CN110819648B

CN110819648B - Immortalized DC cells infected with lentivirus and their use in killing MAGE-A3 expressing tumor cells

Info

Publication number: CN110819648B
Application number: CN201810891711.0A
Authority: CN
Inventors: 邵永平; 胡杨; 田光启
Original assignee: Beijing Huida Biotechnology Co ltd
Current assignee: Beijing Huida Biotechnology Co ltd
Priority date: 2018-08-07
Filing date: 2018-08-07
Publication date: 2022-01-28
Anticipated expiration: 2038-08-07
Also published as: CN110819648A

Abstract

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.

Description

Immortalized DC cells infected with lentivirus and their use in killing MAGE-A3 expressing tumor cells

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:

example 1:

and (3) constructing a vector for the specific antigen sequence, and storing the transformed bacteria for a long time.

The full-length cDNA of MAGE-A3 with restriction enzyme sites (BgIII and NotI) at both ends was synthesized by a whole-gene synthesis method, as shown in SEQ ID No. 1. The target DNA was ligated with lentiviral vector plasmids such as LL3.7, pHD, etc. using restriction enzymes (BgIII and NotI) and DNA ligase to construct MAGE-A3 lentiviral expression plasmids. Together with lentiviral packaging plasmids pLP1(Gag/Pol), pLP2(Rev), pLP-VSVG (VSVG), respectively, E.coli were transformed as follows:

the competent cells (stbl3) were removed from the-80 ℃ freezer, quickly placed on ice until completely thawed; respectively adding 10ng of one of the plasmids into each tube of competent cells in a superclean workbench, and gently and uniformly mixing; placing on ice again for 45-60 min; heat shock at 42 ℃ for 90 sec; quickly putting back on ice for 2 min; adding 800 μ L LB liquid culture medium into each tube in a clean bench, sealing the tube opening with sealing membrane, and shake culturing at 37 deg.C and 250rpm for 45-60 min; uniformly coating 50-200 uL of culture on an LB solid culture medium containing corresponding antibiotics; culturing overnight (12-16 h) in a constant temperature incubator at 37 ℃. Selecting a single clone, and picking the single clone into 5mL of liquid LB culture medium containing corresponding antibiotics, and culturing the single clone at 37 ℃ and 250rpm until OD600 is 0.6-0.8. The identification is carried out by a method of bacteria liquid PCR and plasmid DNA sequencing. If the plasmid sequence is correct, the plasmid is frozen and stored in LB culture medium containing 15% of glycerol for a long time at-80 ℃.

Example 2:

lentiviral packaging, concentration and titer determination were performed on the MAGE-A3 expression vector.

1. The strain obtained in example 1 was extracted with pLP1(Gag/Pol), pLP2(Rev), pLP-VSVG (VSVG) packaging plasmid and MAGE-A3 lentiviral plasmid using endotoxin-free plasmid middle-or large-lift kit, and the plasmid concentration and A260/280 ratio were determined, the A260/280 ratio should be between 1.8-1.9. A small amount of plasmid sample was subjected to agarose gel electrophoresis to verify that the plasmid size was correct, as shown in FIG. 1.

2. One day before plasmid transfection, 293T cells with a degree of fusion of about 80% were digested with 0.25% trypsin 5minutes before the digestion was stopped by adding an equal volume of fetal bovine serum. 20ul of the cell suspension was mixed with 80. mu.L of trypan blue stain and added dropwise to a hemocytometer for counting. 5 to 6 x10⁶Individual 293T cells were suspended in 10ml of culture medium (high-glucose DMEM + 10% FBS, no antibiotics) and plated into 10cm dishes.

3. The following day, the cell culture fluid was aspirated off and 9ml of fresh antibiotic-free medium (high-glucose DMEM + 10% FBS) was added. After the solution change, the cells were cultured in a 5% CO2 incubator at 37 ℃ until the preparation of the DNA-transfection reagent complex was completed.

4. 800. mu.l of the solution was added to a 1.5ml centrifuge tube

The transfection reagent Lipofectamine 3000 was added to 36. mu.L of the culture solution I (Invitrogen), and the mixture was allowed to stand at room temperature (25 ℃ C. + -5 ℃ C. according to the present invention) for 5minutes after mixing by inversion to prepare a transfection reagent mixture. Another 1.5ml centrifuge tube is added with 200ul

Adding 3 mu g of pLP1,3 mu g of pLP2, 3 mu g of pLP-VSVG and 3 mu g of target gene lentiviral plasmid into the culture solution I, mixing uniformly and standing. After 5minutes, the plasmid mixed solution is added into the transfection reagent mixed solution, slowly and uniformly blown, and then kept stand for 20 minutes at room temperature (the room temperature is 25 +/-5 ℃) to prepare the DNA-transfection reagent compound.

5. Adding 1ml of DNA-transfection reagent compound dropwise into the changed 293T cell culture plate prepared in the step 3, shaking the culture dish all around, mixing uniformly, and placing the culture dish into a 37 ℃ and 5% CO2 incubator for overnight culture. The next day, the cell culture was aspirated, 10ml of fresh antibiotic-free medium was added, incubated at 37 ℃ in a 5% CO2 incubator, and virus was collected after 72 hours of transfection. The culture supernatant in the petri dish was collected in a 15ml centrifuge tube, centrifuged at 3000rpm at 4 ℃ for 15minutes, and the supernatant was collected.

6. Centrifuging the collected virus supernatant at 4 deg.C for 2 hr at 66,549 × g, discarding the supernatant, resuspending the virus precipitate in PBS or serum-free medium, preserving at-80 deg.C for a long time, and packaging to avoid repeated freeze thawing.

7. Utilizing Lenti-X of Clonetec corporation^TMThe viral titer was determined with the qRT-PCR Titration kit.

Example 3:

infecting immortalized-DC cells by using MAGE-A3 lentivirus and screening to obtain a cell strain for stably expressing MAGE-A3:

1.2 ml of immortalized DC cell suspension was collected into a 15ml centrifuge tube, centrifuged at 1200rpm for 5 min.

2. Discarding the supernatant, suspending the cells with 2ml of fresh medium, placing 100ul of the cell suspension in an EP tube, adding 100ul of trypan blue into the EP tube, mixing, adding dropwise onto a blood cell technical plate for counting, and inoculating 1X 10⁵Individual cells were plated into one well of a 6-well plate and cell suspension volumes were recorded.

3. According to the result of lentivirus titer determination, the volume of the virus solution added to the cells was calculated according to MOI of 20, the virus supernatant was added to make up the medium to 2ml, and 2. mu.l of a 10mg/ml polybrene solution was added to the wells to ensure a final concentration of 10. mu.g/ml, and the cells were cultured at 37 ℃ in a CO 25% incubator.

After 4.24 hours, the cells were collected in a 15ml centrifuge tube at 1200rpm for 5 min.

5. The supernatant was discarded, and 2ml of fresh medium was used to resuspend the immortalized DC cells and placed in a new well for culture at 37 ℃ in a CO 25% incubator for 2 days.

6. The cells were collected in a 15ml centrifuge tube at 1200rpm for 5min and steps 3-6 were repeated.

7. Discarding the supernatant, resuspending immortalized DC cells in 3ml fresh medium, placing into a new well, placing into a 37 ℃ CO 25% incubator for culture

8. After 2 weeks of virus infection, a portion of the cells were harvested and the expression of the specific antigen was identified by Q-PCR, as shown in Table 1 and FIG. 2.

TABLE 1 differences in expression of MAGE-a3 in emptyDC versus transfected MAGE-A3 antigen

*P＝0.0002

Example 4: cell donor screening and donor PBMC preparation 1. PBMC donor volunteers were drawn 1ml of peripheral blood and stored in EDTA anticoagulant blood collection tubes and mixed well.

2. HLA-A low-typing detection is carried out, and 1-position entry group of the volunteers with serotype HLA-A0201 is selected as PBMC donor of an experimental group and a control group. Serotype non-HLA-a 0201 volunteers, position 1, were selected as PBMC negative control group 1 donors.

3. 10ml of peripheral blood was drawn from the group of volunteers and stored in EDTA anticoagulated blood collection tubes and mixed well.

i. The blood samples were transferred to 50ml centrifuge tubes and PBS equilibrated to room temperature (25 ℃. + -. 5 ℃) was added to a total volume of 35 ml.

To a 50mL centrifuge tube to which 15mL of Ficoll-Paque fraction had been previously added to each tube, 35mL of a diluted blood sample of LPBS was slowly added.

Centrifugation was carried out at 1800 rpm at room temperature (25 ℃ C. + -5 ℃ C.) for 30 minutes using Thermo Scientific Sorvall ST16R or a homocentrifuge, with the centrifuge acceleration parameter set to 1 and the deceleration parameter set to 0.

Transfer the intermediate layer of liquid from each centrifuge tube to a new 50ml centrifuge tube, respectively.

v. add pre-cooled PBS to a final volume of 50mL and centrifuge at 1200rpm for 10 min at 4 ℃. The supernatant was discarded and the cell pellet was resuspended in 1.0mL PBS.

Add pre-chilled PBS to a final volume of 50mL and centrifuge at 1000 rpm for 10 min at 4 ℃. The supernatant was discarded and 1.0ml of LPBS was added to resuspend the cell pellet.

Add pre-chilled PBS to a final volume of 50mL and centrifuge at 800 rpm for 10 minutes at 4 ℃. The supernatant was discarded and 1mL of pre-cooled PBS was added to resuspend the cell pellet.

Complete media was prepared using RPMI1640 medium, 10% FBS and 1% streptomycin, 1.0mL of complete media was added to the centrifuge tube, and the cell pellet was resuspended.

The cell suspension was volumetrically measured with a 2ml serum pipette and 10. mu.l was counted after resuspension.

Preparation of Effector cells 1 resuscitated HLA-A0201, MAGE-A3 expressing immortalized DCs, adjusted to a cell density of 2X10 using RPMI1640 medium and 10% FBS culture system⁵Perml and inoculated in 25 bottles, added 200U/ml IL-2 daily to a density of 5x10⁵/ml。

2. PBMC were cultured at 1-2X10 using RPMI1640 medium and 10% FBS culture system⁶/well in 6-well plates, immortalized DCs expressing MAGE-A3 were incubated with PBMCs at 1: a500 proportion was added to the wells and the cell suspension volume per well was guaranteed to be 2 ml.

IL-2200U/ml was added daily to Day1-Day3 for amplification, and half the change was made when the medium turned yellow.

Day5 cells were transferred to 25 flasks and medium was replenished to 6mL, IL-2200U/mL was added daily for expansion to Day9, and medium was changed half as yellow.

Transferring the cells to 75 culture flasks by Day9 and replenishing the medium to 15mL, adding IL-2200U/mL daily for expansion to Day14, and changing the medium half way or adding medium when the medium turns yellow.

Day14 flow assay CTL cells were analyzed.

Example 5:

the killing effect was verified by killing target cells using CTL as effector cells after transfection of the fluorescein reporter gene into the target cells. On day 10 after mixed co-culture of MAGE-A3 immortalized DCs with PBMCs, 2 lines of luciferase-expressing target tumor cells, HHC1954, were revived for CTLs cell viability assay.

2. After digesting the target cells for 5minutes using 0.25% pancreatin and terminating the digestion, 10mL of the cell suspension was transferred to a 15mL centrifuge tube and centrifuged at room temperature and 1200rpm for 5 minutes.

3. The supernatant was discarded, the cell pellet was resuspended in 2mL of RPMI1640 medium, and the sample was counted.

4. According to 2.5X 10⁴Per 250ul tumor cell density was adjusted and 250ul tumor cell suspension was inoculated into 24-well plates.

5. Killing test cells 2 hours after adherent treatment, following target cell: effector cells (CTL cells) were seeded at a cell density of CTLs adjusted from 1:10 to 1: 1.25. The volume of cell suspension added to each well was guaranteed to be 250. mu.L.

Negative control group PBMC of serotype HLA-a2 and equivalent to each group of effector cells were added, ensuring a cell suspension volume of 250uL per well.

Blank control was added to 250uL RPMI1640 medium.

Co-culturing effector cells with target cells for 16 or 4 hours.

6. Fluorescence detection Using Water for injection, 5 XReporter Lysis Buffer was adjusted to H₂O: RLB ═ 4: 1, preparing a lysis working solution to prepare a cell lysate, and precooling at 4 ℃.

The medium supernatant was aspirated, PBS added using a 10ml serum pipette, gently shaken, PBS aspirated, and PBS added again for washing to ensure that all suspended cells and cell debris were cleared.

Add 100 μ L of cell lysate per well and lyse cells on ice for about 2 hours.

Transfer the lysate to a 1.5mL centrifuge tube, label the sample number, centrifuge at 12000 rpm for 5 min.

v. 20. mu.L of supernatant was taken from each 1.5mL centrifuge tube and added to the 96-well microplate assay well in sequence.

And vi, sequentially adding 100 mu L of Luciferase substrate into the detection holes of the ELISA plate by using a 100 mu L handheld single-channel pipette, and immediately detecting the fluorescence intensity after blowing for 3 times.

The fluorescence intensity of each well was measured sequentially, ensuring that each sample was measured within 10 seconds after addition of substrate.

Calculating the killing rate of the CTL cells to the tumor cells. Percent killing activity [1- (E + T group OD value)/T group OD value ]. times.100%

Effector cells, T: target cell, E + T: effector + target cells

Results of the killing test

The killing experiment of the large-a 3-DC-CTL on the HHC1954 target cells for 4 hours (FIG. 4A) and 16 hours (FIG. 4B) shows that the large-a 3-DC-CTL has the following characteristics in the target cells: killing activity of the effector cells reaches 90.8 percent (4 hours) and 96.6 percent (16 hours) under the condition of 1:10 respectively; in the target cell: killing activity of effector cells reaches 56.2% (4 hours) and 72% (16 hours) under the condition of 1:2.5, respectively.

TABLE 2A mage-a3-DC-CTL 4-hr killing Activity on HHC1954 target cells

TABLE 2B mage-a3-DC-CTL 4 hr killing chemiluminescence assay for HHC1954 target cells

TABLE 3A mage-a3-DC-CTL killing Activity on HHC1954 target cells for 16 hours

TABLE 3B mage-a3-DC-CTL killing chemiluminescence assay of HHC1954 target cells at 16h

Example 6:

flow cytometry identification of CTL cells cultured for 14 days by using DC cell surface marker CD11c antibody with APC fluorescein is performed, as shown in FIG. 5, a CD11c positive cell subset is not found, namely after 14 days of culture, the CTL cell subset has no immortalized DC cells, and the safety of the CTL cells is ensured.

Example 7:

killing experiments were performed using 6 tumor cell lines (see Table 3) of combinations of MAGE-A3 and HLA-A as target cells and specific CTL cells induced by MAGE-A3 expressing DCs as effector cells.

HLA types of target and effector cells, MAGE-A3 expressing DC cells, donor PBMC cells and control cells HHC1954 are as follows:

TABLE 4 PBMC donor, DC cells, target cell HLA and MAGE-A3 phenotypes

Results of the killing experiment:

the results show that DC-CTL has obvious killing effect (P <0.0001) on HHC1954, A, B and C cell strains at the effective-target ratio of 1.25:1 to 10:1, and has no killing effect on D, E and F. (FIG. 6)

Wherein the killing efficiency for HHC1954 target cells is 8.8% to 44.6% at each concentration of 1.25:1 to 10: 1.

Killing efficiency for target cell A is 22.5% to 51.2% at each concentration of 1.25:1 to 10:1 effective target ratio.

Killing efficiency for target cell B at each concentration of 1.25:1 to 10:1 at effective target ratio of 5.8% to 45.2%.

Killing efficiency for target cells C is 11.3% to 38.7% at each concentration of 1.25:1 to 10:1 effective target ratio.

TABLE 5 percent killing of A, B, C and HHC1954 target cells at different target ratios

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing-Hui-Dai Biotech Co., Ltd

<120> lentivirus-infected immortalized DC cells and their use for killing tumor cells expressing MAGE-A3

<130> MP1816384

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1059

<212> DNA

<213> full-Length cDNA of MAGE-A3 carrying restriction sites BgIII and NotI at both ends (MAGE-A3 full-length cDNA cloning sites BgIII and NotI at both ends)

<400> 1

ttaataaaga agaggcggat gaggatccag atctcaatgg cgacggatcc atcgccacca 60

tgcctcttga gcagaggagt cagcactgca agcctgaaga aggccttgag gcccgaggag 120

aggccctggg cctggtgggt gcgcaggctc ctgctactga ggagcaggag gctgcctcct 180

cctcttctac tctagttgaa gtcaccctgg gggaggtgcc tgctgccgag tcaccagatc 240

ctccccagag tcctcaggga gcctccagcc tccccactac catgaactac cctctctgga 300

gccaatccta tgaggactcc agcaaccaag aagaggaggg gccaagcacc ttccctgacc 360

tggagtccga gttccaagca gcactcagta ggaaggtggc cgagttggtt cattttctgc 420

tcctcaagta tcgagccagg gagccggtca caaaggcaga aatgctgggg agtgtcgtcg 480

gaaattggca gtatttcttt cctgtgatct tcagcaaagc ttccagttcc ttgcagctgg 540

tctttggcat cgagctgatg gaagtggacc ccatcggcca cttgtacatc tttgccacct 600

gcctgggcct ctcctacgat ggcctgctgg gtgacaatca gatcatgccc aaggcaggcc 660

tcctgataat cgtcctggcc ataatcgcaa gagagggcga ctgtgcccct gaggagaaaa 720

tctgggagga gctgagtgtg ttagaggtgt ttgaggggag ggaagacagt atcttggggg 780

atcccaagaa gctgctcacc caacatttcg tgcaggaaaa ctacctggag taccggcagg 840

tccccggcag tgatcctgca tgttatgaat tcctgtgggg tccaagggcc ctcgttgaaa 900

ccagctatgt gaaagtcctg caccatatgg taaagatcag tggaggacct cacatttcct 960

acccacccct gcatgagtgg gttttgagag agggggaaga gtaagcggcc gcaaggatct 1020

gctcgacaat caacctctgg attacaaaat tggaaagat 1059

Claims

1. The expression vector is characterized by comprising a lentiviral vector and a sequence of MAGE-A3 full-length cDNA carrying restriction enzyme cutting sites, which is shown as SEQ ID No. 1.

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;

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

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 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.

8. 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.

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