Recombinant plasmid dna pci-ub-polyepi containing epitopes of tumour-associated antigens for colorectal cancer and method of its application for stimulation of specific antitumour immune response against colorectal cancer cells

FIELD: biotechnologies.

SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.

EFFECT: invention allows efficient generation of antigen-specific cytocidal cell with antitumour activity in vitro, required for immune response by the 1-st type T-helper to colorectal cancer antigens.

2 cl, 1 dwg, 4 ex

 

The invention relates to biotechnology, protein engineering and medicine, specific to the creation of recombinant plasmids providing expression polyepitopic tumor-associated antigens in dendritic cells, which are able to stimulate specific T-helper and cytotoxic T-lymphocytes.

The tumor may be a wide range of antigens. Some antigens are presented in all tumors of a particular type, and some antigens are unique and can only be detected in a given patient.

There are many ways to include antigens in the antigenic composition of the vaccine.

Proteins or protein fragments of tumor cells directly injected into the body as a vaccine.

- The body is introduced genetic material encoding these proteins (DNA and RNA vaccines).

- As a "means of delivery" of the antigen in the patient's body can be used virus. The viruses used in this way, is called "viral vectors" and do not have any infectious properties. These viruses in the laboratory to infect cells of the human body and become carriers on the surface of tumor antigens. The virus is able to infect only a small number of cells is sufficient to generate an immune response, but insufficient for stabilizati disease.

Using genetic engineering techniques can also be used viruses to produce cytokines or embed the proteins in the surface of the virus, which contributes to the activation of immunocompetent cells. Thus, the modified viruses can be entered into the patient alone or in combination with a vaccine to enhance the generation of an immune response.

Sometimes as antigens in the vaccine used antibodies. The patient is given antibodies to tumor antigens, then B-lymphocytes produce antibodies to these antibodies, which recognize tumor cells. This so-called "antiidiotypic vaccine that is different from passive antibody therapy.

The described method of generating antigen-specific antitumor cytotoxic cells, taken as a prototype, in which JC was obtained from mononuclear cells (MNCs) in the peripheral blood of patients with cancer of the gastrointestinal tract by cultivation OLS enriched fraction of monocytes for 7 days in the presence of IL-4 (1000 u/ml) and GM-CSF (1000 u/ml). Then in culture derived dendritic cells (DC) were added CEA (peptide) at a concentration of 40 μg/ml and 3 μg/ml β2-macroglobulin, were incubated for 4 h at 20°C. Next peptidylglycine DC were irradiated (55 Gy) and were carried in a ratio of 1:20 with autologous cytotoxic lymphocytes. Qi is amoxicot estimated standard method for the release of 51Cr-tag from the dead cell lines of colorectal cancer. The death of tumor cell lines HT29, WiDr, KM12LM) did not exceed 16% (Matsuda K., Tsunoda T., Tanaka N., Umano Y., Tanimura, N., Nukaya I., K. Takesako, Yamaue H. Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide-pulsed dendritic cells. // Cancer Immunol. Immunther. - 2004. No. 53. - P.609-616.).

The disadvantage of this approach is the long term cultivation of dendritic cells (7 days), which requires the use of large amounts of recombinant cytokines (GM-CSF, IL-4), and the use of additional proteins (β2-macroglobulin), which leads to the considerable increase of technology. Used allogeneic tumor cells are alien antigens that may shape the response, however, remains not known whether the thus activated cells to exhibit cytotoxic activity against tumor antigens and cells, respectively. Uses a tumor-associated protein CEA, which can to varying degrees be expressed on tumor cells. In addition, the efficiency of cytotoxicity of cells achieved in the prototype does not exceed 16%.

The present invention is the creation of a design polyepitopic of immunogens, including epitopes of tumor-associated antigens for colorectal cancer, and the procedure of its use to stimulate specific antitumor immune response against colorectal cancer cells.

The problem is solved by constructing recombinant plasmid DNA pCI-UB-POLYEPI carrying the gene of artificial polyepitope protein under the control of the CMV promoter, and transfection of it in autologous dendritic cells to ensure the expression polyepitopic tumor-associated antigens and stimulation of specific cytotoxic cells.

The essence of the invention consists in the following. The obtained recombinant plasmid DNA pCI-UB-POLYEPI containing 11 epitopes of tumor-associated antigens in colorectal cancer. This polyepitopes design transferout Mature autologous dendritic cells. Then pCI-UB-POLYEPI-transfected DC were cultured with autologous mononuclear cells of peripheral blood of colorectal cancer patients. Mature DC are obtained by adding to immature DC proinflammatory cytokine TNF-α.

The technical result of the invention is to provide polyepitopes construction, transfection of which DK is achieved efficient stimulation of specific cytotoxic lymphocytes against colorectal cancer cells.

The principal difference of the developed method from the prototype is used for activation of the DC polyepitopes design, including epitopes of tumor-associated antigens for colorectalcancer, and application protocols activation and maturation of DC, which also decreases the stage of getting a Mature antigen-transfected DC to 6 nights.

The present invention improves the efficiency of generation of cytotoxic cells, and also allows you to shorten cocultivation DK and MNCs, which ultimately leads to cost reduction method.

The invention is carried out as follows.

For a better understanding of the essence of the present invention the following are examples of its implementation.

Example 1. The preparation of recombinant plasmids pCI-UB.

Plasmid pCI-UB contains ubiquitin, a small protein conservative affecting the cellular localization of the attached protein. Ubiquitin guides attached to protein in the proteasome. The DNA fragment encoding ubiquitin, obtained by PCR of genomic human DNA. Used oligonucleotides containing the recognition sites endonucleases Xhol and EcoRI. The obtained amplication DNA fragment cloned into the vector pCI-neo. In the clone obtained plasmid pCI-UB, the nucleotide sequence of the obtained constructs were checked by sequencing.

Example 2. Design and obtaining tumor polyepitope.

To construct polyepitope protein analyses of amino acid sequences selected proteins (that is L.1) used computational prediction and analysis of existing experimental data.

Table 1.

For prediction of MHC-I epitopes have been using the program (SYFPEITHI; http://www.syfpeithi.de; ProPredl; ; NetMHC-3.0 ). Selected epitopes having the maximum rating for the alleles HLA-A*02, and have the testimony of experimental validation in the literature (if any). While priority was given to the experimental data. For predicting MHC-II epitopes used the program (EpiTOP; http://www.pharmfac.net/EpiTOP/). Selected epitopes, with a total maximum rating for the alleles DRB1-01, 04, 07, 13, 15.

The design was also introduced helper T-cell epitope of diphtheria-tetanus toxoid (QYIKANSKFIGITEL (TT), 830-844 amino acids tetanus toxoid), not specific amplifying the response of CD8+T-lymphocytes. Early work showed that ARY N-terminal amino acids flanking each epitope significantly increase the affinity to TAP the conveyor, and which encourages a more pronounced response of CD8+T-lymphocytes. These sequences are also used to separate epitopes in polyepitopes design. Each epitope requires proteolysis in the specific website above its N-Terminus for efficient translocation, the excess amino acids can be deleted by aminopeptidase. Thus, in the absence of rational design neighboring epitopes can be broken. Design con which e.g. enables separation of epitopes AAY spacer elements sequence, which is the target for proteasome cleavage. DYKDDDDK introduced in polyepitope design for ease of monitoring the expression of polyepitope protein. This peptide is not masked in different positions with the introduction of recombinant proteins and can be effectively detected by using monoclonal antibodies. In polyepitope design was also introduced CTL-peptide from ovalbumin (SIINFEKL) for monitoring immunization. Design and amino acid sequence developed polyepitope protein is shown in figures 1 and 2, respectively. For artificial gene encoding polyepitopic protein, its amino acid sequence was subjected to reverse-transcribed with regard to the frequency of occurrence of codons in humans, for cloning 5'-end was added to the EcoRI site at the 3'-end was added XbaI site, containing a stop codon in frame. The gene was synthesized by PCR with overlapping oligonucleotides (length of 70-80 N.O.) and cloned in plasmid pMTL22 sites EcoRI and XbaI. The nucleotide sequence of the obtained clone was verified by sequencing Tengeru. As a result, the selected clones with the desired nucleotide sequence. The result obtained plasmid DNA pMTL22-POLYEPI.

MUC4-1-EpCAM-1-EpCAM-2-EpCAM-3-CEA-1-CEA-2-CEA-3-MUC1-TT-OVA-MUC4-2 - EpCAM-4-CEA-4

1. Schematic structure polyepitope the CSOs protein.

DYKDDDDK-LLGVGTFW-ADRM-GLKAGVIAV-AAMEY-VLAFGLLLA-ADRIW-

YQLDPKFITSI-

AAYARY-IMIGVLVGV-ADRTW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-

LLLLTVLTV-ADRM-QYIKANSKFGIGITEL-ANIY-SINFEKL-ARY-

SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-

IQNDTGFYTLHVIKSDLVNEE

2. Amino acid sequence polyepitope protein. Bold tumor-specific epitopes, underlining auxiliary motives for processing.

Example 3. The preparation of recombinant plasmids pCI-UB-POLYEPI Plasmid DNA pCI-UB and pMTL22-POLYEPI was subjected to hydrolysis with restriction endonucleases EcoRI and XbaI in series with intermediate presidenial DNA with isopropanol. Restrictive DNA fragments obtained by the hydrolysis of pMTL22-POLYEPI, were separated in agarose 1% gel using gel electrophoresis. Cut a strip of the gel containing the DNA fragment encoding polyepitope. Was suirable DNA from the gel by adsorption of DNA on the soft-feel. Gidralizovanny DNA pCI-UB was extracted with phenol-chloroform mixture and subsequent presidenial isopropanol. Next was performed ligation of the resulting DNA fragments with subsequent transformation of E. coli cells strain XLBlue ligase mixture. Selected clones containing polyepitope. The nucleotide sequence of the obtained clone was verified by sequencing Tengeru.

Example 4. Obtaining dendritic cells, transfected with the plasmid pCI-UB-POLYEPI

Isolated from the peripheral blood of patients colorectal the cancer adherent fraction of mononuclear cells cultivated at a concentration of 1 million/ml in complete medium containing 10% fetal calf serum, 2 mm L-glutamine, 10 mm HEPES, 5×10-5mm 2-mercaptoethanol, 80 μg/ml gentamicin, 100 μg/ml ampicillin in an atmosphere of 5% CO2at 37°C with the addition of rcgm-CSF (50 ng/ml) and rcil-4 (100 ng/ml). After 96 hours of cultivation to derived immature DC is added to maturation paid rcfn-α at a dose of 25 ng/ml After 24 hours of cultivation was performed procedure magnetic transfection of Mature dendritic cells using reagents firm "Homoki", the research Protocol was based on the proposed manufacturer of the scheme. After 24 hours was carried out by the joint cultivation of transfected dendritic cells and unattached fraction of mononuclear cells at a ratio of 1:10/ 96 hours test conducted on the cytotoxicity of culture of mononuclear cells against autologous tumor cells.

To determine the modulation of the cytotoxic activity of T-lymphocytes using the derived DC were evaluated by the death of tumor cells in the cytotoxic test. For this to mononuclear cells of the patient, who had previously acultural transfected with plasmids DK, were added to autologous tumor cells. Cytotoxicity was assessed by the increase in the content of the intracellular enzyme lactate dehydrogenase in conditioned medium resulting from the loss of tumor cells. Shown significant improvement values cytot is sicnosti, that is an indicator of activation of antitumor cytotoxic cells, necessary for the destruction of tumor cells (figure 1).

Joint cultivation of MNCs with DC transfected with a recombinant polyepitope the plasmid pCI-UB-POLYEPI, is an effective method of generating specific cytotoxic cells mononuclear origin in patients with colorectal cancer that is manifested in the strengthening of their cytotoxic anti-tumour response. Thus, as a result of the research has developed a method of stimulation of antitumor cytotoxic immune response using autologous dendritic cells transfected polyepitopes recombinant plasmid providing the expression polyepitopic tumor-associated antigens for colorectal cancer in dendritic cells and stimulation of specific cytotoxic cells.

1. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumor-associated antigens, colorectal cancer, has a size of 6 355 BP and expresses the following amino acid sequence:
DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGITEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE;
recombinant plasmid pCI-UB-POLYEPI contains the CMV enhancer (5251-5910 P.N.), the CMV promoter (5919-6001),T7 promoter (6317-6336 P.N.), ubiquitin (1-229), also contains a unique restriction sites EcoRI (233), XbaI(891), NotI (908), BamHI (3164), NdeI (5638), SacI (5980), XhoI (6342).

2. The method of generating antigen-specific antitumor cytotoxic cells, which consists in co-cultivation of mononuclear cells (MNCs) in peripheral blood of colorectal cancer patients with dendritic cells (DC), activated by tumor antigens, characterized in that cultivation of use Mature DK, obtained by adding to immature DC proinflammatory cytokine TNF-α, activated by transfection in them recombinant plasmid DNA pCI-UB-POLYEPI claimed in claim 1.



 

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