In addition, expression of these TAA heterogeneous in different tumors from different patients and can vary even among metastases obtained from one patient. However, in the last couple of years identified a number of specific tumor peptide antigens, which are expressed in several malignant tumors, i.e. HER-2, Muc-1 and telomerase.

Apoptosis is a genetic program suicide cells, and it has been suggested that inhibition of apoptosis is an important mechanism involved in the formation of malignant tumors by lengthening the life span of cells, contributing to the accumulation of transforming mutations. Survivin is a newly identified member of this family of inhibitors of apoptosis proteins (IAP). In General, the analysis of gene expression, covering about 4 million transcripts, survivin identified as one of the major genes invariably subjected to increasing regulation in many types of malignant tumors but not in normal tissue. Solid malignancy, sverkhekspressiya survivin include cancer of the lung, colon, breast, pancreas and prostate, as well as the blood is € malignancy. In addition, it was reported that the number of melanoma and non-melanoma skin cancer are invariably survivin-positive. Overexpression of survivin in the majority of human malignancies indicates the main role of inhibition of apoptosis in the progression of the tumor, this view is confirmed by the observation that in the case of colorectal and bladder cancer, and neuroblastoma expression of survivin associated with poor prognosis. In contrast, survivin not detected in normal tissues of an adult organism. These characteristics qualify survivin as suitable TAA for both diagnostic and therapeutic purposes.

Thus in the last decade identified a large number of TAA that are recognized CTL restricted by the major histocompatibility complex (MHC). As survivin sverkhekspressiya in most malignant human tumors and inhibition of its function leads to increased apoptosis, this protein may serve as targets for therapeutic CTL responses.

Protein survivin and its possible diagnostic and therapeutic applications described in (1) and U.S. patent 6245523, which are included in this description by reference. Survivin is a cytoplasmic Bel is to with a molecular mass of 16.5 KD, contains a single BIR and vysokozaraznoy double-stranded region at the carboxyl end instead of a finger RING, which inhibits apoptosis induced by cancellation growth factor (IL-3), when moving in precursor B-cells. The gene encoding survivin, almost identical sequence proteasome receptor 1 effector cells (EPR-1), but oriented in the opposite direction, thus indicating the existence of two separate genes duplicated in the configuration of the "head to head". Accordingly, survivin can be described as antisense product EPR-1. Functionally, inhibition of expression of survivin increasing regulation of its natural antisense transcript EPR-1 leads to massive apoptosis and reduced cell growth.

In the US 6245523 described the selection is cleared of survivin and provides nucleic acid molecules that encode a protein of survivin, and antibodies and other molecules that are associated with surviving. In the US 6245523 also described active against apoptosis protein fragments of survivin and its variants in which amino acid residue was built with N - or C-Terminus or within the described sequence of survivin. In particular, declared that such peptides must contain a key functional residues required for apoptosis, such as Trp at position 67, Pro at position 73 and Cy in position 84.

In the last decade, numerous clinical trials have demonstrated the possibility of specific vaccination with peptides for the induction of antitumor T-cell responses in patients with malignant tumor. However, the disease course in patients in most cases did not improve. Such an inconsistency in many cases was explained by mechanisms of escape of tumor cells from immune surveillance. In the case of therapeutic techniques targeted to antigens, which play a minor role in malignant growth, breeding deficient antigen of malignant cells is a well-known constraint.

However, in the case of patients with breast cancer was observed paradoxical role of the protein Bcl-2. In primary breast tumors negative for Bcl-2 was associated with poor clinical outcome. In addition, it was reported that overexpression of the protein Bcl-2 correlates with tumors positive for estrogen receptor and mediated elements of response to the estrogen receptor in the promotor region of the gene Bcl-2. The prognosis of estrogen receptor-positive tumors is more favorable than the negative Outlook on the estrogen receptor tumors. It was suggested several possible explanations for these apparently paradoxical results, such as the inhibitory effect of Bcl-2 on ProLife is the situation of the cells, regulation of Bcl-2 expression by estrogen and/or presence of antagonists of Bcl-2, which inhibit its cytoprotective function.

However, the above studies have also shown that overexpression of Bcl-2 in malignant breast tumors correlates with drug resistance and that decreasing regulation of Bcl-2 antimyeloma the oligonucleotides modulate drug sensitivity due to apoptosis. In addition, transfection of Bcl-2 gene in lines of cancerous breast cells consistently resulted in increased resistance to apoptosis. It also describes that the presence of another inhibitor of apoptosis protein of survivin in carcinoma of the breast, is closely linked with the expression of Bcl-2 and reduced apoptosis index (AI) and poor overall survival. A similar relationship between survivin and Bcl-2 are described in neuroblastoma, stomach cancer, cancer of rectum and colon, Hodgkin's disease with a high degree of malignancy. Thus, when carcinoma of the breast, as in the case of most other human malignant tumors, inhibition of apoptosis is a common feature, and expression protivopolozhnyh genes, for example genes survivin and/or Bcl-2, can cause more pronounced protivoponosnye effects, which are reflected in the form of reduced apoptotic index. It was recently shown that survivin is a target for spontaneous T-cell reactivity in patients with various malignant tumors. These initial findings were later confirmed and supported (by the authors of the present invention and by other researchers).

The INVENTION

The present invention is based on the discovery that peptides restricted by class I MHC, can be derived from another class of apoptosis-regulatory proteins other than surviving, i.e. family proteins Bcl-2, which are able to bind with HLA-class I molecules MHC and thus to cause a CTL immune responses in patients suffering from malignant diseases. These observations suggest that proteins belonging to the family of proteins Bcl-2, act as TRAP molecules, which are processed by cells in vivo in the peptides, which have the function of TRA. These observations open the way for new therapeutic and diagnostic methods that can be applied in the fight against malignant diseases.

In the present invention revealed that Bcl-2 is a suitable target for immunotherapy of a number of malignant diseases. Bcl-2 is an important cellular factor, and its expression is essential for the viability of tumor cells. Thus, Bcl-2 is an attractive target for vaccination, because the evasion from immune surveillance during down-regulation or loss of expression of this protein would violate abutment is ivy tumor growth. In addition, the research that led to the present invention, the authors conducted a search and found spontaneous T-cell reactivity in PBL versus derived from Bcl-2 peptides in patients with breast cancer using ELISPOT analysis.

Accordingly, the present invention in the first aspect relates to an isolated protein, belonging to the family of proteins Bcl-2, or immunogene active peptide fragment for use as a drug for prevention or treatment of a malignant tumor. In particular, the invention relates to isolated immunogen active peptide fragments derived from protein, belonging to the family of proteins Bcl-2, for use as a drug for prevention or treatment of malignant tumors.

The following aspect of the invention relates to pharmaceutical compositions containing the above protein and/or peptide fragment according to the invention.

Also the aspect of the invention relates to a vaccine composition containing the isolated protein, belonging to the family of proteins Bcl-2, or immunogene active peptide fragment or nucleic acid encoding this protein or the peptide fragment for use as a drug for prevention or treatment of slocate the state of the tumor.

The following aspects the invention relates to a diagnostic kit for the diagnosis of ex vivo or in situ presence of the patient with malignant tumor T cells in PBL or in tumor tissue, which interact with a representative of a family of proteins Bcl-2, while the set contains a peptide fragment according to the invention, which is defined above; a complex of the peptide fragment according to the invention and molecules HLA class I or a fragment of such molecule.

The aim of the invention is also a method of detecting in a patient with a malignant tumour in the presence of T-cells that interact with a representative of a family of proteins Bcl-2, the method includes effecting contact of a sample of tumor tissue or blood sample with a complex according to the invention, which is defined above, detection of binding of the complex with tissue or blood cells.

In addition, a molecule that can specifically bind to a peptide fragment according to the invention, and the molecule that is able to block this binding.

In another aspect the invention relates to a method for the treatment of malignant disease, the method includes the administration to a patient suffering from a disease, an effective amount of the pharmaceutical composition according to the invention, the molecules according to the invention, Kotor, which can specifically bind with a peptide fragment according to the invention, and/or molecules according to the invention, which is able to block this binding.

In another aspect the invention relates to the use of a protein or peptide fragment, which is defined herein, in the manufacture of drugs for the treatment of malignant disease.

DETAILED description of the INVENTION

The main purpose of the present invention to provide isolated proteins belonging to the family of proteins Bcl-2, or their immunologically active peptide fragments for use as a drug for prevention or treatment of malignant tumors.

Family proteins Bcl-2 includes several proteins that regulate apoptosis. To this family belong as proapoptotic and protivoponosnye representatives. This paper presents a study of the potential of this family of proteins as pharmaceutical or diagnostically active substances in malignant tumors, with particular reference to protein Bcl-2. In addition, described the potential of Bcl-XLand Mcl-1 as pharmaceutically and diagnostically active substances. However, it is likely that immune responses, such immune responses observed against protein Bcl-2 or its fragments, there are or may be beyond the ENES patients with malignant tumor against other members of the family proteins Bcl-2, for example, other protivopolozhnyh proteins, such as Mcl-1 or Bcl-XLthat are also associated with drug resistance and sverkhekspressiya in malignant tumors. Accordingly, the invention relates to any representative of the families of proteins Bcl-2, preferably any protivopolozhnomu representative, which can cause immune responses in patients with malignant tumor, for example to a protein selected from the group consisting of Bcl-2, Bcl-w, Mcl-1, Bfl-1/A1, Bcl-b, Bcl2-L-10 and Bcl-XLpreferably selected from the group consisting of Bcl-2, Mcl-1, Bcl-w and Bcl-XLmore preferably selected from the group consisting of Bcl-2, Mcl-1 and Bcl-XL.

Representatives protivopolozhnogo of Bcl-2 are its oncogenic effects through inhibition of apoptosis in cells, which in norm to perish, thus contributing to the accumulation of cells in vivo.

All members of the family proteins Bcl-2 contain at least one of the four conservative motifs, known as homology domains of Bcl-2 (BH) (BH1, BH2, BH3 and BH4). In addition to the presence of domains BH preferred protivoponosnye molecules have zakariayi in the membrane domain at the carboxyl end (TM). Protivoponosnye representatives, such as Bcl-2 and Bcl-XLcontain all four BH domain along with the transmembrane domain. Multi-domain Proporta the major proteins, such as Bax and Bak contain everything except the BH4 domain. The second subgroup proapoptotic protein, known as protein-only with BH3-domain (e.g., Bad, and Bid), consists of molecules that contain only the BH3 domain and no other BH domains. In proapoptotic proteins, such as Bcl-XSand Mcl-1S representing alternative splanirowannya forms of genes bcl-x and mcl-1, respectively, there are no domains BH1 and BH2. In addition, Mcl-1S lacks a transmembrane domain. Proteins belonging to the family of Bcl-2, are described, for example, in reference 6.

Although it is preferable that a protein belonging to the family of proteins Bcl-2, had protivopoloznymi properties, the present invention also assumes that a protein belonging to the family of Bcl-2, can be proapoptotic protein, such as protein selected from the group consisting of Bax, Bok/Mtd, Bad, Bik/Nbk, Bid, Hrk/DP5, Bim, Noxa, Bmf and PUMA/bbc3.

In one preferred embodiment of the invention a protein belonging to the family of proteins Bcl-2, is Bcl-2, preferably Bcl-2, more preferably Bcl-2 having the sequence from the source number of access P10415 in the SwissProt database.

In another preferred embodiment of the invention a protein belonging to the family of proteins Bcl-2, Bcl is-XLpreferably Bcl-XLman, more preferably Bcl-XLand Audi sequence with the source number of access Q07817 in the SwissProt database.

In yet another preferred embodiment of the invention a protein belonging to the family of Bcl-2, is Mcl-1, preferably Mcl-1, more preferably Mcl-1 having the sequence from the source number of access Q07820 in the SwissProt database.

As in several human malignant tumors expressed high levels of Bcl-2 and other members of Bcl-2, immunotherapy methods that target specific antigens, can have wide clinical application. The main concern with this approach may cause the introduction of self-reactive immune responses. Thus, the future of vaccination, based on the representatives of this protein family, will depend on therapeutic efficacy, and the type of side effects that can follow after immunization. When first used the peptides derived from antigens of melanocyte differentiation, for the treatment of patients with melanoma in stage IV, suggested that this may result in a significant destruction of the melanocytes, which, in turn, would have revealed clinically, such as vitiligo or retinitis. However, clinical experience has shown that the frequency of vitiligo in patients receiving vaccination was not significantly higher than the frequency associated with melanoma of hypopigmentation in patients receiving other the forms of therapy. In addition, there were no reports of serious side effects in different trials of a vaccine against autoantigens.

In one applicable option offers new peptide fragments, restricted by class I MHC (also referred to in this invention as "peptides"), which are characterized by the presence of at least one of the several signs, one of which is the ability to bind to a molecule HLA class I, which he limited to, affinity, measured by the number of peptide that is capable of half the maximum capture molecules HLA class I (a value of C50), which is not more than 50 μm, as determined by analysis of binding in the Assembly, which is described in this publication. The specified Assembly analysis carried out as described previously (2), and the analysis is based on stabilization of HLA molecules after loading peptide scarce on the carrier peptides cell line T2. Then with proper stacking stable heavy chain HLA subjected thus, using conformation-dependent antibodies, and quantify the binding of the peptide.

The analysis provides a simple way of screening selected for studies of peptides for their ability to bind with a molecule of a given HLA allele with the above affinity. In preferred embodiments, peptide the fragment according to the invention is a fragment, having a value of C50that does not exceed 30 μm, for example, the value of C50that does not exceed 20 μm, including the values of C50not exceeding 10 microns, not greater than 5 μm and not greater than 2 μm.

However, the preferred peptides according to the present invention are peptides capable of inducing specific T-cell response, which is determined by ELISPOT analysis, for example by ELISPOT analysis described in this publication below in example 1, section 4. Some peptides, although not linked to the MHC with high affinity, can cause T-cell response, as defined in the ELISPOT. Other peptides capable of binding to MHC with high affinity, also called T-cell response, as defined in the ELISPOT. Both peptides are the preferred peptides according to the invention.

Therefore, the preferred peptides according to the present invention are peptides capable of inducing specific T-cell response, as measured in the ELISPOT analysis, commonly used to measure more than 50 specific for peptide spots on the 108cells, more preferably 107even more preferably 106even more preferably 105cells, such as 104cells.

As indicated above, the HLA system is a major system of the human histocompatibility (MHC). In General, system HC control a number of parameters: transplantation antigens, thymusdependent immune responses, some complement factors and predisposition to certain diseases. More specifically, MHC encodes three different types of molecules, i.e. molecules of class I, II and III, which define the more General characteristics of the MHC. Of these molecules, the molecules of the class I represent so-called molecules HLA-A, HLA-B and HLA-C, which are presented on the surface of most okleinowany cells and platelets.

The peptides according to the present invention are characterized by their ability to communicate (which are limited) specific molecule HLA class I MHC. Thus, in one embodiment, the peptide is a peptide which is limited by the molecule HLA-A class I MHC, including HLA-A1, HLA-A2, HLA-A3, HLA-A9, HLA-A10, HLA-A11, HLA-Aw19, HLA-A23(9), HLA-A24(9), HLA-A25(10), HLA-A26(10), HLA-A28, HLA-A29(w19), HLA-A30(w19), HLA-A31(w19), HLA-A32(w19), HLA-Aw33(w19), HLA-Aw34(10), HLA-Aw36, HLA-Aw43, HLA-Aw66(10), HLA-Aw68(28), HLA-A69(28). In the literature also use a simpler notation, using only basic numerical designation, for example HLA-A19 or HLA-A24 instead HLA-Aw19 and HLA-A24(49), respectively. In specific embodiments, the peptide according to the invention is limited to the type of molecule HLA class I MHC selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24.

The peptides according to the invention can be obtained, for example, from the known sequences of the representative family proteins Bcl-2 (3. In a preferred embodiment of the invention, the peptide contains (or more preferably consists of) not more than 200, preferably not more than 100, more preferably not more than 50, even more preferably not more than 25, even more preferably not more than 20, and more preferably, not more than 15, for example not more than 10, for example, in the range from 9 to 10 consecutive amino acids of one of the above representatives of the family of proteins Bcl-2, preferably of Bcl-2 with the original the access number P10415, Mcl-1 with the source number of access Q07820 or Bcl-XLwith the source number of access Q07817 in the SwissProt database.

Selection of peptides, potentially with the ability to communicate with a particular HLA molecule, can be done by alignment of known sequences that are associated with particular HLA molecule, so as to identify the prevalence of several related amino acids in specific positions of the peptides. Such predominant amino acid residues is also called herein "anchor residues" or "motives anchor residues". Following this relatively simple method, based on known data sequences, which can be found in the available databases, it is possible to obtain peptides of the molecules of the family of proteins Bcl-2, to the which probably associated with a particular HLA molecule. Typical examples of such analyses for a number of HLA molecules to the following:

* In one embodiment, there is no specific anchor residue for this provision, however, in the preferred embodiment, the anchor residue is R or A.

Thus, as an example, nonapeptide, potentially having the ability to bind to HLA-A1, could have one of the following sequences: Xaa-T-D-Xaa-Xaa-Xaa-L-Xaa-Y-Xaa-T-E-Xaa-Xaa-Xaa-L-Xaa-Y; Xaa-S-D-Xaa-Xaa-Xaa-L-Xaa-Y or Xaa-S-E-Xaa-Xaa-Xaa-L-Xaa-Y (Xaa is any amino acid residue). This way can be constructed in sequence, potentially with the ability to communicate with any other HLA molecule.

It will be clear that the person skilled in the art will be able to identify additional motifs anchor residues of the HLA molecules.

Thus, in applicable embodiments, the peptides according to the invention include peptides, sequences which contain in the case of each of the specific HLA alleles that are listed in the table, any of the amino acid residues listed in the table.

Thus, the peptides according to the invention can be any of the above peptides containing a continuous sequence of PR which some members of the Swiss family proteins Bcl-2, in the range from 1 to 10, preferably in the range from 1 to 5, more preferably in the range from 1 to 3, more preferably in the range from 1 to 2, more preferably 1 if the amino acid substituted by another amino acid, preferably so that the peptide contains one or more, preferably all of the anchor residues of this specific peptide-HLA-A, which is indicated in the table above.

A non-limiting example of how to obtain peptides representatives of the family of proteins Bcl-2, containing the anchor residues of this specific for HLA-A peptide described in example 3, in the section "response to modified peptides". Accordingly, in one embodiment of the invention, the peptide may be any peptide containing not more than 200, preferably not more than 100, more preferably not more than 50, even more preferably not more than 25, even more preferably not more than 20, even more preferably not more than 15, even more preferably not more than 10 amino acids and containing (or more preferably consisting of) a sequence selected from the group consisting of RLKRDWLVK (SEQ ID NO: 62), QSDEIISRY (SEQ ID NO: 63) and QSEEIISRY (SEQ ID NO: 64), more preferably selected from the group consisting of RLKRDWLVK (SEQ ID NO: 62).

Thus, a simple method for the identification of peptides according to ISO is retenu includes the following stages: the choice of a particular HLA molecules, for example, molecules with high frequency in this population, the implementation of the analysis based on the alignment, which is described above to identify motives anchor residues in the protein family proteins Bcl-2, the selection or design of peptides suitable size, which contain one or more of the identified anchor residues, and testing the resulting peptides in respect of (i) the ability to communicate with a particular HLA molecule, using the analysis of assemblies, which are described in this publication, (ii) the ability of the peptides to cause INF-γ-producing cells in a PBL population of a patient with malignant tumor with a frequency component of at least 1 in 104PBL defined in ELISPOT analysis described in this publication, and/or (iii) the ability of the peptides to identify in situ in the tumor CTLs that react to the test epitope peptides.

In specific embodiments, the peptide according to the invention is restricted by HLA-A2 peptide derived from Bcl-2 having the sequence selected from the following sequences: ALVGACITL (SEQ ID NO:1), ALSPVPPVV (SEQ ID NO:2), SLALVGACI (SEQ ID NO:3), KTLLSLALV (SEQ ID NO:4), LLSLALVGA (SEQ ID NO:5), WLSLKTLLSL (SEQ ID NO:6), AAAGPALSPV (SEQ ID NO:7), PLFDFSWLSL (SEQ ID NO:8), FTARGRFATV (SEQ ID NO:9), YLNRHLHTWI (SEQ ID NO:10), NIALWMTEYL (SEQ ID NO:11).

In one preferred embodiment, the peptide can be any pepti is, consisting of not more than 200, preferably not more than 100, more preferably not more than 50, even more preferably not more than 25, even more preferably not more than 20, even more preferably not more than 15, even more preferably not more than 10 amino acids and containing (or more preferably consisting of) a sequence selected from the group consisting of ALVGACITL (SEQ ID NO:1), ALSPVPPW (SEQ ID NO:2), SLALVGACI (SEQ ID NO:3), KTLLSLALV (SEQ ID NO:4), LLSLALVGA (SEQ ID NO:5), WLSLKTLLSL (SEQ ID NO:6), AAAGPALSPV (SEQ ID NO:7), PLFDFSWLSL (SEQ ID NO:8), FTARGRFATV (SEQ ID NO:9), YLNRHLHTWI (SEQ ID NO:10), NIALWMTEYL (SEQ ID NO:11), more preferably selected from the group consisting of NIALWMTEYL (SEQ ID NO: 11), YLNRHLHTWI (SEQ ID NO: 10), PLFDFSWLSL (SEQ ID NO: 8) and WLSLKTLLSL (SEQ ID NO: 6), even more preferably selected from the group consisting of PLFDFSWLSL (SEQ ID NO: 8) and WLSLKTLLSL (SEQ ID NO: 6).

In another preferred embodiment, the peptide may be any peptide, consisting of not more than 200, preferably not more than 100, more preferably not more than 50, even more preferably not more than 25, even more preferably not more than 20, even more preferably not more than 15, even more preferably not more than 10 amino acids and containing (or more preferably consisting of) a sequence selected from the group consisting of EMQVLVSRI (SEQ ID NO: 44), TAYQSFEQV (SEQ ID NO: 43), YLNDHLEPWI (SEQ ID NO: 42), RIAAWMATYL (SEQ ID NO: 45), WMATYLNDHL (SEQ ID NO: 46), VLVSRIAAWM (SE ID NO: 48) and VAFFSFGGAL (SEQ ID NO: 49), more preferably from the group consisting of TAYQSFEQV (SEQ ID NO: 43), YLNDHLEPWI (SEQ ID NO: 42), RIAAWMATYL (SEQ ID NO: 45), WMATYLNDHL (SEQ ID NO: 46), VLVSRIAAWM (SEQ ID NO: 48) and VAFFSFGGAL (SEQ ID NO: 49), even more preferably selected from the group consisting of TAYQSFEQV (SEQ ID NO: 43), VAFFSFGGAL (SEQ ID NO: 49), VLVSRIAAWM (SEQ ID NO: 48) and RIAAWMATYL (SEQ ID NO: 45) or selected from the group consisting of TAYQSFEQV (SEQ ID NO: 43) and WMATYLNDHL (SEQ ID NO: 46) or selected from the group consisting of YLNDHLEPWI (SEQ ID NO: 42).

In another preferred embodiment, the peptide may be any peptide, consisting of not more than 200, preferably not more than 100, more preferably not more than 50, even more preferably not more than 25, even more preferably not more than 20, even more preferably not more than 15, even more preferably not more than 10 amino acids and containing (or more preferably consisting of) a sequence selected from the group consisting of RIAAWMATY (SEQ ID NO: 50) and ALCVESVDK (SEQ ID NO: 51), more preferably selected from the group consisting of RIAAWMATY (SEQ ID NO: 50).

In another preferred embodiment, the peptide may be any peptide, consisting of not more than 200, preferably not more than 100, more preferably not more than 50, even more preferably not more than 25, even more preferably not more than 20, even more preferably not more than 15, even more preferably, not b is more than 10 amino acids and containing (or, more preferably consisting of) a sequence selected from the group consisting of YLREQATGAK (SEQ ID NO: 52), SITDVLVRTK (SEQ ID NO: 53), LISFGAFVAK (SEQ ID NO: 54), RLLFFAPTR (SEQ ID NO: 55), RTKRDWLVK (SEQ ID NO: 56) and DIKNEDDVK (SEQ ID NO: 57), more preferably selected from the group consisting of RLLFFAPTR (SEQ ID NO: 55) and RTKRDWLVK (SEQ ID NO: 56).

In another preferred embodiment, the peptide may be any peptide, consisting of not more than 200, preferably not more than 100, more preferably not more than 50, even more preferably not more than 25, even more preferably not more than 20, even more preferably not more than 15, even more preferably not more than 10 amino acids and containing (or more preferably consisting of) a sequence selected from the group consisting of PAEEEEDDLY (SEQ ID NO: 58), SPEEELDGY (SEQ ID NO: 59), QSLEIISRY (SEQ ID NO: 60) and AGVGAGLAY (SEQ ID NO: 61), more preferably selected from the group consisting of PAEEEEDDLY (SEQ ID NO: 58) and QSLEIISRY (SEQ ID NO: 60).

In the following applicable embodiments, the peptide according to the invention is a peptide, which is limited in the molecule HLA-B class I MHC, including any of the following molecules: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17, HLA-B18, HLA-B21, HLA-Bw22, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-Bw41, HLA-Bw42, HLA-B44, HLA-B45, HLA-Bw46 and HLA-Bw47. In specific embodiments, the type HLA-B class I MHC, with which the peptide according to the invention are able to bind selected from HLA-B7, HLA-B35, HLA-B4, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.

In the following applicable embodiments, the peptide according to the invention is a peptide, which is limited in the molecule HLA-C class I MHC, including the following molecules: HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw7 and HLA-Cw1.

Preferably, the peptide fragment according to the invention contains less than 50 amino acid residues and more preferably it contains no more than 20 amino acid residues, for example not more than 10 amino acid residues. In specific embodiments, the peptide is heptapeptides, octapeptides, nonapeptide, Decapeptide or undecapeptide.

The peptide according to the invention, as described above, obtained from a representative of the family of proteins Bcl-2 or its fragment. Protein, from which can be derived peptide may be any member of the family proteins Bcl-2 from any animal species from which the protein is expressed. In preferred embodiments, the source of protein derived from mammals, including species of rodents, rabbit, and species of primates, such as man. Based on the sequence of the selected protein peptide according to the invention receive through any suitable chemical or enzymatic treatment of raw protein, which results in peptide suitable size, which is specified above, or peptide can be synthesized by any conventional with the persons synthesis of peptides which well-known specialist in this field.

The peptide according to the invention may have a sequence that is native sequence representative of a family of proteins Bcl-2, from which it is derived. However, peptides with higher affinity to any given HLA molecule, can be obtained from such native sequence by modifying the sequence by the substitution, deleterevision or adding at least one amino acid residue, for example, on the basis of the method described above, whereby identify motives anchor residues for a given HLA molecules.

An important characteristic of the peptide according to the invention is its ability to recognize or to cause INF-γ-producing immunoreactive T cells, the cytotoxic T-cells (CTL), which specifically recognize specific peptide, in a PBL population or tumor cells of the patient with malignant tumor (target cells). Specified activity is easy to determine, putting PBL or tumor cells from the patient ELISPOT analysis, which is described in reference (4) and the following example. Before analysis, it may be preferable to encourage the analyzed population PBL or tumor cells by contacting cells with a test peptide. Preference is sustained fashion peptide is able to cause or to recognize INF-γ-producing T cells with a frequency of at least 1 in 10 4PBL, which is determined by ELISPOT analysis used in this invention. More preferably the frequency is at least 5 to 104PBL, most preferably at least 10 to 104PBL, for example at least 50 or 100 to 104PBL.

The ELISPOT analysis is a powerful tool to monitor the responses of T-cells specific for a peptide derived from Bcl-2. However, although it has been shown that the reactivity in the ELISPOT in most cases correlates with the ability of CTL to lyse target cells, the final proof for this view can only be obtained directly. Therefore, the main conclusion is given in this description of the observations is that the peptides according to the invention can be expressed and to be in complex with HLA molecules on malignant cells. This makes these malignant cells sensitive to destruction by CTL cells and makes applicable immunization proteins of Bcl-2 to control the growth of neoplasm. The presence of spontaneous CTL responses in PBL from patients with breast cancer restricted by HLA epitopes peptides derived from Bcl-2, proves the possibility of immunotherapy based on these tumor antigens, not only for patients with breast cancer, but also the Kolka representatives of the family proteins Bcl-2 sverkhekspressiya in many malignant tumors, including lung cancer, colorectal, prostate, and leukemia and lymphoma, in the case of a wide range of malignant diseases.

Accordingly, in another preferred embodiment, the peptide according to the invention can cause the appearance of INF-γ-producing cells in a PBL population of a patient having a malignant disease in which is expressed family of proteins Bcl-2, including hematopoietic malignancies such as chronic lymphocytic leukemia and chronic myelogenous leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, cancer of the colon, pancreas cancer and prostate cancer.

In addition to the ability to induce immune responses in populations of PBL, it is also assumed that the peptides according to the invention is able to induce cytolytic immune responses in situ, i.e. in the tissues of solid tumors. It can be shown, receiving the complexes of HLA-peptide, for example multipersoona and provided with registered label, and using such complexes for immunohistochemical stains to identify tumor tissue CTLs that are reactive against a peptide epitope according to the invention. Accordingly, the next important characteristic of a peptide according to the invention is that it provides identification in situ in the tumor CTL, which are aktivnye against the peptide epitope.

It is also assumed that the peptides according to the invention, in addition to ability to bind to HLA molecules, which leads to the presentation of HLA complexes and peptides on the cell surface, and these complexes, in turn, act as epitopes or targets for cytolytic T-cells, can cause other types of immune responses, such as B-cell responses, leading to the production of antibodies against complexes, and/or hypersensitivity reaction of the delayed type (RGST). The last type of immune response is determined in the form of redness and palpable seals in the injection of the peptide according to the invention.

The vaccine composition according to the present invention may contain a nucleic acid encoding a protein belonging to the family of proteins Bcl-2, or peptide fragment. The specified nucleic acid thus can encode any of the above proteins and peptide fragments. The nucleic acid may represent, for example DNA, RNA, LNA, HNA, PNA, preferably the nucleic acid is a DNA or RNA.

Nucleic acids according to the invention may be in any suitable vector, such as expressing vector. There are many vectors, and the specialist will be able to choose a suitable vector for a particular purpose. The vector may be, for example, in the form of plasma is water, Comedy, viral particles, or artificial chromosome. A suitable nucleic acid sequence may be incorporated into the vector in a number of ways, for example, DNA can be integrated into a suitable site(s) of restriction enzyme using methods known in this field. In addition to the nucleic acid sequence according to the invention, the vector may further comprise one or more of the following: a signal sequence, the beginning of replication, one or more marker genes, an enhancer element, a promoter and termination sequence transcription. The vector may also contain additional sequences. When the construction of suitable vectors containing one or more of these components, use the standard methods of ligation, which are known to the person skilled in the art. The vector preferably is expressing a vector containing a nucleic acid operatively linked with a regulatory sequence of the nucleic acid control its expression in a suitable cell. In the scope of the present invention specified regulatory sequence of nucleic acids in General must be capable of driving expression in a cell of a mammal, preferably a human cell, more predpochtitel is but in antigen presenting cell.

In one preferred embodiment, the vector is a viral vector. Specified viral vector may, in addition to the nucleic acid that encodes a member of the family proteins Bcl-2 or its peptide fragment to contain a second nucleic acid sequence encoding a stimulating T-cell polypeptide. Stimulating T-cells, the polypeptide is preferably selected from the group consisting of B7.1, ICAM-1 and LFA-3.

The vector may also be a bacterial vector, such as vector based on the attenuated bacteria. Attenuated bacterial vectors can be used to induce long-term immune responses mucosa at sites of infection and persistence. As vectors can be used in a variety of recombinant bacteria, for example, a bacterial vector may be selected from the group consisting of Salmonella, Lactococcus and Listeria. In General it can be demonstrated induction of immunity to heterologous antigen L1 or HPV16 E7 with a strong induction of CTL and tumor regression in mice.

The invention also relates to a set of components, containing

(i) any of the vaccine compositions described in this publication, and/or

ii) any of the proteins belonging to the family of proteins Bcl-2, described in this publication, and/or

(iii) any of peptide fragments of proteins under item (ii), described what this publication and/or

(iv) any of the nucleic acids encoding the proteins under item (ii) or peptides under item (iii), and

additional anti-tumor agent.

The components of the kit are preferably included in a separate composition, however, the scope of the present invention includes a variant in which all the components of the kit are included in the same composition. Thus, the components of the kit can be introduced simultaneously or sequentially in any order.

Antitumor agent can be an agent used in chemotherapy or gene therapy, immune-stimulating substances or antibodies. Immunostimulating substances can be, for example, cytokines, such as cytokines selected from the group consisting of GM-CSF, IFN type I, interleukin 12 and interleukin 15. The antibody is preferably an immunostimulating antibody, such as anti-CD40 or anti-CTLA-4 antibodies. Immunostimulating substance may be a substance capable of Deplete inhibiting immune cell (e.g., regulatory T cells) or factors specified substance can be, for example, of ubiquitin ligase E3. Of ubiquitin ligase E3 (HECT, RING and U-box proteins) has emerged as a key molecular regulators of immune cells, and each of them may be involved in the regulation of immune responses during infection by targeting implementation specification is cnie inhibiting molecules for proteolytic destruction. Currently, several proteins HECT and RING E3 are also associated with the induction and maintenance of immune autotolerance: c-Cbl, Cbl-b, GRAIL, Itch and Nedd4, each of which negatively regulates the production of the growth factor for T cells and proliferation.

It is obvious that the received data according to the present invention provide the basis for therapeutic and diagnostic applications of protein or peptide fragment according to the invention.

Accordingly, in this aspect the present invention relates to pharmaceutical compositions containing the protein or peptide fragment according to the invention, in particular to pharmaceutical compositions, which when administered to a patient with a malignant tumor is able to induce an immune response against malignant diseases, including products from vaccinated patients effector T cells with cytotoxic activity against malignant cells.

As it is well known that different HLA molecules are differently distributed in the major populations of mankind, there is a need to identify peptide epitopes that are restricted by several molecules of class I HLA to expand the category of patients that can be treated according to the methods proposed in the present invention. Feature multiple epitopes of Bcl-2 with different elements of the exhaust gas is anichini HLA expands clinical capabilities of the target antigen in two important areas: (i) it increases the number of patients suitable for immunotherapy based on peptides derived from Bcl-2. Antigen HLA-A2 is expressed in approximately 50% of individuals in populations of Europeans and Asians, both antigen HLA-A1 and HLA-A3 is expressed in approximately 25% Caucasoids, and 5% of Asians, whereas antigen HLA-A11 is expressed in approximately 15% Caucasoids, and 30% of Asians. Even though these numbers cannot be collapsed due to the common expression, a combination of peptides that are limited to many of these antigens, of course, could cover the majority of patients with malignant tumor. (ii) Joint purposeful impact on several elements of the limitations in each patient, probably reduces the risk of escape from immune surveillance in case of loss of HLA allele. The loss of one HLA allele is an important component changes MHC described for malignant cells, whereas the total loss of expression of class I molecules is a fairly rare event. Thus, when identifying epitopes Bcl-2, restricted by different HLA alleles, can be targeted to affect more than one HLA molecule simultaneously in patients with overlapping alleles.

Thus, it is possible to develop highly immunogenic multiepitope vaccines. Preferably such vaccines should be designed to facilitate the simultaneous delivery of the best on the walking peptides derived from Bcl-2, optionally in combination with other suitable peptides and/or adjuvants, which are described next. The present invention covers such multiepitope vaccine containing the result of the Bcl-2 peptides optionally in combination with additional proteins or peptide fragments that are not relevant or is not received from the family of proteins Bcl-2, and/or adjuvants, which are described below, and/or restricted by class II MHC-epitopes, which are described below.

Increased attention has been focused on how to call specific to the tumor cell T-helper immunity, i.e. the vaccine epitopes restricted by class II MHC, despite the fact that tumors usually do not Express MHC class II. This is based on recently obtained evidence that induction and effectiveness of vaccine-induced antitumor response in many cases requires cooperation is specific to the tumor CD4-positive Th cells. Thus, an important driver for the development of vaccines that have a more complex structure, is the desire to purposefully affect numerous tumor antigens, for example, through the creation of vaccines containing or encoding a set of carefully selected CTL-epitopes and Th-cell epitopes.

Obviously, multiepitope vaccines which are an effective way to enhance immunity against epitopes, derived from several different antigens, without the necessity of introducing potentially harmful proteins, such as oncoproteins (or their coding genes). Such vaccines also can selectively induce immunity against subdominant and cryptic T-cell epitopes, which can be especially important in the case associated with tumors of autoantigens, in which case there may be a tolerance against epitopes, which are largely represented in normal tissues. In addition, antigen cells may not be able to provide some epitopes that are expressed on tumor cells, due to functional differences between immunoproteasome antigen presenting cells and "constitutive" proteasomes present in most tumor cells. In the case of vaccines based on peptides, such epitopes can be entered in the "MHC-ready" form, which provides a view through the exogenous load regardless of the capture and processing of antigen by antigen presenting cells of the host.

Because the peptides according to the invention are relatively small molecules, such compositions may require a combination of peptides with various substances such as adjuvants to get HAC the ins, immunogenic compositions, etc. Adjuvants in a broad sense are substances that stimulate immune responses. Often the preferred adjuvant is complete or incomplete beta-blockers or killed B. pertussis organisms used, for example, in combination with precipitiously alum antigen. General discussion adjuvants are presented in Goding, Monoclonal Antibodies: Principles & Practice (2ndedition, 1986), p.61-63. However, Goding notes that in the case when interest antigen has a low molecular weight or is subimmunogenic, it is recommended that bind to immunogenic carrier. Examples of such carrier molecules include hemocyanin marine saucer "keyhole", bovine serum albumin, ovalbumin and avian immunoglobulin. It was also noted that different extracts of saponin applicable as adjuvants in immunogenic compositions. The recently proposed the use of adjuvant colony-stimulating factor granulocyte-macrophage (GM-CSF), a well-known cytokine (WO 97/28816).

The vaccine composition according to the invention preferably contain an adjuvant and/or carrier. Examples of applicable adjuvants and carriers are given below in this description. Thus, a protein belonging to the family of proteins Bcl-2, or peptide fragment that is present in the composition is, may be associated with a carrier such as a protein or antigen presenting cell such as a dendritic cell (DC)capable of expressing a protein family proteins Bcl-2 or its peptide fragment of the T-cell.

Adjuvants are any substance, the mixing of which with the composition of the vaccine increases or otherwise modifies the immune response to protein family proteins Bcl-2 or its peptide fragment. The carriers are skeletal structures, such as a polypeptide or a polysaccharide, which can be associated protein family proteins Bcl-2 or its peptide fragment.

Adjuvants can be selected, for example, from the group consisting of AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3Ca3(PO4)2, kaolin, coal, aluminum hydroxide, muramyldipeptide, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetylmuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxyphosphonic) - ethylamine (CGP 19835A, also referred to as MTP-PE), and RIBI (MPL+TDM+CWS) in the emulsion 2% squalene/tween-80, RTM, lipopolysaccharides and their various derivatives, including lipid A, complete adjuvant's adjuvant (FCA), incomplete adjuvant's adjuvant, adjuvant Merck Adjuvant 65, polynucleotides (in the example, poly IC and poly AU acids), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOM, Quil A, ALUN (see US 58767 and 5554372), derivatives of lipid A, derivatives of cholera toxin, derived HSP-derived LPS, synthetic matrices or GMDP, interleukin 1, interleukin 2, Montanide ISA-51 and QS-21. Preferred adjuvants used in the invention include adjuvants on the basis of oil/surfactant, such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants is based on bacterial DNA adjuvants, such as adjuvants containing CpG-oligonucleotide sequence. Other preferred adjuvants adjuvants are based on viral dnrc, such as poly-I:C. Imidazopyridine are another example of preferred adjuvants. In addition, the preferred adjuvant are liposomes. Preferred adjuvants adjuvants are suitable for use on humans.

Adjuvants Montanide (available from Seppic, Belgium) can be selected from the group consisting of Montanide ISA-51, Montanide ISA-50, Montanide ISA 70, Montanide ISA 206, Montanide ISA-25, Montanide ISA-720, Montanide ISA-708, Montanide ISA-763A, Montanide ISA-207, Montanide ISA-264, Montanide ISA-27, Montanide ISA-35, Montanide ISA 51F, Montanide ISA 016D and Montanide IMS, p is edocfile from the group consisting of Montanide ISA-51, Montanide IMS and Montanide ISA-720, more preferably from the group consisting of Montanide ISA-51. Montanide ISA-51 (Seppic, Inc.) is adjuvant based on a mixture of oil/surfactant, in which different surfactants combined with nametablecolumn mineral oil, metabolisable oil or their mixture. Adjuvants are prepared for use in the form of an emulsion with an aqueous solution containing a protein belonging to the family of proteins Bcl-2, or peptide fragment. Surface-active agent is an oleate manned. QS-21 (Antigenics; Aquila Biopharmaceuticals, Framingham, MA) is a highly water-soluble saponin, which is used in aqueous solution. Adjuvants QS-21 and Montanide ISA-51 can be supplied in sterile, single use vials.

General discussion adjuvants are presented in Goding, Monoclonal Antibodies: Principles & Practice (2ndedition, 1986), p.61-63. However, Goding notes that in the case when interest antigen has a low molecular weight or is subimmunogenic, it is recommended that bind to immunogenic carrier. Examples of such carrier molecules include hemocyanin marine saucer "keyhole", bovine serum albumin, ovalbumin and avian immunoglobulin. It was also noted that different extracts of saponin applicable as adjuvants in immunos is the R compositions. The recently proposed the use of adjuvant colony-stimulating factor granulocyte-macrophage (GM-CSF), a well-known cytokine (WO 97/28816).

The required functional properties of adjuvants that can be used according to the present invention are listed in table 1 below.

Table 1. The modes of action of adjuvants.

ActionThe type of adjuvantAdvantage
1. ImmunomodulirutayaTypically, small molecules or proteins that modify the network of cytokines.Increasing regulation of the immune response. Selection of Th1 or Th2
2. PresentationAs a rule, amphipatic molecules or complexes that interact with the immunogen in its native conformation.The increased response in the form of neutralizing antibodies. The long duration of response
3. Induction of CTL- Particles that can bind or enclose the immunogen and which may be incorporated into cell membranes or destroy them;Processing of protein in tsito the OLE, leading to the right is limited to class I peptides
- water-oil emulsion for direct binding of peptide to MHC-1 on the cell surface.Easiest way, if you know a random peptide(dy)
4. Purposeful action- Adjuvants in the form of particles, which bind the immunogen. Adjuvants, which saturate the cells of Kupffer;Effective use of adjuvant and immunogen
- carbohydrate adjuvants, which purposefully act on the receptors of lecithin on macrophages and DC.As specified above. Can also determine the type of response, if purposeful exposure selectively
5. Education depot- water-oil emulsion for short-term actionsEfficiency
- microspheres or nanospheres for long-termThe ability disposable vaccine

Source: Cox, J. C. and Coulter, A. R. (1997). Vaccine 15, 248-56.

The vaccine composition according to the present invention may include a few different adjuvants. In addition, the invention encompasses Ter is non-therapeutic composition, additionally contain any adjuvant substance, including any of the above substances and their combinations. It is also assumed that a protein belonging to the family of proteins Bcl-2, or peptide fragments and adjuvant can be entered separately in any suitable sequence.

The media may be present regardless of the adjuvant. Media feature can for example be to increase the molecular weight, in particular, peptide fragments, to increase their activity or immunogenicity, to give stability, to increase the biological activity or to increase the half-life in serum. In addition, the media can help to provide a protein belonging to the family of Bcl-2, or peptide fragments to T cells. The carrier can be any suitable carrier known to the person skilled in the art, for example a protein or antigen presenting cell. Protein carrier can be, without limitation, hemocyanin marine saucer "keyhole", serum proteins, such as transferrin, bovine serum albumin, serum albumin human thyroglobulin or ovalbumin, antibodies or hormones, such as insulin or palmitic acid. For immunization of people the media can be physiologically acceptable medium that is acceptable to humans and safe. But who in one embodiment of the invention suitable carriers are the tetanus toxoid and/or diphtheria toxoid. Alternative media can be dextrans, such as sepharose.

Accordingly, the invention relates to therapeutic compositions, optionally containing adjuvant substance, including any of the above substances or their combinations. It is also assumed that the antigen, i.e. the peptide according to the invention and an adjuvant, can be introduced simultaneously or separately in any suitable sequence.

The choice of antigen in the pharmaceutical compositions according to the invention will depend on the settings defined by the person skilled in the art. As indicated, each of the different peptides according to the invention is presented on the cell surface specific HLA molecule. Essentially, if subjected to the treatment of the subject tiponut against HLA phenotype, then choose this peptide/peptide, which is known to be associated with that particular HLA molecule.

Alternative interest antigen is chosen based on the prevalence of different HLA phenotypes in this population. As an example, HLA-A2 is the most common phenotype in the population of Europeans, and therefore, the composition containing from survivin peptide binding to HLA-A2, will be active in a large proportion of this population. However, the composition according to the invention may also contain a combination of the Vuh or more from survivin peptides which specifically interact with different HLA molecules, so as to cover a large part of the target population. Thus, as an example, the pharmaceutical composition may contain a combination of peptide restricted by HLA molecule is A peptide, a limited molecule, HLA-B, for example, including such molecules HLA-A and HLA-B, which correspond to the predominant HLA phenotypes in the target population, such as HLA-A2 and HLA-B35. In addition, the composition may contain a peptide restricted by HLA molecule.

It is assumed that appropriate immunogenic composition according to the invention, in addition to the peptide derived from the representative of the family of proteins Bcl-2, which is defined in this description may contain immunogene effective amount of the representative family proteins Bcl-2, which is defined herein, or immunogenic fragment.

The number of immunogenic peptide according to the invention in the pharmaceutical composition may vary depending on the particular application. However, a single dose of immunogen everywhere preferably is from about 10 μg to about 5000 μg, more preferably from about 50 μg to 2500 μg, for example, from about 100 μg to 1000 μg. Methods of administration include intradermal, subcutaneous and intravenous injection, implantation in the form of preparation is the long-term release, etc. In this case, carry out any and all forms of introduction, well-known in this field. Also provided for any and all conventional dosage forms, which are known in this area as suitable for preparation of injection immunogenic peptide compositions, such as liofilizovannye forms and solutions, suspensions or emulsion form, optionally containing conventional pharmaceutically acceptable carriers, diluents, preservatives, adjuvants, buffer components, etc.

The pharmaceutical compositions can be prepared and entered using any conventional Protocol known to the person skilled in the art. Example 5 shows a non-limiting example of getting the vaccine composition according to the invention, as well as a non-limiting example, the introduction of such vaccines. The person skilled in the art it will be clear that the Protocol can be easily adapted to any of the vaccine compositions described in this specification.

In the next version according to the invention the pharmaceutical composition according to the invention is applicable to treatment of a patient with a malignant tumour, when during the progression of malignant tumors in this patient in malignant cells developed reduced sensitivity to chemotherapeutic active anticancer drug is the agent and/or radiation therapy.

The pharmaceutical composition according to the invention can mainly contain at least one additional immunogenic protein or peptide fragment selected from a protein or peptide fragment, not belonging to the family of proteins Bcl-2 or not derived from this protein, including a protein involved in the regulation of apoptosis, or its derived peptide fragment. As one example of such a protein or peptide is survivin, which is defined above, or its peptide fragment. In specific embodiments, additional immunogenic obtained from survivin peptide is restricted by HLA-A2 peptide having a sequence selected from the following sequences: FLKLDRERA (survivin101-109) (SEQ ID NO: 12), TLPPAWQPFL (survivin5-14) (SEQ ID NO: 13), ELTLGEFLKL (survivin95-104) (SEQ ID NO: 14), LLLGEFLKL (SEQ ID NO: 15) and LMLGEFLKL (SEQ ID NO: 16). (Symbols in parentheses indicate positions of residues in the protein survivin, which are described in US 6245523). LLLGEFLKL (SEQ ID NO: 15) is the sequence obtained from survivin96-104replace the "T" in position 2 of the peptide residue "L", and LMLGEFLKL (SEQ ID NO: 16) derived from survivin96-104replace the "T" in position 2 the remainder of "M". In the following specific embodiments, additional immunogenic obtained from survivin peptide is restricted by HLA-B35 polucen the th of survivin peptide, having a sequence selected from the following sequences: CPTENEPDL (survivin46-54) (SEQ ID NO: 17), EPDLAQCFF (survivin51-59) (SEQ ID NO: 18), CPTENEPDY (SEQ ID NO: 19) and EPDLAQCFY (SEQ ID NO: 20). (Symbols in parentheses indicate positions of residues in the protein survivin, which are described in US 6245523). CPTENEPDY (SEQ ID NO: 19) is the sequence obtained from survivin46-54replace "L" on the C-end of the peptide residue "Y", and EPDLAQCFY (SEQ ID NO: 20) derived from survivin51-59the replacement residue "F" to C-end 2 the remainder of the "Y".

In the following embodiments, the additional peptide is restricted by HLA-A1 peptide having a sequence selected from the following sequences: survivin38-46(Sur38Y9) (C replaced by Y in P9, MAEAGFIHY) (SEQ ID NO: 21), survivin47-56(Sur47Y10) (Q replaced by Y in P10, PTENEPDLAY (SEQ ID NO: 22)), survivin92-101(Sur92-101) (QFEELTLGEF) (SEQ ID NO: 23) and

survivin93-101(Sur93T2 (E replaced by T in P2, FTELTLGEF (SEQ ID NO: 24)). The peptide according to the invention can also be restricted by HLA-A3 peptide, such as

survivin18-24(Sur18K10) (F replaced by K in P10, RISTFKNWPK (SEQ ID NO: 25) and/or restricted by HLA-A11 peptide, such as survivin53-62(Sur53-62) (DLAQCFFCFK) (SEQ ID NO: 26), and/or restricted by HLA-A2 peptide, such as survivin18-28(Sur18-28) (RISTFKNWPFL) (SEQ ID NO: 27).

However, in one preferred embodiment of the invention the composition of the vaccine does not contain survivin or it is fragments.

Other applicable additional peptides include known polypeptide inhibitor of apoptosis ML-IAP, which has a rather selective expression detected in melanomas. Thus fragments of ML-IAP, is able to induce specific T-cell response, i.e. cytotoxic T-cell response or helper T-cell response, may not necessarily be included in the composition according to the present invention. Applicable peptide fragments of ML-IAP include any of the fragments of ML-IAP, as described in the application for the grant of a patent WO 2004/089980, which is hereby incorporated by reference in full, preferably ML-IAP245(RLQEERTCKV)(SEQ ID NO:28), ML-IAP280(QLCPICRAPV)(SEQ ID NO:29), ML-IAP90(RLASFYDWPL)(SEQ ID NO:30), ML-IAP154(LLRSKGRDFV)(SEQ ID NO:31), ML-IAP230(VLEPPGARDV)(SEQ ID NO:32), ML-IAP98(PLTAEVPPEL)(SEQ ID NO:33), ML-IAP34(SLGSPVLGL)(SEQ ID NO:34), ML-IAP54(QILGQLRPL)(SEQ ID NO:35), ML-IAP99(LTAEVPPEL)(SEQ ID NO:36), ML-IAP83(GMGSEELRL)(SEQ ID NO:37) and ML-IAP200(ELPTPRREV)(SEQ ID NO:38).

Other suitable additional peptides include TRAG-3 and its peptide fragments. TRAG-3 there are at least two alternative splaisiruemym forms, and peptides all forms of splicing TRAG-3 is applicable as an additional peptides. In particular, fragments of any of the forms of splicing TRAG-3 in the case when these fragments are able to induce specific T-cell response, i.e. cytotoxic T-Claix the full answer, or helper T-cell response, may not necessarily be included in the composition according to the present invention.

In addition, the composition according to the present invention can be presented in the form of multiepitope vaccine containing a limited class I epitope and limited class II epitopes, which are defined in the description above.

Immunoprotective effect of the composition according to the invention can be identified using several methods, for example as described in WO 97/28816 above. Successful immune response can also be identified by the appearance of reactions RGST after immunization and/or identification of antibodies specifically recognizing the peptide(dy) composition of the vaccine.

In preferred embodiments, the pharmaceutical composition according to the invention is a vaccine composition. Thus, the pharmaceutical composition may be an immunogenic composition or a vaccine that can induce an immune response against malignant disease. Used in this sense, the terms "immunogenic composition or vaccine" refers to a composition that causes at least one type of immune response against malignant cells. Accordingly, such an immune response can be any of the types listed above: CTL-response, in which are formed CTL, which are able to identify the complex of HLA/peptide, presented at the cell surface, leading to lysis of the cells, because the vaccine triggers the production of the vaccinated subject effector T cells with cytotoxic activity against malignant cells; B-cell response, causing the production of antitumor antibodies; and/or RGST-type immune response.

In applicable embodiments, the immunogenic response directed against malignant diseases cause the introduction of a peptide according to the invention or by means of load molecules MHC class I antigen presenting cells (APC) from the patient, the allocation of PBL from patient and incubation of the cells with the peptide prior to injecting the cells back into the patient's body, either through the allocation predecessors APK from the body of the patient and cell differentiation in vocational agriculture with the use of cytokines and antigen before injecting the cells back into the patient's body.

Thus, in one aspect of the invention provides a composition of a vaccine containing antigen cells containing protein belonging to the family of Bcl-2, or peptide fragment or nucleic acid encoding this protein or the peptide fragment. Antigen presenting cell can be any cell capable of expressing the antigen to the T cell. Preferred Antiherpes the influencers cells are dendritic cells. Dendritic cells (DC) can be obtained and used in therapeutic method according to any suitable Protocol, for example, as described in this publication below. The person skilled in the art it will be clear that the Protocol can be adapted for use in patients with different HLA type and various diseases.

On dendritic cells (DC) pulsed act 50 μg/ml restricted by HLA peptide (synthesized as GMP) for 1 hour at 37°C and 5 x 106cells injected subcutaneously in 1 and day 14, and then every 4 weeks, additional leikaferez after 5 vaccinations. Create a DC for clinical use and quality control can be performed essentially as described in reference 5.

Thus, in one embodiment of the present invention is a method of treatment of patients with malignant tumor is a way in which the peptide is administered through the presentation of peptide antigen presenting cells (APC) of the patient ex vivo with subsequent injection of the thus treated APK again in the body of the patient. There are at least two alternative ways of exercising. One alternative is the separation of agriculture from the body of the patient with malignant tumor and incubation (load) molecules MHC class I peptide. Load molecules MHC class I means incubation with peptide for in order for agriculture to molecules MHC class I specific peptides is contacted with the peptide and therefore were able to present it to T cells. Then AIC again injected to the patient. Another alternative method is based on recent discoveries in the biology of dendritic cells. In this case, monocytes (which are the precursors of dendritic cells) are separated from the body of the patient and differentiate in vitro in professional APC (or dendritic cells) with the use of cytokines and antigen. Then educated in vitro DC pulse is affected by the peptide and injected into the patient.

Due to the fact that representatives of the family proteins Bcl-2 appears to be expressed in some forms of malignant tumors, it is highly likely that can be derived vaccine according to the invention for combating any type of malignant disease, in which these proteins are expressed. Thus, as examples of the composition of the vaccine according to the invention is immunologically active against hematopoietic malignancies, including chronic lymphocytic leukemia and chronic myelogenous leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, cancer of the colon, pancreas cancer and prostate cancer.

From the above description specifications the sheet is easy to see what proteins and/or peptides according to the invention is applicable as a means of diagnosis of a malignant tumor. Therefore, the peptides according to the invention provide the basis for the development of widely applicable diagnostic and prognostic methods related to malignant diseases. Thus, other applicable embodiments, the composition according to the invention is a composition for diagnosing ex vivo or in situ presence of the patient's malignant tumors, for example, based on the detection of T-cells reactive against the members of the family proteins Bcl-2, among PBL or in tumor tissue.

Accordingly, in the following aspects of the proposed diagnostic kit for the diagnosis of ex vivo or in situ presence of the patient with malignant tumor T-cells reactive against the representatives of Bcl-2, among PBL or in tumor tissue containing one or more peptides according to the invention, and a method of detecting in a patient with a malignant tumour of the presence of such reactive T-cells, the method involves contacting the tumor tissue or blood sample with a complex of the peptide according to the invention and molecules HLA class I or a fragment of such molecule and detecting binding of the complex with tissue or blood cells.

Other applicable diagnostic or prognostic the RCM method is based on the formation of antibody in a heterologous species of animal, for example, murine antibodies directed against a peptide derived from a representative of the family of proteins Bcl-2, according to the invention, which can then be used, for example, for diagnosing the presence of malignant cells presenting the peptide. For immunization purposes, the number of peptide may be less than the number used in the course of therapy in vivo, such as described above. In General, the preferred dose may be in the range of from about 1 μg to 750 μg of peptide. You can also get a monoclonal antibody-based immunization peptide according to the invention. Accordingly, the present invention also relates to a molecule, in particular a monoclonal or polyclonal antibody, including a fragment that can specifically bind to a peptide according to the invention, and to the molecule, which is capable of blocking this binding, for example the antibody produced against monoclonal or polyclonal antibodies directed against the peptide according to the invention. The invention also relates to isolated receptor of T-cells that can specifically bind to a peptide or protein according to the invention, and coded them isolated nucleic acids. Such receptors of T-cells can be, for example, a clone of it is specific to the protein or Pat the DN T-cells using standard methods, well-known specialist.

In one aspect, the invention also relates to isolated T cells containing the receptor of the T cells can specifically bind to any of the proteins belonging to the family of Bcl-2, and/or their peptide fragments described in this publication. Isolated T-cells are preferably T-cells that have been propagated in vitro. The ways of propagation of T-cells in vitro are well known to the specialist. Such T cells may be particularly applicable for the treatment of malignant tumors by using adaptive transfer or autologous cell transfer. Thus, the invention also relates to methods of treatment, which consists in the introduction of T-cells containing the receptor of the T cells can specifically bind to a protein belonging to the family of Bcl-2, or peptide fragments of the person, such as someone suffering from a malignant tumor. The invention also relates to the use of T-cells containing the receptor of the T cells can specifically bind to a protein belonging to the family of Bcl-2, or peptide fragments, to obtain drugs for the treatment of malignant tumors. Autologous cell transfer can be performed essentially as described in reference 7.

In one aspect the invention relates to the complex is the peptide according to the invention and molecules HLA class I or a fragment of such molecule, which is applicable as a diagnostic reagent, such as described above. Such a complex may be Monomeric or multimeric.

The present invention relates to a method of relief or cure of malignant diseases. Accordingly, the following aspect of the invention features a method of treating a malignant disease associated with the expression of a representative family proteins Bcl-2, including as examples: hematopoietic malignancy, including chronic lymphocytic leukemia and chronic myelogenous leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, cancer of the colon, pancreas cancer and prostate cancer, and this method is the introduction to a patient suffering from a disease, an effective amount of the pharmaceutical composition according to the invention, a molecule that can specifically bind to a peptide according to the invention, and/or molecules, which able to block the binding of such molecules.

In some cases, suitable is the combination of the method of treatment according to the invention with the conventional treatment of malignant tumors, such as chemotherapy, radiation therapy, treatment immunostimulating substances, gene therapy, treatment with antibodies and treatment using dendritic cells is K. Because increased expression of members of the family proteins Bcl-2 in tumor cells correlates with drug resistance, the combination is based on Bcl-2 immunotherapy, which is disclosed in the present invention, the cytotoxic immunotherapy may be an effective method for the treatment of malignant tumors.

In one aspect the invention relates to a method of controlling immunization, while this method includes a stage

i) obtaining a blood sample from the individual;

ii) obtaining a protein belonging to the family of proteins Bcl-2, or peptide fragment, with the specified protein or peptide can be any of the proteins or peptides are mentioned in this description;

iii) determine whether the specified sample of blood antibodies or T-cells containing the receptor of T-cells, specific binding protein or peptide;

iv) thus determining whether there was an increased immune response to a specific protein or peptide in the specified individual.

The individual preferably is, for example, a person who has been immunized with a protein belonging to the family of proteins Bcl-2, or peptide fragment, or nucleic acid that encodes this protein or peptide.

Hereinafter the invention will be illustrated in the following non-limiting examples of the drawings, where

in Fig. 1 shows the identification of peptides that bind HLA-A2 derived from Bcl-2. Strip the heavy chain of MHC class I quantitatively assessed using the device for imaging the luminescence. The number of stable heavy chain HLA-A2 is directly dependent on the affinity of binding of the added peptide. The binding is limited by HLA-A2 positive control peptide Pol476HIV (black squares) compared with peptides Bcl172(black triangles), Bcl180(black circles) and Bcl200(white circles); and

Fig. 2 illustrates the T-cell response against peptides Bcl172, Bcl180, Bcl208and

Bcl214. Analyzed PBL from 15 patients with breast cancer. T-lymphocytes once stimulated by the peptide before sowing 105cells per well in three repetitions either in the absence or in the presence of peptide. The average number of specific peptide spots (after subtracting spots without added peptide) was calculated for each patient using the analyzer ImmunoSpot® series 2.0 (CTL Analyzers, LLC, Cleveland, US).

Fig. 3 illustrates the T-cell responses against Bcl-2, which are measured in the ELISPOT analysis INF-γ. Analyzed PBL from ten positive for HLA-A2 patients with CLL, three positive for HLA-A2 patients with AML and two patients with pancreatic cancer (PC). Investigated peptides Bcl208(A) and Bcl214(B). T-lymph is the ITA's once stimulated by the peptide before sowing 10 5cells per well in three repetitions either in the absence or in the presence of peptide. The average number of specific peptide spots (after subtracting spots without added peptide) was calculated for each patient using the analyzer ImmunoSpot® series 2.0 (CTL Analyzers, LLC, Cleveland, US). Responsible individuals (defined by the average number of antigen-specific spots ± ½ standard deviation > 25 105lymphocytes) are marked with black squares, while not meeting the individuals marked with white squares.

Fig. 4 illustrates the detection of Bcl-2-specific CTL in the ELISPOT analysis of granzyme B. T-lymphocytes from four patients with breast cancer in different final phase (b19, b20, b22, b16) and healthy control donors (h1) once stimulated by the peptide before sowing 105cells per well in three repetitions either in the absence or in the presence of peptide Bcl208(A) or Bcl214(B). The average number patinopecten spots granzyme B (after subtracting spots without added peptide) was calculated for each patient using the analyzer ImmunoSpot® series 2.0 (CTL Analyzers, LLC, Cleveland, US). Responsible individuals (defined by the average number of antigen-specific spots ± ½ standard deviation > 25 105lymphocytes) are marked with black squares, while not meeting the individuals marked with white kV is the include.

Fig. 5 illustrates the cytolytic ability of Bcl-2-specific CTL. Bcl208-reactive CTL were isolated from PBL from patients with breast cancer using magnetic beads coated with HLA-A2/Bcl208. A) the Bulk cultures were analyzed in relation to specific lysis of T2 cells with peptide Bcl208(black squares) or without (white squares). B) Lysis Bcl208-selected T-cells HLA-A2-positive cell line breast cancer MDA-MB-231 (black circles) and HLA-A2-negative cell line breast cancer ZR75-1 (white circles).

6 illustrates restricted by HLA-A2 T-cell responses against Bcl-XLmeasured in ELISPOT analysis INF-γ. Analyzed PBL from twelve healthy person, eighteen patients with breast cancer (BC patients), six patients with melanoma and two patients with pancreatic cancer (PC-patients). All the people were HLA-A2 positive. Investigated peptides Bcl-XL173-182(YLNDHLEPWI) (SEQ ID NO: 48) (A), Bcl-XL141-150(VAFFSFGGAL) (SEQ ID NO: 49) (B), Bcl-XL161-170(VLVSRIAAWM) (SEQ ID NO: 48) (C) and Bcl-XL165-174(RIAAWMATYL) (SEQ ID NO: 45) (D). T-lymphocytes once stimulated by the peptide before sowing 105cells per well in three repetitions either in the absence or in the presence of an appropriate peptide. The average number of specific peptide spots (after subtracting spots without added peptide) races who were cityval for each patient, using the analyzer ImmunoSpot® series 2.0 (CTL Analyzers, LLC, Cleveland, US). Responsible individuals (defined by the average number of antigen-specific spots ± ½ standard deviation > 25 105lymphocytes) are marked with black squares, while not meeting the individuals marked with white squares.

Fig. 7 illustrates the detection of Bcl-XL-specific CTL in the ELISPOT analysis of granzyme Century T-lymphocytes from three different patients with breast cancer (BC35, BC36 and BC17) once stimulated by the peptide before sowing 3 x 105cells per well in three repetitions either in the absence or in the presence of peptide Bcl-XL173-182(YLNDHLEPWI). The average number patinopecten spots granzyme B (after subtracting spots without added peptide) was calculated for each patient using the analyzer ImmunoSpot® series 2.0 (CTL Analyzers, LLC, Cleveland, US). Responsible individuals (defined by the average number of antigen-specific spots ± ½ standard deviation > 25 105lymphocytes) are marked with black squares, while not meeting the individuals marked with white squares.

Fig. 8 illustrates the analysis of Bcl-XL-specific CD8-positive cells in PBL from a patient with breast cancer. PBL from patient BC36 once stimulated Bcl-XL173-182in vitro and before analysis was isolated cells are CD8+. FACS staining of culture using an and-CD8 antibodies and pentamers of the complex of HLA-A2/Bcl-X L173-182revealed that 95.5% of the cells were CD8-positive and 0.24% of them were positive in respect of pentamera (A). Pentamer HLA-A2/HIV was used as negative control (B). The cell culture was additionally analyzed using ELISPOT (C).

Fig. 9 illustrates restricted by HLA-A2 T-cell responses against Bcl-XLmeasured in ELISPOT analysis INF-γ. Analyzed PBL from twelve healthy person, eighteen patients with breast cancer (BC patients), six patients with melanoma and two patients with pancreatic cancer (PC-patients). All the people were HLA-A2 positive. Investigated peptides Bcl-XL118-12(TAYQSFEQV) (SEQ ID NO: 43) (A) and Bcl-XL169-178(WMATYLNDHL) (SEQ ID NO: 46) (B). T-lymphocytes once stimulated by the peptide before sowing 105cells per well in three repetitions either in the absence or in the presence of an appropriate peptide. The average number of specific peptide spots (after subtracting spots without added peptide) was calculated for each patient using the analyzer ImmunoSpot® series 2.0 (CTL Analyzers, LLC, Cleveland, US). Responsible individuals (defined by the average number of antigen-specific spots ± ½ standard deviation > 25 105lymphocytes) are marked with black squares, while not meeting the individuals marked with white squares.

Fig. 10 illustrates restricted by HLA-A3 T-cell is occurred against Bcl-X Lmeasured in ELISPOT analysis INF-γ. T-lymphocytes once stimulated by the peptide before sowing 105cells per well in three repetitions either in the absence or in the presence of peptide Bcl-XL165-173(RIAAWMATY) (SEQ ID NO: 50). Analyzed PBL from seven healthy people, five patients with breast cancer, four patients with melanoma, two patients with pancreatic cancer and five patients with multiple myeloma. All the people were HLA-A3-positive. The average number of specific peptide spots (after subtracting spots without added peptide) was calculated for each patient using the analyzer ImmunoSpot® series 2.0 (CTL Analyzers, LLC, Cleveland, US).

Fig. 11 illustrates restricted by HLA-A3 T-cell responses against Mcl-1, measured in the ELISPOT analysis INF-γ. T-lymphocytes once stimulated by the peptide before sowing 3 x 105cells per well in three repetitions either in the absence or in the presence of peptide. Researched PBL from ten healthy people, six patients with breast cancer (BC), two patients with pancreatic cancer (PC) and six patients with CLL against peptide Mcl-195-103(left) and peptide Mcl-1300-308(on the right). All the people were HLA-A3-positive. The average number of specific peptide spots (after subtracting spots without added peptide) was calculated for each patient using the analyzer ImmunoSpot® series 20 (CTL Analyzers, LLC, Cleveland, US). Responsible individuals (defined by the average number of antigen-specific spots ± ½ standard deviation > 25 105lymphocytes) are marked with black squares, while not meeting the individuals marked with white squares.

Fig. 12 illustrates restricted by HLA-A1 T-cell responses against Mcl-1, measured in the ELISPOT analysis INF-γ. T-lymphocytes once stimulated by the peptide before sowing 3 x 105cells per well in three repetitions either in the absence or in the presence of peptide Mcl-1166-175or Mcl-1177-185. Researched PBL from six healthy individuals, four patients with breast cancer (BC) and seven patients with melanoma against peptide Mcl-195-103(left) and peptide Mcl-1300-308(on the right). All the people were HLA-A1-positive. The average number of specific peptide spots (after subtracting spots without added peptide) was calculated for each patient using the analyzer ImmunoSpot® series 2.0 (CTL Analyzers, LLC, Cleveland, US). Responsible individuals (defined by the average number of antigen-specific spots ± ½ standard deviation > 25 105lymphocytes) are marked with black squares, while not meeting the individuals marked with white squares.

EXAMPLES

Example 1

Immune responses against Bcl-2 in patients with breast cancer.

Materials and methods

1. Patients

Peripheral blood lymphocytes (PBL) were collected from patients with breast cancer. PBL were isolated using Lymphoprep separation, were identified by HLA (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark) and frozen in FCS with 10% DMSO. None of the patients had not received immunotherapy before sampling of blood.

2. Analysis of the Assembly in relation to the binding of peptide to MHC molecules of class I

The binding affinity of synthetic peptides (Invitrogen, Carlsbad, CA, USA) with molecules HLA-A2, metabolically labeled with [35S]-methionine was measured in the analysis of assemblies, which are described previously. The analysis is based on mediated peptide stabilization "empty" HLA molecules released by lysis of the cells of the deficit on the TAP cell line T2. HLA-molecules with stable stacking was immunoprecipitated using specific for HLA class I conformation-dependent mate W6/32, and separated by electrophoresis in gel isoelectric focusing (IEF). Strip the heavy chain MHC quantitatively evaluated using the program for visualization device luminescence ImageGauge (FUJI photo film Co., Carrollton, TX, USA). The intensity of the band is in direct proportion to the number associated with the peptide complex of MHC class I extracted during the analysis. Then the degree of stabilization of HLA-A2 is directly connected with the binding affinity of the added peptide. Removing HLA-A2 and the measure, in the presence of 50, 5, 0.5, and 0.05 µm of the corresponding peptide. The value of C50was calculated for each peptide in the form of a concentration of a peptide sufficient to premaxillae stabilization.

3. Antigenic stimulation of PBL

To increase the sensitivity of the ELISPOT analysis, PBL once stimulated in vitro prior to analysis. 0-day PBL or crushed lymph nodes were thawed and were sown in 2 ml/well at a concentration of 2 x 106cells in 24-well plates (Nunc, Denmark) in X-vivo (Bio Whittaker, Walkersville, Maryland), 5% inactivated by heating human serum and 2 mm L-glutamine in the presence of 10 μm of peptide. Two days later the cultures were added with 20 units/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany). Cultured cells were tested in respect of reactivity in the ELISPOT at day 12.

4. Analysis of the ELISPOT

The ELISPOT analysis was used to quantify effector cells, releasing it is specific to the peptide epitope of interferon-γ, as described previously (4). Briefly, 96-well tablets with nitrocellulose bottom (MultiScreen MAIP N45, Millipore, Hedehusene, Denmark) were coated with anti-IFN-γ antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked by X-vivo and was added to the cells two times with two different concentrations of cells. Then to each well was added peptides and the plates were incubated over night. The next day, the medium was discarded and l the NCI washed before the addition of biotinylated second antibody (7-B6-1-Biotin, Mabtech). The plates were incubated for 2 hours, washed, and to each well was added conjugate avidin-enzyme (AP-Avidin, Calbiochem, Life Technologies). The plates were incubated at room temperature for 1 hour and to each well was added the enzyme's substrate NBT/BCIP (Gibco, Life Technologies) and incubated at room temperature for 5-10 minutes the Reaction was stopped by washing with tap water when dark purple spots. Spots were counted using an ImmunoSpot analyzer® series 2.0 (CTL Analyzers, LLC, Cleveland, US), and frequency specific CTL peptide could be calculated based on the number of cells forming the spot. All analyses were carried out in three repetitions for each peptide antigen.

5. Results

Linking derived from Bcl-2 peptides with HLA-A2

Organized the screening of the amino acid sequence of the protein Bcl-2 in the case of most possible Nona - and decameric peptide epitopes HLA-A2, using the master it is specific to the HLA-A2 anchor residues (2). Synthesized thirteen originating from Bcl-2 peptides and examined in relation to binding to HLA-A2 compared to the positive high-affinity relative to HLA-A2 control epitope of pol476-484HIV-1 (ILKEPVHGV) (SEQ ID NO: 39) through the analysis of the Assembly. The Assembly analysis is based on the stabilization of molecules of class I after load different concentration of the mi peptide deficient in TAP cell line T2. Then subjected to the correct installation of stable heavy chain MHC was immunoprecipitated using conformation-dependent antibodies. The degree of stabilization of the MHC molecules of class I is directly dependent on the affinity of binding of the added peptide, as shown in Fig. 1. The concentration of peptide required for premaxillae extract molecules MHC class I (the value of C50), was 0.7 μm for pol476-484HIV-1 (table 2). Eight derived from Bcl-2 peptides was associated with almost the same high affinity, as a positive control; Bcl224, Bcl85, Bcl222, Bcl218, Bcl220, Bcl214, Bcl124and Bcl172(C50= 0,7, 1, 1, 2, 1, 3, 1 2 mcm respectively) (table 2). Peptides Bcl80, Bcl208and Bcl180been associated only with intermediate or weak affinity (C50= 36,7 and 20 μm, respectively. Two of the investigated peptides (Bcl216, Bcl200) was not associated with HLA-A2. The list of peptides included in this study are shown in table 2:

Table 2. The peptides examined in this study

aThe number of values listed in the form of a subscript indicates the position of the first amino acid in the sequence

bThe value of C50indicates the concentration of peptide needed the th for premaxillae binding HLA-A2.

CTL responses against peptides derived from Bcl-2, subjected to chemotherapy in patients with breast cancer

Using the analysis of the secretion of INF-γ ELISPOT, the authors investigated the presence of specific T-cell responses against derived from Bcl-2 peptides to T-cells in peripheral blood from patients with breast cancer. This method has previously been highly effective in the identification of specific tumor CTL in patients with malignant tumor.

PBL from 15 positive for HLA-A2 patients with breast cancer once stimulated in vitro prior study by ELISPOT analysis. This method was selected in order to increase the sensitivity of the ELISPOT as described in (4). As many described CTL epitopes are actually low-affinity peptides, the authors have included all thirteen calculated on the basis of Bcl-2 peptides in the first stage of the experiments. Identified individuals with responses against Bcl172, Bcl180, Bcl208and Bcl214and the data only for these peptides are shown in Fig. 2. Revealed spontaneous CTL responses against Bcl172in PBL from eight patients (50%) and against Bcl180four patients (≈25%) (Fig. 2). However, the most frequent responses were detected against Bcl208and Bcl214because twelve (≈80%) of patients had registered CTL-response against Bcl208and eleven patients (≈75%) had Bcl 214response (Fig. 2).

Example 2

The immunogenicity of Bcl-2 in patients with malignant tumor

The essence

In this example, the authors describe the spontaneous T-cell reactivity against Bcl-2 in peripheral blood of patients suffering as a result of tumors unrelated types, i.e. pancreatic cancer, AML and CLL. In addition, the authors show that these Bcl-2-reactive T cells are indeed specific to the peptide cytotoxic effector cells. Thus, Bcl-2 can serve as an important and widely applicable target for ways antitumor immunotherapy, for example, in combination with conventional radiotherapy and chemotherapy.

Introduction

The family of Bcl-2 includes several key artists involved in the regulation of apoptosis, and includes both proapoptotic and protivoponosnye molecules. Bcl-2 is an important cellular factor contributing to the pathogenesis and progression of malignant tumors. In this study the authors investigated the natural cellular immunogenicity of Bcl-2 in patients with malignant tumor.

Methods

Patients

PBL were isolated using Lymphoprep separation, were identified by HLA (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark) and frozen in FCS with 10% DMSO. None of the patients do not floor the al immunotherapy before sampling of blood. Patients received consent on the basis of the information provided before any of the specified measurements. Peripheral blood lymphocytes (PBL) were collected from thirteen positive for HLA-A2 patients with breast cancer, representing progressive disease with distant metastases, with a certain disease stage IV; the majority of patients had more than one tumor localization (8/13 patients). Previous treatment included chemotherapy, endocrine therapy and radiation therapy. Eight patients previously treated with chemotherapy, while five patients received only endocrine therapy and had not received chemotherapy before inclusion in the study. Also included are twelve positive for HLA-A2 patients with localized resectable malignant tumor of the breast, and blood samples were collected before primary surgery and chemotherapy. In addition, PBL were collected from two positive for HLA-A2 patients with pancreatic cancer, representing progressive disease with distant metastases, with a certain disease stage IV. Finally, PBL collected from ten HLA-A2-patients with newly diagnosed CLL and three with AML before treatment. PBL from twelve HLA-A2-positive healthy individuals served as controls.

ELISPOT of granzyme B

Analysis of the ELISPOT Grasim the B (GrB) was used to measure the cytotoxicity of antigen-specific CTL, as described. Briefly, 96-well tablets with nitrocellulose bottom (MultiScreen MAIP N45, Millipore) were coated catching GrB antibody (BD Biosciences, Brondby, Denmark). The wells were washed and blocked medium X-vivo with 5% human serum. Was added to the cells with different concentrations of cells. Then to each well was added to the cells T2 and peptides and the plates were incubated for 4 hours, the medium was discarded and the wells were washed before the addition of antibodies for the detection of GrB (BD Biosciences). The plates were incubated for 2 hours, washed, and to each well was added avidin-horseradish peroxidase (BD Biosciences). The plates were incubated at room temperature for 1 hour, each well was added a reagent - substrate AEC (BD Biosciences) and incubated at room temperature for 5-10 minutes the Reaction was stopped by washing with tap water with the appearance of red spots. Spots were counted and the frequency is specific to the CTL peptide was calculated similarly to the analysis of the ELISPOT INF-γ. All the analysis was carried out in two or three repetitions for each peptide antigen.

The selection is specific to the peptide T-cell

Antigen-specific cells were isolated using magnetic beads coated with Bcl208/HLA-A2, as described previously. Biotinylated monomers (Prolmmune, Oxford, UK) was connected with magnetic beads coated with streptavidin (Dynabeads M-280, Dynal A/S, Oslo, Norway), through Incubus and 2.5 ág of monomers with 5×10 6balls in 40 μl PBS for 20 min at room temperature. Magnetic complexes were washed three times in PBS in a magnetic field (Dynal A/S, Oslo, Norway) and then mixed with PBL in the ratio of 1:10 in PBS with 5% BSA and rotated very gently for 1 hour. Antigen-specific T-cells CD8+, communicating with magnetic complexes, gently washed three times. Isolated cells resuspendable many times in X-vivo with 5% HS and incubated for 2 hours, then magnetic beads are released and removed from the cell suspension. Isolated cells were cultured in 48-hole tablet in X-vivo, 5% HS and 106coated with anti-CD28, anti-CD3 artificial cell-based antigen presenting cells (K32/41BBL)that Express the ligand, 4-1BB (4-1BBL) (courtesy of Dr. Carl H. June, Department of Pathology and Laboratory Medicine, University of Pennsylvania). The day after the selection was added 20 units/ml IL-2, and on day 5 tested the ability of these cells to kill target cells in any of the standard tests release51Cr.

Cloning by limiting dilution

CTL clones were established from selected cultures way limiting dilution in 96-well tablets using irradiated PBMC as feeder cells in the presence of 40 units/ml IL-2 and 1 μg/ml PHA in X-vivo with 5% HS. Fresh medium and IL-2 was added to the clones every 3-4 days.

the analysis of cytotoxicity

Conventional analyses of the release of [51Cr] in respect mediated CTL cytotoxicity was carried out as described in other publications. Target cells were T2 cells in the presence or in the absence of peptide, HLA-A2-positive cell line breast cancer MDA-MB-231 and HLA-A2-negative cell line breast cancer ZR75-1. Both lines of breast cancer cells expressed Bcl-2, as investigated by reverse transcription-PCR (data not shown).

Results

CTL responses against derived from Bcl-2 peptides

To investigate whether Bcl-2-specific T cells in PBL from patients with leukemia, the authors investigated PBL from ten HLA-A2-positive patients with CLL and three patients with AML in relation to reactivity against two peptides bcl208and bcl214. Bcl-2 responses were observed in five patients with CLL and two patients with AML (figure 3). In addition, the authors investigated PBL from two malignant tumors of the pancreas and found that both patients had been a CTL-response against peptides bcl208and bcl214(figure 3). Similarly investigated PBL from 12 healthy HLA-A2-positive people. Unexpectedly revealed a weak CTL response against peptide bcl208one of the healthy individuals (data not shown).

Bcl-2-specific release of granzyme B in PBL

Using GrB ELISPOT, the author is investigated, whether bcl-2-specific T cells detected in PBL, the cytotoxic function. Accordingly PBL from three bcl-2-reactive patients with breast cancer (patients No: 19, 20, and 22) were analyzed in relation to the reactivity against two epitopes bcl208and bcl214(figure 4). All three patients responses against both peptides can be identified with a frequency of approximately 50-140-specific CTL peptide at 105PBL. As a control, the authors have included patient (patient No. 16), in which the authors were able to identify only answer against bcl172but not against bcl208and bcl214in the analysis of the ELISPOT INF-γ, and control in the form of a healthy person (h1). As expected, not revealed the release of GrB against bcl208or bcl214no patient with breast cancer No. 16, or in the case of healthy control.

The functional ability of the Bcl-2-reactive CTL

To further characterize the functional ability of the Bcl-2-reactive CTL were enriched in these cells using magnetic beads coated complexes of HLA-A2/bcl208as described. Cells once stimulated with peptide in vitro prior to extraction. A small part of the selected cells were cloned by limiting dilution. Breeding culture explored in relation to the recognition of T2 cells either in the absence of peptide, or a pulse treatment is tannich bcl 208in GrB ELISPOT. Several of the obtained clones showed specific recognition pulse processed

bcl208the T2 cells (data not shown). However, unfortunately, the authors could not reproduce these clones for further analysis.

The day after allocation to the remaining cells were added to IL-2 and on day 5 tested the ability of cells to kill the loaded peptide T2 cells in the standard analysis release51Cr. With this purpose, or non-loaded T2 cells or T2 cells loaded with peptide bcl208served as targets. The specified analysis revealed that were killed cells T2, the pulse-treated bcl208(figa). These enriched and stimulated in vitro bcl208-reactive T-cells are then used to test the ability to kill HLA-A2-positive expressing Bcl-2 breast cancer cell line MDA-MB-231. Enriched T cells were effectively literally cells MDA-MB-231, while in contrast, were not observed cytotoxicity directed against expressing Bcl-2, HLA-A2-negative breast cancer cell line ZR75-1 (fig.5b).

Example 3

The immunogenicity of Bcl-X(L) in patients with malignant tumor

The essence

In this example, the authors show that Bcl-XLis a target for recognition by T-cells in patients with malignant the second tumor. Accordingly, the authors describe spontaneous restricted by HLA-A2 and HLA-A3 cytotoxic T-cell responses against peptide epitopes derived from Bcl-XLusing ELISPOT and staining with flow cytometry. Thus, the cellular immune responses against inhibitors of apoptosis, similar to the proteins of Bcl-2 seems to represent a General phenomenon in malignant tumors, and therefore, this group of proteins is an attractive universal protein targets for antitumor immunotherapy. In addition, because increased expression of these proteins in cells correlates with drug resistance, the combination of immunotherapy with cytotoxic chemotherapy is a very attractive method for the treatment of malignant tumors.

Introduction

Protivopotochny protein Bcl-XLis produced from the long form of alternative splicing of the gene bcl-x, whereas proapoptotic Bcl-XScomes from a short form of alternative splicing of the same gene. Bcl-XLplays an important role in malignant tumors, as it was directly linked with resistance to conventional forms of therapy and poor prognosis. Functional inhibition of Bcl-XLrestores the apoptotic process and makes the neoplastic cells sensitive the chemical and radiation therapy, while the treatment of malignant cell lines so that they expressed high levels of Bcl-XLthat leads to the phenotype of multidrug resistance. It was reported enhanced expression of Bcl-XLin many different malignancies, including AML and multiple myeloma, and solid malignant tumors such as cancer of the bladder, breast cancer, pancreas cancer and melanoma.

The ideal targets for immunotherapy are the products of genes silent in normal tissues, sverkhekspressiya in malignant cells and directly involved in the viability of malignant cells and the progression.

Materials and methods

Patients

Peripheral blood lymphocytes (PBL) were collected from patients suffering from malignant tumors of different origin, and from healthy individuals as control, and were isolated using Lymphoprep separation, were identified by HLA (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark) and frozen in FCS with 10% DMSO. None of the patients had not received immunotherapy before sampling of blood. Patients received consent on the basis of the information provided before any of the specified measurements.

Flow cytometry (FACS)

PBL from a patient with breast cancer once stimulirovalo vitro appropriate peptide and on the seventh day was isolated cells CD8+ PBL, using CD8-negative set to highlight Dynal (Dynal Biotech ASA, Oslo, Norway). The resulting CD8-positive culture T-cells were dyed associated with PE pentamerone Pro5 MHCTM(Prolmmune, Oxford, UK) followed by staining with antibodies using related fluorochroman Mat: CD8-APC and CD3-FITZ (Becton Dickinson, Immunocytometry Systems, San Jose, CA). Both staining was performed in PBS + 2% FCS for 30 min at 4°C in the dark. Used complexes of pentameron Pro5 MHCTM: HLA-A2/Bcl-XL173-182(YLNDHLEPWI) (SEQ ID NO: 42) and HLA-A2/pol476-484HIV-1 (ILKEPVHGV) (SEQ ID NO: 39). The samples were analyzed in a BD FACS using a computer program DIVA (BD, San Jose, CA).

Results

Spontaneous CTL responses against derived from Bcl-XLpeptides

Gene bcl-x transcribed in two mRNA through alternative splicing. Protivopotochny protein Bcl-XLis produced from the long form of alternative splicing, whereas proapoptotic Bcl-XScomes from a short form of alternative splicing of this gene. The protein product of the larger BCL-XLdiffers from protein Bcl-XSregion insertions (amino acids 126-188). Thus, to determine whether Bcl-XLnatural target for T-cells in patients with malignant tumors, the authors have thoroughly researched this area insertions (including nine amino acids at each end) from which Oseni prospective HLA-A2 epitopes, using basic specific for HLA-A2 anchor residues. The authors then synthesized seven calculated on the basis of Bcl-XLpeptides (Bcl-XL158-166(EMQVLVSRI) (SEQ ID NO: 44), Bcl-XL118-126(TAYQSFEQV) (SEQ ID NO: 43), Bcl-XL173-182(YLNDHLEPWI) (SEQ ID NO: 42), Bcl-XL165-174(RIAAWMATYL) (SEQ ID NO: 45), Bcl-XL169-178(WMATYLNDHL) (SEQ ID NO: 46), Bcl-XL161-170(VLVSRIAAWM) (SEQ ID NO: 48), Bcl-XL141-150(VAFFSFGGAL) (SEQ ID NO: 49)) and thoroughly researched PBL from patients HLA-A2+ with malignant tumors of different origin using the ELISPOT against these peptides. Previously it was shown that this method is highly effective for the identification of tumor-specific CTL in patients with malignant tumor. Indeed, strong and has a high frequency of CTL responses were detected against four of the investigated peptides (Bcl-XL173-182, Bcl-XL141-150, Bcl-XL161-170and Bcl-XL165-174in patients with malignant tumors of different origin (6). In total, fifteen of the eighteen patients HLA-A2+ breast cancer occurred immune response against at least one of these four peptides Bcl-XL(responsible individuals was determined by the average number of antigen-specific cells ± ½ standard deviation > 25 105lymphocytes). Similarly, four of the six patients with melanoma and one of the two investigated patsie the tov with pancreatic cancer had an immune response against at least one of these four peptides. Thus, nine of the eighteen studied patients with breast cancer had an immune response against Bcl-XL173-182, while two of the six patients with melanoma HLA-A2+ had an immune response against this peptide (figa). Four of the eighteen studied patients with breast cancer had an immune response against Bcl-XL141-150at the same time , the authors showed responses in PBL from one of the two studied patients with pancreatic cancer. The authors were unable to detect a response in PBL from any of the five investigated patients with melanoma against this peptide (fig.6b). Similarly, the authors found the answer in PBL from six patients with breast cancer and one investigated a patient with cancer of the pancreas against Bcl-XL161-170(figs). Finally, four patients with breast cancer, two patients with melanoma and one patient with pancreatic cancer had the answer against Bcl-XL165-174(fig.6d). As a control were investigated PBL from 12 healthy people HLA-A2+. Importantly, there were no answers against any of the peptides of Bcl-XL173-182, Bcl-XL141-150,

Bcl-XL161-170or Bcl-XL165-174none of the healthy individuals (6).

Specific for Bcl-XLthe release of granzyme B in PBL

Using GrB ELISPOT, the authors evaluated whether Bcl-XL-specific T-the yrs, detected in PBL, the cytotoxic function. Thus, PBL from two Bcl-XL-reactive patients with breast cancer (patients No. 35 and 36) were analyzed in relation to the reactivity against Bcl-XL173-182(Fig.7). Both patients it was possible to identify responses against Bcl-XL173-182with a frequency of approximately 50-100 specific CTL peptide 3 x 105cells. As a control, the authors included patient (patient No. 17), in which the authors were able to identify only against Bcl-XL141-150but not against Bcl-XL173-182in the ELISPOT INF-γ. As expected, not revealed the release of GrB against Bcl-XL173-182in a patient with breast cancer No. 17.

FACS analyses of specific Bcl-XLT-cells

Spontaneous emergence of specific Bcl-XL173-182CTL in PBL from patients with breast cancer was further evaluated using FACS analysis and staining pentameron Pro5 MHCTM. PBL from a patient with breast cancer No. 36 once stimulated in vitro with peptide and was isolated CD8-positive cells. The resulting culture was stained with complex pentamera HLA-A2/BCL-X. FACS analysis showed easily detectable population positive for pentamer T-cells, components of 0.24% T-cells CD8+ (figa). For comparison, among the same T-cells CD8+ watched about 1.4% specific Bcl-XL173-182secreting IFN-γ T-cell D8+ when parsing using ELISPOT (figs).

Additional restricted by HLA-A2 epitopes Bcl-X(L)

The authors carefully examined PBL from patients HLA-A2+ with malignant tumors of different origin using the ELISPOT against Bcl-XL118-126(TAYQSFEQV) (SEQ ID NO: 43) (figa) and Bcl-XL169-178(WMATYLNDHL) (SEQ ID NO: 46) (fig.9b), identifying weak spontaneous CTL response in patients with malignant tumors of different origin against both peptides.

Restricted by HLA-A3 responses against Bcl-X(L)

In addition, the authors carefully examined the scope insertions (including nine amino acids at each end) in respect of prospective HLA-A3 epitopes, using the master HLA-A3-specific anchor residues. The authors then synthesized two peptides: Bcl-XL165-173(RIAAWMATY) (SEQ ID NO: 50) and Bcl-XL149-157(ALCVESVDK) (SEQ ID NO: 51). The authors then examined carefully the PBL from patients HLA-A3+ with malignant tumors of different origin using the ELISPOT against peptide Bcl-XL165-173(RIAAWMATY) (SEQ ID NO:50) and Bcl-XL149-157(ALCVESVDK) (SEQ ID NO: 51). Previously it was shown that this method is highly effective for the identification of specific tumor CTL in patients with malignant tumor. Indeed, strong and has a high frequency of CTL responses were detected against Bcl-XL165-173(RIAAWMATY) (SEQ ID N0: 50) in patients with malignant tumors of different origin. The authors were able to identify the answer is Rotel Bcl-X L165-173in PBL HLA-A3+ in four of the five studied patients with breast cancer (responsible individuals is determined by the average number of antigen-specific cells ± ½ standard deviation > 25 105lymphocytes), in four out of four investigated patients with melanoma, two of the two investigated patients with pancreatic cancer, and one out of four investigated patients with multiple myeloma (figure 10). It is important that the authors could not identify the answer is none of the seven healthy people HLA-A3+, which were studied as controls (figure 10).

Example 4

The immunogenicity of Mcl-1 in patients with malignant tumor

The essence

In this example, the authors show that Mcl-1 is a target for recognition by T-cells in patients with malignant tumor. Accordingly, the authors describe spontaneous restricted by HLA-A1 and HLA-A3 cytotoxic T-cell responses against peptide epitopes derived from Mcl-1, using ELISPOT.

Introduction

Myeloid cell factor-1 (Mcl-1) is inhibiting the death of the representative of the family of Bcl-2, which is expressed in early differentiation of monocytes and may increase the viability for transfection in immature myeloid cells. Mcl-1 transgenic mice stimulates survival of several types of hematopoetic the cell and the immortality of myeloid cells. It was reported elevated levels of Mcl-1 in several human malignant tumors, including patients with prostate cancer, pancreatic cancer, melanoma, breast cancer, ovarian cancer and cervical cancer, as well as with B-cell chronic lymphocytic leukemia (B-CLL) and AML and ALL at relapse. In patients with B-CLL high levels of Mcl-1 is strictly correlated with the inability to achieve complete remission after therapy with one drug. In multiple myeloma Mcl-1 plays an important role in the viability of malignant cells. In this regard, it is shown that in mice expressing the transgene, mcl-1 under the control of its own promoter, with a high incidence of developing B-cell neoplasia in the range from follicular lymphoma to diffuse both lymphoma.

Restricted by HLA-A3 responses against Ml-1

To determine whether Mcl-1 natural target for T-cells in patients with malignant tumors, the authors investigated the protein sequence against most possible Nona - and decameric peptide epitopes HLA-A3, using the master HLA-A3-specific anchor residues. Then the authors have synthesized six calculated on the basis of Mcl-1 peptides (Mc1-1185-194(YLREQATGAK) (SEQ ID NO: 52), Mcl-1293-302(SITDVLVRTK) (SEQ ID NO: 53), Mcl-1267-276(LISFGAFVAK) (SEQ ID NO: 54), Mcl-195-103(RLLFFAPTR) (SEQ ID NO: 55), Mcl-1300-308(RTKRWLVK) (SEQ ID NO: 56), Mcl-1236-244(DIKNEDDVK) (SEQ ID NO: 57)) and thoroughly researched PBL from patients HLA-A3+ with malignant tumors of different origin in respect of reactivity against these peptides, giving advantage to ELISPOT analysis. Previously it was shown that this method is highly effective for the identification of specific tumor CTL in patients with malignant tumor. Indeed, strong and has a high frequency of CTL responses revealed against two derived from Mcl-1 peptides in patients with malignant tumors of different origin (Mcl-195-103and Mcl-1300-303) (11). In total, five of the six patients HLA-A3+ with breast cancer had an immune response against one of these two peptides Mcl-1. Accordingly, five patients with breast cancer had a response against Mcl-195-103(responsible individuals is determined by the average number of antigen-specific cells ± ½ standard deviation > 25 105lymphocytes) and three patients had a response against Mcl-1300-308(figure 11). Furthermore, two of the two studied patients HLA-A3+ with pancreatic cancer had an immune response against peptide Mcl-195-103while one of them also reacted against Mcl-1300-308. In addition, the authors investigated PBL from six patients suffering from B-CLL, and identified the CTE is against Mcl-1 95-103two of these patients. As a control were investigated PBL from 10 healthy people HLA-A3+. Importantly, there were no answers or against peptide Mcl-195-103or against peptide Mcl-1300-308none of the healthy donors (11). Similarly, there were no answers against any of the additional four derived from Mcl-1 peptides none of the patients with malignant tumors and not in healthy donors in the control (data not shown).

Restricted by HLA-A1 answers against Mcl-1

To determine whether Mcl-1 natural target for T-cells in patients with malignant tumors, the authors investigated the protein sequence against most possible Nona - and decameric peptide epitope HLA-A1, using the master HLA-A1-specific anchor residues. Then the authors have synthesized four calculated on the basis of Mcl-1 peptide (Mcl-1166-175(PAEEEEDDLY) (SEQ ID NO: 58), Mcl-1121-129(SPEEELDGY) (SEQ ID NO: 59), Mcl-1177-185(QSLEIISRY) (SEQ ID NO: 60), Mcl-1339-347(AGVGAGLAY) (SEQ ID NO: 61)) and thoroughly researched PBL from patients HLA-A1+ with malignant tumors of different origin in respect of reactivity against these peptides, giving advantage to ELISPOT analysis. Indeed, CTL responses were detected against two derived from Mcl-1 peptides in patients with malignant tumors of different origin (Mcl-1166-175 177-185) (Fig). And overall, three out of four investigated patients HLA-A1+ with breast cancer had an immune response against Mc1-1177-185and one of them, moreover, had the answer against Mcl-1166-175(Fig). In addition, one of the seven patients with melanoma had an immune response against peptide Mcl-1177-185and the other of them had the answer against Mcl-1166-175. As a control were investigated PBL from six healthy people HLA-A1+. Importantly, there were no answers or against peptide Mc1-1166-175or against peptide Mcl-1177-185none of the healthy donors (Fig).

Responses to modified peptides

Immunogenicity restricted by HLA-A3 peptide Mcl-1300-308increased substitution of threonine at position 2 more good anchor residue of HLA-A3, namely leucine (Mcl-1300-308L2 (RLKRDWLVK) (SEQ ID NO: 62)). Spontaneous immune responses revealed two patients with breast cancer against Mcl-1300-308L2 (data not shown). Similarly, to create a more immunogenic epitope, the authors modified restricted by HLA-A1 peptide Mcl-1177-185(QSLEIISRY)(SEQ ID NO: 60) in position 3, creating two peptide

Mcl-1177-185D3 (QSDEIISRY) (SEQ ID NO: 63) and Mcl-1177-185E3 (QSEEIISRY) (SEQ ID NO: 64).

Discussion

Almost all malignant neoplasms are characterized by defects in the signaling of apoptosis. This makes cancer cells resist ntime to endogenous apoptotic stimuli, as well as to exogenous stimuli, such as chemotherapeutic drugs and irradiation. Defective apoptosis observed in malignant human tumors, often caused by overexpression protivopolozhnyh protein family proteins Bcl-2, i.e. Bcl-2, Bcl-XLand Mcl-1, Bcl-w, Bfl-1A1, Bcl-b and Bcl2-L-10. The use of inhibitors such apoptotic proteins for vaccination has the advantage due to down-regulation or absence of expression of these proteins, so some form of escape from immune surveillance can disrupt the steady growth of the tumor, as for the survival of tumor cells requires functionally active members of the family of Bcl-2. For methods of treatment, purposefully affecting antigens, which play an important role in the growth and viability of tumor cells, selection deficient antigen tumors is a well-known limitation. In addition, because increased expression of proteins of Bcl-2 in cells correlates with drug resistance, combination based on the family of Bcl-2 immunotherapy with cytotoxic chemotherapy is a very attractive method for the treatment of malignant tumors.

The authors scanned proteins Bcl-2, Bcl-X(L) and Mcl-1 in the presence of a binding peptide motifs and used them to search for the specific T-cell responses in patients with malignant tumor. This purpose was revealed spontaneous T-cell reactivity against all representatives of the family of Bcl-2 in patients suffering from tumors unrelated types, i.e. pancreatic cancer, breast cancer, melanoma, AML and CLL, with ELISPOT. The presence of specific Bcl-XLcells CD8+ PBL from patients with malignant tumors was confirmed colourings CD8/pentameron when FACS-analysis. The obtained data, taken together, show that CTL-specific epitopes of these proteins can be widely applicable for therapeutic vaccination against malignant tumors and are therefore important immunotherapy value.

In addition, eleven patients with breast cancer had a Bcl-2-specific CTL, eight of these patients had previously received treatment for at least one type of chemotherapy. Two patients (patients No. 14 and 17) were not detected CTL responses to four different peptides to Bcl-2. Both patients had previously received antihormone therapy, but had not received chemotherapy. In this way, the authors could not identify any responses in patients with primary localized malignant breast cancer before chemotherapy. Thus, in patients with breast cancer Bcl-2 responses were detected only in patients who received chemotherapy. Although tumor load can game the ü important role this may indicate that the immune responses induced or increased as a consequence induced by treatment increased the expression of Bcl-2. This indicates a scenario in which the combining is based on the family of Bcl-2 immunotherapy with cytotoxic chemotherapy may synergistically improve the degree of this response. Data about the condition of the patients investigated in relation to Bcl-X(L)and Mcl-1 responses.

In this study the authors have given the advantage to the analysis of the GrB ELISPOT, to show that Bcl-2 or Bcl-X(L)-specific CTL in PBL of patients really are cytotoxic effector cells. To further confirm this view, the authors conducted enrichment Bcl-2-reactive T cells from PBL of the patient, and showed that the resulting line T-cells were able to lyse pulse processed peptide T2 cells-cells in the normal analysis release51Cr. In addition, this line of Bcl-2-reactive T-cells were able to kill HLA matching line of breast cancer cells without killing while HLA-A2-negative target cells. The data obtained show that malignant cells do ProcessInput and present peptide Bcl-2 in the context of a molecule HLA-A2. Finally, the authors were able to clone these selected cells and is provided, they highly specific reacted against the peptide epitope Bcl-2.

In the case when the peptides derived from antigens of melanocyte differentiation, first used to treat patients with stage IV melanoma, it was assumed that this could result in a significant destruction of the melanocytes, which, in turn, could manifest clinically, such as vitiligo or retinitis. However, clinical experience has shown that the frequency of occurrence of vitiligo in patients receiving vaccination was not significantly higher than the frequency associated with melanoma of hypopigmentation in patients receiving other forms of therapy. In addition, there were no reports of serious side effects when various trials of vaccination against autoantigens. These data, taken together, confirm that cellular immune responses against a group of proteins of Bcl-2 are a common feature of malignant tumors. When attempting to maximize the impact of immunotherapy attractive way could be to take into account the profile of expression and prognostic value of the selected target for a particular disease or pathological condition being treated. Thus, while in some malignant tumors or specific disease observed joint e is xpressia Bcl-2, Mcl-1 and Bcl-XLin other malignant tumors observed the expression of only one or the other protein. Thus, in some diseases, such as cancer of the ovary, the expression of Mcl-1, but not the expression of Bcl-2, is associated with advanced stage and poor survival, and for this reason, Mcl-1 may be the primary antigen, whereas for diseases such as CLL, in which Bcl-2 and Mcl-1 together sverkhekspressiya, simultaneous interventions at both the protein may be a more effective way than purposeful exposure to any one molecule. Similarly Tanaka et al. he described that the presence of another protein inhibitor of apoptosis - survivin in carcinoma of the breast is strictly linked with the expression of Bcl-2 and low apoptotic index (AI) and poor overall survival. A similar relationship between survivin and Bcl-2 are described in neuroblastoma, cancer stomach cancer colorectal and non-Hodgkin's lymphoma high malignancy. The safety and potential efficacy obtained from survivin peptides for therapeutic vaccination against malignant tumors is currently under investigation in phase I/II clinical trials (J. Becker, personal communication). Thus, attractive immunotherapy method can be purposeful impact as asemasta proteins Bcl-2, and survivin, especially as they perform their protivopoloznuju function through different cellular pathways.

Example 5

Peptide vaccine

Peptides family proteins Bcl-2 can be, for example, synthesized in the device UVA Biomolecular Core with the free amide NH2-the end and the free acid COOH-end. Each comes in the form of liofilizirovannogo peptide, which is then pererastayut in sterile water and diluted with a solution of lactate ringer's (LR, Baxter Healthcare, Deerfield, IL) as a buffer to final concentration of 67-80% of lactate ringer in the water. Then the resulting solution is sterile filtered, placed in vials from borosilicate glass and subjected to a series of studies in relation to quality assurance, including proof of identity, sterility, General safety and purity in accordance with FDA guidelines, as defined in IND 6453. Tests on the stability of the peptides did not show any decrease in the purity or concentration of the peptide when the above-mentioned solutions of the peptides were stored at -20°C within 3 years.

In practice, patients will receive the vaccine, containing about 100 µg restricted by HLA class I peptide with limited HLA class II helper peptide or without him. Patients, for example, Vaccinium about 100 μg of peptide HLA class I in Freund separately or, for example, Vaccinium p is IMEMO 100 mcg restricted by HLA class I peptide plus 190 µg limited class II helper peptide. Expect a higher dose helper peptide to provide equimolar amount of helper and cytotoxic epitopes. In addition, patients can be vaccinated with a longer peptide containing the amino acid sequences of both peptides.

The above peptides in aqueous solution in a volume of 1 ml can be entered either in the form of a solution/suspension of approximately 100 ág of QS-21, or in the form of an emulsion with approximately 1 ml of the adjuvant Montanide ISA-51.

Patients subjected to immunization, for example, at day 0 and 1, 2, 3, 6, 9, and 12 month peptides plus adjuvant, using only seven immunizations. With rare exceptions, vaccination is carried out in the same hand each vaccine. The peptides preferably injected subcutaneously.

LINKS

1. Altieri, D. S., Marchisio, P. C., and Marchisio, C. Survivin apoptosis: an interloper be-tween cell death and cell proliferation in cancer. Lab Invest 79: 1327-1333, 1999.

2. Andersen, M. H., L. Tan, I. Sondergaard, J. Zeuthen, T. Elliott. and J. S. Haurum. 2000. Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules.Tissue Antigens 55:519.

3. Reed, J. C. 1998. Bcl-2 family proteins.Oncogene 17:3225.

4. Andersen, M. H., L. O. Pedersen, J. C. Becker, and P. thor Straten. 2001. Identification of a Cytotoxic T Lymphocyte Response to the Apoptose Inhibitor Protein Survivin in Cancer Patients.Cancer Res. 61:869.

5. Thumer, C., Roder, C., Dieckmann, D., Heuer, M., Kruse, M., Glaser, A., Keikavoussi, P., Kampgen, E., Bender, A., and Schuler, G. (1999) Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. J.Immunol.Methods 223, 1.

6. Shangary, S. and Johnson, D.E. (2003). Recent advances in te development of anticancer agents targeting cell death inhibitors in the Bcl-2 protein family. Leukemia17:1470-1482.

7. Rosenberg S.A. and M.E. Dudley (2004). Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes.PNAS101:14639-14645.

1. The vaccine composition containing an isolated immunogen active peptide of no more than 15 amino acid residues, obtained from a family member proteins Bcl-2, where the specified peptide contains a sequence selected from the group consisting of SEQ ID NO: 6, 8,10, 11, 42, 43, 45, 46, 48, 49, 50, 55, 56, 58, 60 and 62, for use as a drug for the treatment of malignant tumors expressing family proteins Bcl-2.

2. The composition according to claim 1, which when administered to a patient with a malignant tumor is able to induce an immune response against malignant diseases.

3. The composition according to claim 1, which when administered to a patient with a malignant tumor, in what toroi is expressed member of a family of proteins Bcl-2, able to induce an immune response against malignant diseases.

4. The vaccine composition according to claim 3, in which the protein is Bcl-2.

5. The vaccine composition according to claim 3, in which the protein is Bcl-XL.

6. The vaccine composition according to claim 3, in which the protein is Mcl-1.

7. Isolated immunogen active peptide fragment of no more than 15 amino acid residues, obtained from a family member proteins Bcl-2, where the specified peptide contains a sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 11, 42, 43, 45, 46, 48, 49, 50, 55, 56, 58, 60 and 62, for use as a drug for the treatment of malignant tumors expressing a protein member of the protein family of Bcl-2.

8. Peptide fragment according to claim 7, in which the protein is Bcl-2.

9. Peptide fragment according to claim 7, in which the protein is Bcl-XL.

10. Peptide fragment according to claim 7, in which the protein is Mcl-1.

11. Peptide fragment according to any one of claims 7 to 10, which can cause cellular immune response in patients with malignant tumor.

12. Peptide fragment according to any one of claims 7 to 10, which is a peptide, a limited MHC class I with at least one of the following characteristic features:
(i) capable of contacting molecule HLA class I, which he limited to, affinity, measured by the number of peptide that sposobnosti premaxillae removing molecules HLA-class (value 50)constituting not more than 50 μm, based on the definitions described here, the analysis of binding in the Assembly,
(ii) can cause cells producing INF-γ, in a population of peripheral blood lymphocytes (PBL) of a patient with a malignant tumour, with a frequency of at least 1 in 104PBL, which is determined by the ELISPOT analysis, and/or
(iii) capable of detecting in situ in the tumor tissue of cytotoxic lymphocytes (CTL)that are reactive against a peptide epitope.

13. Peptide fragment according to item 12, which has the value50that is not more than 30 μm.

14. Peptide fragment according to item 12, having a value of C50that is not more than 20 μm.

15. Peptide fragment according to item 12, limited molecule HLA-A class I MHC.

16. Peptide fragment according to item 15, the limited type of HLA class I MHC selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24.

17. The peptide fragment of claim,16, which is restricted to HLA-A2.

18. Peptide fragment according to any one of claims 7 to 17, which contains a sequence selected from the group consisting of WLSLKTLLSL (SEQ ID NO: 6), PLFDFSWLSL (SEQ ID NO: 8), YLNRHLHTWI (SEQ ID NO: 10) and NIALWMTEYL (SEQ ID NO: 11).

19. Peptide fragment according to any one of claims 7 to 17, where the peptide contains a sequence selected from the group consisting of TAYQSFEQV (SEQ ID NO: 43), YLNDHLEPWI (SEQ ID NO: 42), RIAAWMATYL (SEQ ID NO: 45), WMATYLNDHL (SEQ ID NO: 46), VLVSRIAAWM (SEQ ID NO: 48) and VAFFSFGGAL (SEQ ID O: 49).

20. Peptide fragment according to any one of claims 7 to 17, where the peptide contains the sequence RIAAWMATY (SEQ ID NO: 50).

21. Peptide fragment according to any one of claims 7 to 17, where the peptide contains a sequence selected from the group consisting of RLLFFAPTR (SEQ ID NO: 55) and RTKRDWLVK (SEQ ID NO: 56).

22. Peptide fragment according to any one of claims 7 to 17, where the peptide contains a sequence selected from the group consisting of PAEEEEDDLY (SEQ ID NO: 58) and QSLEIISRY (SEQ ID NO: 60).

23. Peptide fragment according to any one of claims 7 to 17, where the peptide is selected from the group consisting of RLKRDWLVK (SEQ ID NO: 62).

24. Peptide fragment according to any one of claims 7 to 17, which is nonapeptides or decapeptides.

25. Protein or peptide fragment according to any one of claims 7 to 24, which is the native sequence, isolated or obtained from a mammal.

26. Protein or peptide fragment according to any one of claims 7 to 24, which is the native sequence, isolated from the human protein.

27. Peptide fragment according to any one of claims 7 to 26, which is derived from the native sequence of a member of a family of proteins Bcl-2 by substitution, deletion or addition of at least one amino acid residue.

28. Peptide fragment according to any one of claims 7 to 27, which can cause cells producing INF-γ, in a PBL population of a patient with a malignant tumour with a frequency of at least 10 to 104PBL.

29. Peptide f is agent according to any one of claims 7 to 28, which can cause cells producing INF-γ, in a PBL population of a patient having a malignant disease in which is expressed protein belonging to the family of proteins Bcl-2.

30. Peptide fragment by clause 29, in which the malignant disease is selected from the group consisting of hematopoietic malignancies, including chronic lymphocytic leukemia and chronic myelogenous leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer.

31. The vaccine composition according to claim 1, containing a peptide fragment according to any one of claims 7 to 30.

32. The vaccine composition according to claim,31, causing the person to be vaccinated patient's production of effector T cells with cytotoxic activity against malignant cells.

33. The composition of the vaccine for p, optionally containing immunogenic protein or peptide fragment selected from a protein or peptide fragment that are not relevant or is not received from the family of proteins Bcl-2.

34. The composition of the vaccine for p, where the protein or peptide fragment, not belonging to the family of proteins Bcl-2 or received from this family, is a protein involved in the regulation of cell apoptosis, or its derived peptide fragment.

35. The composition of the vaccine for p, immunogenic where the protein or peptide fragment, selected from a protein or peptide fragment, not belonging to the family of proteins Bcl-2 or received from this family is survivin or peptide fragment.

36. The composition of the vaccine for p, where the immunogenic protein or peptide fragment selected from a protein or peptide fragment, not belonging to the family of proteins Bcl-2 or received from this family, is a ML-IAP or peptide fragment.

37. The composition of the vaccine for p containing adjuvant.

38. The vaccine composition according to clause 37, where the adjuvant is selected from the group consisting of adjuvants, based on bacterial DNA adjuvants based on oil/surfactants, adjuvants on the basis of viral dnrc and imidazoquinolines.

39. The composition of the vaccine for p containing antigen cells containing peptide fragment.

40. The vaccine composition according to § 39, where the antigen presenting cell is a dendritic cell.

41. The composition of the vaccine for p containing the liposome.

42. The set containing the vaccine composition according to any one of claims 1 to 6 and 31-41 and additional anti-cancer agent.

43. Set in § 42, in which the anticancer agent is an antibody.

44. Set in § 42, in which the anticancer agent is a cytokine.

45. Composition for diagnosing ex vivo or in situ presence of the patient from placecast is authorized tumor T cells in PBL or in tumor tissue, which are reactive against a representative of a family of proteins BC1-2, and the composition comprises a peptide fragment according to any one of claims 7 to 30.

46. Diagnostic kit for the diagnosis of ex vivo or in situ presence of the patient with malignant tumor T cells in PBL or tumor cells that are reactive against a representative of a family of proteins l-2, this set contains a peptide fragment according to any one of claims 7 to 30.

47. The complex peptide fragment according to any one of claims 7 to 30 and molecules HLA class I or a fragment of such molecule for use as a diagnostic reagent.

48. Complex p, which is Monomeric.

49. Complex p,47, who is multimeric.

50. A method of detecting in a patient with a malignant tumour in the presence of T-cells reactive against the member of the protein family l-2, providing reduction of tumor tissue or blood sample into contact with the complex p and detecting binding of the complex with tissue or blood cells.

51. Molecule that can specifically bind to a peptide fragment according to any one of claims 7 to 30, where this molecule is an antibody or its fragment, or a receptor of T cells.

52. A method for the treatment of malignant disease, which is expressed member of a family of proteins l-2, providing the rst is giving to the patient, suffering from said disease an effective amount of a composition according to any one of claims 1 to 6 and 31-41 or kit according to any one of p-44.

53. The method according to paragraph 52, where the malignant disease is selected from the group consisting of hematopoietic malignancies, including chronic lymphocytic leukemia and chronic myelogenous leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer.

54. The method according to paragraph 52, which combined with additional treatment of malignant tumors.

55. The method according to item 54, where additional treatment selected from the group consisting of chemotherapy, radiation therapy, treatment immunostimulating substances, gene therapy, antibody therapy and treatment using dendritic cells.

56. The use of the peptide fragment according to any one of claims 7 to 30, or a vaccine composition according to any one of claims 1 to 6 and 31-41 in the production of pharmaceuticals for the treatment or prevention of malignant diseases, which is expressed member of a family of proteins Bcl-2.

57. Use p, according to which the malignant disease is selected from the group consisting of hematopoietic malignancies, including chronic lymphocytic leukemia and chronic myelogenous leukemia, melanoma, breast cancer, cervical cancer, RA is as ovarian, lung cancer, colon cancer, pancreas cancer and prostate cancer.

58. The application of § 57, which combined with additional treatment of malignant tumors.

59. The application of § 58, where additional treatment selected from the group consisting of chemotherapy, radiation therapy, treatment immunostimulating substances, gene therapy, antibody therapy and treatment using dendritic cells.

60. Method of monitoring the immunization peptide fragment according to any one of claims 7 to 30 or vaccine composition according to any one of claims 1 to 6 and 31-41, providing the stage
i) obtaining a blood sample from the individual,
ii) obtaining a protein belonging to the family of proteins Bcl-2, or peptide fragment,
iii) determine whether the specified sample of blood antibodies or T-cells containing the receptor of T cells, specific binding protein or peptide,
iv) determine, therefore, also raised whether the immune response to a specific protein or latido the specified individual.

61. The method according to p, where the peptide fragment is a peptide fragment according to any one of claims 7 to 30.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to paediatrics. The method of prediction of mitochondrial maladies in infants with undifferentiated forms of neuropsychic delay is ensured by cytochemical analysis of enzyme strength of biological lymphocytic metabolism in peripheral blood. Further, strength of SDH, GPDH, GDH, MDH, LDH, ratio of GPDH to SDH strength, sigmal deviation of quantitative character of enzyme strength, and age-specific ratio of GPDH to SDH strength are calculated. Or space ratio of all SDH enzyme granules, ellipse index of GPDH clusters, integrated optical density of all GDH granules, area of all small LDH granules and age-specific sigmal deviations of each enzyme index. On the basis of findings, mitochondrial cytochemical factor (MCF) is evaluated. Increase in MCF shows potential development of mitochondrial malady.

EFFECT: accessing the adequate information on energy metabolism state using minimum material required and ensured of sparing (minimally invasive) conditions of the patient's examination.

2 cl, 7 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to laboratory diagnostics, and can be used for early detection of infection-caused placental insufficiency (PI) development. To implement the method, the patient's biological fluids are analysed, including blood serum or cervical canal mucus to determine neopterin content. In case neopterin concentration exceeds 9 nmol/l in blood serum or 2 nmol/l in cervical canal mucus, placental insufficiency development is predicted women of high-risk groups with markers of urinogenital infections.

EFFECT: placental insufficiency at early pre-clinical stage of disease that enables timely medical and preventive measures that finally yields in favourable perinatal outcomes and reduced disease of newborns.

2 cl, 1 dwg, 2 tbl, 1 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to field of biochemistry, cytochemistry, physiology and clinical medicine and is intended for quantitative characteristic if energy process in mitochondria - succinic acid oxidation and its signal action. Essence of cytobiochemical method lies in the following: determination of succinate dehydrogenase (SDG) activity by measuring on blood smear of value of reduced nitro blue tetrazolium (NBT) in lymphocytes is carried out in sample complex, using composition of media, with reagent concentrations brought close to their content in cell. On the basis of said determination also claimed is method of quantitative determination of level of adrenegetic regulation in organism. In order to realise claimed method standard set of dry reagents allowing to unify measurements has been created. Said invention considerably increases sensitivity of measurement of SDG activity in lymphocytes in case of change of state in organism induced by adrenergetic regulation.

EFFECT: increasing sensitivity of measurement of SDG activity in lymphocytes in case of change of state in organism induced by adrenergetic regulation.

4 cl, 2 tbl, 11 ex, 11 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to gynecology. The way of diagnostics of an external genital endometriosis is offered. Content of the vascular-endothelial growth factor (VEGF) is defined in the peritoneal liquid received at a laparoscopy, and in blood serum and if the relation of VEGF content in blood serum to VEGF content in a peritoneal liquid is more than 4.35, presence of an external genital endometriosis is diagnosed.

EFFECT: high accuracy of diagnosing of an external genital endometriosis at patients.

3 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine area, namely to pediatrics, and can be used for diagnostics of degree of disturbance of cardiovascular system function at newborns from groups of perinatal risk. Essence of the way: peptide fraction of "N-trailer fragment (or a N-terminal fragment) of the precursor of cerebral sodium-uretic peptide" - level NT-pro-BNP is defined at newborns in the first days of a life in blood serum. Detection in blood serum of NT-pro-BNP level testifies: to 440 fmol/ml - there is no disturbance of function, from 441 to 603 fmol/ml - adaptic dysfunction, from 604 to 1636 fmol/ml - easy degree from 1636 to 2465 fmol/ml - intermediate degree, more than 2465 fmol/ml - serious degree the obtained data allow to make the clinical diagnosis with the greatest accuracy in as much as possible early terms. Application of a way allows prescribing pathogenetically caused therapy.

EFFECT: simple and effective way of differential diagnostics of organic and functional damages of cardiovascular system at newborns from group of perinatal risk.

5 ex

FIELD: medicine.

SUBSTANCE: invention concerns ophthalmology and is intended for forecasting of advance of proliferative complications at peripheric uveites at children. The way is characterised that at children with peripheric uveites the content of interleukin 2 (IL-2) in blood serum is determined, and if the level equal or above 100 pkg/ml increase of membrane creation is prognosticated in vitreous body and-or in an epiretinal manner.

EFFECT: invention provides rising of accuracy of forecasting.

4 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to oncology. The essence of the way of diagnostics of recurring hemoblastoses forecasting at children consists in the following: level of interleukin 6 in blood serum is defined at patients in the course of treatment. If in the acute period of disease level of cytokine is higher than 30 pg/ml, and at repeated research - depression to less than 12 pg/ml - recurrence-free hemoblastosis course is prognosticated. At increase of interleukin 6 from 12 to 20 pg/ml - probability of relapse is improbable, and at rising more than 20 pg/ml recurring of malignant process is prognosticated.

EFFECT: use of the way allows prognosticating duration and fastness of remission perform preclinical diagnosis of a basic disease.

2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine area, namely to oncology. Breast cancer (BC) outcome prediction method involves biochemical examination of tumour tissue for activity of aromatase, 2,4-oestrogenhydroxylase and glutathione-S-transferase. If activity of aromatase is less than 20 fmole/mg of protein per hour, activity of 2,4-oestrogenhydroxylases is more than 230 pmole/mg of protein per hour regardless activity of glutathione-B-transferase, high probability of recurrence is predicted. If activity of glutathione-S-transferase is less than 40 mkmol/mg of protein per minute without changes in other indicators, high probability of distant metastases are predicted. If activity of aromatase is more than 20 fmol/mg of protein per hour, activity of 2,4-oestrogenhydroxylases is less than 230 pmol/mg of protein per hour, activity of glutathione-S-transferase is more than 40 mkmol/mg of protein per minute, favourable clinical outcome of the disease in recurrence development and/or remote metastases is predicted.

EFFECT: high-accuracy and informative prediction of disease progressing in the form of tumour recurrences and/or metastases within first 2 years in BC patients after radical surgical treatment.

2 ex, 3 dwg

FIELD: medicine.

SUBSTANCE: invention refers to physiology and pharmaceutics. The method is ensured by preparing survival brain section with registering the initial and following parametres after incubation of the sections cuts in the medium with the analysed protein to determine its action as compared to the initial parametres and those after protein action. The method involves registration of initial bioelectric activity of the section cells and incubation of the sections in the protein medium concentrated 1 mg/ml within 20 minutes. Then protein action is stopped, while the sections are exposed to pentylenetetrazol (chemoconvulsant) in epileptogenic concentration 1-10 mkg/ml within 40 minutes. Then the sections are washed with physiologic saline, and bioelectric activity of cells is registered. The absence or presence of epileptic categories shows action of HSP-70 on protection of nervous cells in simulated epilepsy in experiment.

EFFECT: possibility for analysis HSP-70 of specific set for its activity.

2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely an otorhinolaryngology. Substance of the diagnostic technique for inflammatory destruction of paranasal and ear sinuses consists in analysis of ferritin in exudate from puncture of paranasal sinuses or middle ear lavage, as well as in blood serum. Rank score is evaluated for each ferritin indicator in exudate and blood serum. Total score 5 and more indicates destructive inflammatory process, while total 3 and less shows nondestructive inflammatory process. Application of the method allows for higher accuracy of diagnostics of inflammatory destruction of paranasal and ear sinuses.

EFFECT: higher accuracy of diagnostics of inflammatory destruction of paranasal and ear sinuses.

3 ex

FIELD: medicine.

SUBSTANCE: method involves determining glypicane-3(GPC3) quantity in blood serum, blood plasma or whole blood sample taken from organism patient having hepatopathy or belonging to hepatocellular carcinoma risk group and interpreting GPC3 availability data for diagnosing hepatocellular carcinoma cases. Antibodies are produced that show properties of specific binding to GPC3.

EFFECT: rapid noninvasive diagnosis method of improved specificity.

23 cl, 5 dwg, 4 tbl

FIELD: medicine.

SUBSTANCE: method is divided into several stages like determining one or several markers level associated with state under diagnosis; determining one or several normalization markers usable instead of information describing morphological aspects of given sample; comparing and/or joining data describing marker levels as disease sign and normalization markers; and setting diagnosis of clinically relevant state.

EFFECT: high accuracy of diagnosis.

51 cl, 3 dwg, 7 tbl

FIELD: medicine.

SUBSTANCE: illustrative compositions include one or several polypeptides of pulmonary tumor, their immunogenic parts, polynucleotides which code such polypeptides, an antigen-presenting cell that expresses such polypeptides, and T-cells being specific to cells that express such polypeptides. The compositions described could be applied, for example, during diagnostics, prophylaxis and/or treating diseases, especially pulmonary cancer.

EFFECT: increased specificity of diagnostics.

11 cl, 10 ex, 5 tbl

FIELD: medicine, oncology.

SUBSTANCE: the present innovation deals with cytological study of ascitic fluid before therapy, smears should be stained according to Felgen's technique to determine tumor ploidy. The values of gynecological status of a patient (reproductive period - 1, postmenopausal - 2), the presence (1) or absence (0) of hydrothorax, the size of residual tumor up to 2 cm (1) or above 2 cm (2) and tumor ploidy (aneuploid - 1 or diploid -2) should be applied for calculating canonic linear discriminant function (CLDF) by the following formula: CLDF=-4.35465+status code*1.28017+hydrothorax code*0.64462+residual tumor code*1.55890-ploidy code*0.65926. In case, if rating value is below discriminant value (DV=-0.94584) one should predict favorable prognosis of the disease mentioned; if this value is above DV - unfavorable prognosis. The method is simple and enables to carry out prediction at high accuracy.

EFFECT: higher efficiency of prediction.

1 dwg, 2 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: method involves determining concentrations of carcinogens like aniline, toluyline, diethyl alanine, diphenyl and 1-naphtholamine in urine and urodynamic disorders and proliferative urothelium activity from Ki-67 expression degree data. High carcinogen concentrations and urodynamic disorders being detected apart from the fact of tumor removal and unchanged mucosa biopsy, transurethral prostate resection or urinary bladder neck resection is carried out. Next, liquid consumption increase in postoperative period and carcinogen contact reduction are advised. Three months later, control study is carried out. Carcinogen concentration level remaining high and no proliferative urothelium activity reduction being observed on the background of urodynamic disorders, cystectomy is to be repeatedly carried out.

EFFECT: enhanced effectiveness of treatment; high accuracy of diagnosis.

FIELD: medicine.

SUBSTANCE: method involves studying nuclear Ki-67 or P-63 marker expression localization in epidermis and derma layers. Marker being basally localized, keratoacanthoma is to be diagnosed. Marker being diffusely localized, squamous cell skin carcinoma is to be diagnosed.

EFFECT: high accuracy of diagnosis.

4 dwg, 2 tbl

FIELD: biotechnology, peptides.

SUBSTANCE: invention relates to a method for preparing antibodies raised to human leukocyte differentiation factor (HLDF) or to HLDF fragment (31-38) representing peptide of the following structure: Arg-Arg-Trp-His-Arg-Leu-Glu-Lys possessing with antigenic and nucleic acids-hydrolyzing properties, and for diagnostic aims also. Antibodies are prepared from rabbit plasma blood immunized with three injections of antigens wherein synthetic HLDF factor or conjugate is used as antigens. Diagnosis of anaplastic state of human cells is carried out by using solutions of antibodies to HLDF factor or HLDF fragment (31-38) in the concentration 0.0013 mg/ml as biological markers. Invention provides carrying out the differential diagnosis of tumors and normal organs and effective detecting initial stages in cell differentiation disturbances.

EFFECT: improved preparing method of antibody, improved method for diagnosis.

6 cl, 21 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: binding structure is to be bound in tumor cells and/or to tumor cell surface. Target structure available and/or expressing in the tumor cells and/or on tumor cell surface. The binding structure recognizes and blocks the target structure. Substance binding to the target structure or blocks the target structure expression is described. Pharmaceutical compositions comprise the binding structure, target structure or the substance as active principle. Methods for making phage selection, and methods for making in-vitro and in vivo diagnosis and prognosis and methods for treating malignant human diseases provide for the materials usage.

EFFECT: enhanced effectiveness in treating tumor diseases.

42 cl, 14 dwg, 4 tbl

FIELD: medicinal biochemistry.

SUBSTANCE: the present innovation deals with detecting oncoprotein E7 of human papilloma virus (HPV) in biopsy sample with the help of the pairs of monoclonal antibodies referring to IgG2a and IgG2b groups chosen out of the following groups: 716-321, 716-325, 716-332, 716-343, 716-281, 716-288 one of which is indicated for primary protein binding and another, being the antibody conjugate with enzymatic label - to detect the complexes developed.

EFFECT: higher sensitivity of the method.

5 cl, 4 dwg, 4 ex, 2 tbl

FIELD: medicine, oncology.

SUBSTANCE: the suggested method deals with biochemical studying in malignant tumor and endometrial tissues, moreover, both before and after carrying out complex therapy one should detect activity of cathepsin D and acid-stable inhibitors, calculate the coefficient for the ratio of cathepsin D to acid-stable inhibitors and at coefficient values being above those characteristic for the tissue of intact endometrium by more than 2.4-2.8 times it is possible to predict relapse development for the terms of 6 mo. Thus , the present innovation enables to detect the development of pathological process before its clinical manifestation.

EFFECT: higher efficiency of prediction.

2 ex

FIELD: medicine.

SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.

EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.

4 cl, 6 dwg, 22 tbl, 19 ex

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