阅读说明：本技术 感染有慢病毒的永生化dc细胞及其在杀伤表达mage-a3肿瘤细胞中的应用 (Immortalized DC cells infected with lentivirus and their use in killing MAGE-A3 expressing tumor cells ) 是由 邵永平 胡杨 田光启 于 2018-08-07 设计创作，主要内容包括：本发明涉及免疫领域,特别涉及感染有MAGE-A3慢病毒的永生化DC细胞及其在杀伤表达MAGE-A3肿瘤细胞中的应用。本发明开发了一种将永生化树突状细胞进行基因改造,使之能持续表达并提呈MAGE-A3抗原肽,进而激活CD8+T细胞,对表达MAGE-A3的肿瘤细胞进行特异性杀伤的肿瘤免疫疗法。(The invention relates to the field of immunity, in particular to an immortalized DC cell infected with MAGE-A3 lentivirus and application thereof in killing and expressing MAGE-A3 tumor cells. The invention develops a tumor immunotherapy which carries out gene modification on immortalized dendritic cells, enables the immortalized dendritic cells to continuously express and present MAGE-A3 antigen peptide, further activates CD8+ T cells and carries out specific killing on tumor cells expressing MAGE-A3.)

1. An expression vector comprising a lentiviral vector and a vector having any one of the nucleotide sequences set forth below:

I. has a nucleotide sequence encoding MAGE-a 3;

II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases in the nucleotide sequence shown as I;

III, a nucleotide sequence with at least 80 percent of homology with the nucleotide sequence shown in the I;

IV, a nucleotide sequence having the same functional fragment or functional variant as the sequence described in I;

v, a complementary sequence of the nucleotide sequence shown as I, II, III or IV.

2. The method for constructing an expression vector according to claim 1, wherein a MAGE-A3 full-length cDNA carrying a restriction enzyme site is obtained, and the MAGE-A3 full-length cDNA is ligated to a lentiviral vector using the restriction enzyme and a DNA ligase to obtain the expression vector.

3. A strain transfected with the expression vector of claim 1 and a lentiviral packaging vector.

4. A cell transfected with the expression vector of claim 1 and a lentiviral packaging vector.

5. MAGE-A3 lentivirus obtained after culturing the cells of claim 4.

6. Immortalized DC cells infected with MAGE-a3 lentivirus according to claim 5.

7. Use of immortalised DC cells according to claim 6 infected with a MAGE-A3 lentivirus according to claim 5 for expressing MAGE-A3.

8. Use of immortalised DC cells infected with the MAGE-A3 lentivirus of claim 5 according to claim 6 for activating cytotoxic CD8+ T cells specifically recognizing MAGE-A3.

9. Use of immortalised DC cells infected with the MAGE-A3 lentivirus according to claim 6 for screening T cell receptors specifically recognizing MAGE-A3 antigenic peptides and/or HLA complexes; or

Use of immortalized DC cells infected with the MAGE-a3 lentivirus according to claim 6 in the manufacture of a medicament for killing tumor cells; the tumor cells express MAGE-a 3.

10. A medicament comprising immortalised DC cells infected with the MAGE-a3 lentivirus according to claim 6.

Technical Field

The invention relates to the field of immunity, in particular to an immortalized DC cell infected with lentivirus and application thereof in killing tumor cells.

Background

Immunotherapy (immunotherapy) refers to a treatment method for artificially enhancing or suppressing the immune function of the body to treat diseases in response to a low or high immune state of the body.

Infection with tumor cells or pathogens activates T lymphocytes in the human body to mount an immune response against these diseases. Activation of T lymphocytes requires the involvement of a class of cells called Antigen Presenting Cells (APCs). Antigen-presenting cells include primarily Dendritic Cells (DCs), macrophages and B lymphocytes, with dendritic cells being the most important and most efficient antigen-presenter. Antigen presenting cells digest antigenic proteins from tumors or pathogens, which are then presented as small peptides to the cell surface, where the antigen signals are activated and amplified once they are recognized by T lymphocytes. A large number of activated T cells attack and kill tumor cells or cells infected by pathogens, thereby performing an immune function.

In some chronic diseases such as tumor or pathogen-induced chronic inflammatory diseases, the immune function of T lymphocytes is gradually inactivated with time, and the killing effect on tumor cells is lost. In addition, the infiltration of T lymphocytes in tumor tissues is often low and does not achieve sufficient killing effect, which is the main reason for tumor immune escape. The in vitro activation and mass culture of T cells with tumor specific killing capability, and the transfusion back to the body of a patient to play the immune function is a general effective strategy of the current cellular immunotherapy.

Ideally, a good immunotherapy strategy should satisfy the following conditions: firstly, the anti-tumor effect is better at a lower ratio of effector cells to tumor cells (E: T ratio), so that the number of cells injected into a patient is reduced to the maximum extent, and the cost and the risk can be reduced simultaneously; second, in a "emerging" model, anti-tumor immune cells can be derived from healthy donors without causing graft versus host disease complications (GVHD), thereby providing economical, effective treatment and significantly reducing patient waiting times; thirdly, toxic and side effects possibly brought by gene modification are avoided as much as possible, and CAR-T cells can be remained in a patient body for a long time through gene engineering modification, so that unknown risks exist; fourth, the optimization of the quality of immune cells, although there are various methods for activating immune cells, the activation of immune cells by dendritic cells, which conforms to the natural laws of the body, is considered to be one of the best methods.

The current clinical immunotherapy for tumor is mainly based on T lymphocytes from patients, including four steps of T lymphocyte isolation, in vitro activation of antigen-specific T cells, expansion and reinfusion. Where the in vitro expansion of antigen-specific T cell populations is a very critical and challenging step in this therapeutic strategy. The currently used approaches are three 1) approaches that stimulate T cells with a mixture of CD3, CD28 antibody and IL 12. The method provides three key signals of activation, amplification and survival for T cells, but the presentation mode of the signals is completely different from the natural antigen presentation process of in vivo APC, which can cause the slow amplification speed, limited function or dysfunction of the T cells; 2) the Car-T technology or the TCR-T technology is used for preparing the CD8+ T cells with the specific antigen peptide recognition capability. The advantage of this approach is that specific CD8+ T cells still have strong killing activity at lower effector/tumor cell ratios (E: T ratio). However, single antigenic peptide recognition has some disadvantages. For example, the non-specific killing to normal tissues except tumor tissues is possible, so that the potential safety hazard is generated; in addition, immunotherapy against a single target is likely to result in immune tolerance after the tumor actively loses antigen. 3) The DC cells separated from the patient or differentiated by the stimulation of the mononuclear cells are utilized to obtain and present the antigen by phagocytosis of apoptotic tumor cell fragments, transduction of antigen peptide genes and other modes, and the activation and the amplification of the T cells are induced. This approach is similar to in vivo antigen presentation, but is limited by the extremely low DC cell content in the patient's blood and does not meet the expanding clinical needs. Moreover, these DC cells cannot survive in vitro for a long period of time and often die after a short period of culture.

Therefore, the method for stably expressing the tumor specific antigen by the immortalized DC cell line by the gene modification has important practical significance.

Disclosure of Invention

In view of the above, the present invention provides a method for stably and continuously expressing MAGE-A3 tumor antigen in immortalized DC cells. The method enables the immortalized DC cells to process and present MAGE-A3 tumor antigen in a way as close to nature as possible, and can efficiently induce the activation and the amplification of CD8+ T lymphocytes in vitro, thereby solving an important problem restricting the T cell immunotherapy.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides a lentiviral vector and a lentiviral vector having any one of the nucleotide sequences shown below:

I. has a nucleotide sequence encoding MAGE-a 3;

II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases in the nucleotide sequence shown as I;

III, a nucleotide sequence with at least 80 percent of homology with the nucleotide sequence shown in the I;

IV, a nucleotide sequence having the same functional fragment or functional variant as the sequence described in I;

v, a complementary sequence of the nucleotide sequence shown as I, II, III or IV.

The invention also provides a construction method of the expression vector, the full-length cDNA of MAGE-A3 carrying restriction enzyme sites is obtained, the full-length cDNA of MAGE-A3 is connected with a lentiviral vector by using the restriction enzyme and DNA ligase, and the expression vector is obtained.

In the present invention, the sequence of the full-length cDNA of MAGE-A3 carrying restriction enzyme sites is shown in SEQ ID No. 1.

The invention also provides a bacterial strain transfected with the expression vector and the lentivirus packaging vector.

In some embodiments of the invention, the strain is constructed by: coli was transformed with the expression vector and lentiviral packaging plasmids pLP1(Gag/Pol), pLP2(Rev), pLP-VSVG (VSVG).

The invention also provides cells transfected with the expression vector and the lentiviral packaging vector.

In some embodiments of the invention, the transfection method of the cell is: the expression vector was transfected into 293T cells together with lentiviral packaging plasmids pLP1(Gag/Pol), pLP2(Rev), pLP-VSVG (VSVG).

On the basis, the invention also provides MAGE-A3 lentivirus obtained after the cell culture.

In some embodiments of the invention, the MAGE-A3 lentivirus is prepared by: : the expression vector and lentiviral packaging plasmids pLP1(Gag/Pol), pLP2(Rev), pLP-VSVG (VSVG) were transfected into 293T cells, cultured, and virus was collected.

The invention also provides immortalized DC cells infected with the MAGE-A3 lentivirus.

In some embodiments of the invention, the immortalized DC cells infected with the MAGE-a3 lentivirus are prepared by: infecting an immortalized DC cell with the MAGE-A3 lentivirus; preferably, the MAGE-a3 lentivirus is added at MOI ═ 20, based on lentivirus titer determination.

The invention also provides the application of the immortalized DC cell infected with MAGE-A3 lentivirus in expressing MAGE-A3.

The invention also provides the use of immortalised DC cells infected with MAGE-A3 lentivirus for activating cytotoxic CD8+ T cells specifically recognising MAGE-A3.

The invention also provides application of immortalized DC cells infected with MAGE-A3 lentivirus in screening T cell receptors for specifically recognizing MAGE-A3 antigen peptide and/or HLA complex.

The invention also provides the application of the immortalized DC cell infected with MAGE-A3 lentivirus in the preparation of the drug for killing tumor cells; the tumor cells express MAGE-a 3.

Based on the above studies, the present invention also provides a medicament comprising immortalized DC cells infected with MAGE-A3 lentivirus.

The present invention provides a method for genetically modifying immortalized dendritic cells (DC cells) to a cell line that stably expresses MAGE-A3-specific antigen for a long period of time. The cell line can induce CD8+ T cell activation and large-scale expansion in vitro, so that the cell line has killing effect on tumor cells expressing MAGE-A3.

1. An immortalized dendritic cell is provided that has the ability to persistently express, process and present the MAGE-A3 antigen.

2. The initial T cell can be domesticated to become cytotoxic CD8+ T Cell (CTL) with MAGE-A3 specificity, and tumor cells with high MAGE-A3 expression are killed.

3. Can be used for screening T Cell Receptor (TCR) which can specifically recognize MAGE-A3 antigen peptide/HLA complex.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the correct size of the plasmid nucleic acid sequence as detected by amplification electrophoresis of packaged plasmids;

FIG. 2 shows MAGE-A3 expression levels in immortalized and no-load immortalized DCs after introduction of the MAGE-A3 lentivirus; qPCR detection with beta-actin as an internal reference shows that the no-load immortalized DC hardly expresses MAGE-A3, and the mRNA expression level of the transfected MAGE-a3 is obviously improved (P is 0.0002);

FIG. 3 shows flow cytometry detection of CTL cell subsets; CTL detection by flow cytometry showed NK, NKT, CD8+ T cells and CD4+ T cells in subsets of 9.53%, 4.17% and 47.6% (82.4% x 57.8%), respectively;

FIG. 4 shows the results of the killing experiment of large-a 3-DC-CTL against HHC1954 target cells; wherein, FIG. 4(A) shows the results of a 4-hour killing experiment of mage-a3-DC-CTL against HHC1954 target cells; FIG. 4(B) shows the results of a 16-hour killing experiment of mage-a3-DC-CTL against HHC1954 target cells;

FIG. 5 shows the CTL subpopulation CD11c flow cytometry detection;

FIG. 6 shows that the killing effect for A, B, C and HHC1954 target cells is 4 hours at different effective target ratios, showing that the killing effect is similar to that of HHC1954 at A, B, C three target cells, and the killing ratio is more than 40% at an effective target ratio of 10: 1.

Detailed Description

The invention discloses a preparation method of an immortalized DC cell line capable of stably expressing MAGE-A3 antigen and application thereof in killing tumor cells. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The MAGE-A3 lentivirus vector, the immortalized DC cell infected with MAGE-A3 lentivirus and the raw materials, strains, cells and reagents used in the application of the immortalized DC cell in killing tumor cells can be purchased from the market.

The invention is further illustrated by the following examples: