CN114410689A - Preparation method for enhancing lethality of tumor infiltrating lymphocytes - Google Patents
Preparation method for enhancing lethality of tumor infiltrating lymphocytes Download PDFInfo
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Abstract
The invention belongs to the field of preparation of anti-tumor cells, and particularly relates to a preparation method of tumor infiltrating lymphocytes, which comprises the following steps: preparing a recombinant lentivirus containing a target gene, wherein the target gene comprises an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence; separating infiltrating lymphocytes from the tumor tissue, adding IL-2, and culturing the cells to logarithmic phase; adding the recombinant lentivirus into infiltrating lymphocytes cultured to a logarithmic phase, culturing for 2-4 days, adding a screening agent for screening culture, and culturing until all cells are transgenic positive cells; and (3) using a Tet-on system, adding an inducer to induce the expression of IL-7 or/and IL-15, and maintaining the amplification culture of the cells. The amplification efficiency of the TIL cells obtained by the preparation method is obviously improved and is about 5-10 times higher than that of the common TIL amplification method, and the amplified lymphocytes have higher tumor killing effect.
Description
Technical Field
The invention relates to a preparation method of anti-tumor cells, in particular to a preparation method for enhancing lethality of tumor infiltrating lymphocytes.
Background
Tumor Infiltrating Lymphocytes (TILs) have been developed for more than 30 years as a personalized Adoptive Cell Therapy (ACT) and show great clinical application prospects. The TIL has TCR clonal diversity, superior tumor homing ability, significant efficacy and good safety in treating solid tumors, and has incomparable advantages over other ACT methods. To date, TIL therapy is currently undergoing clinical trials, primarily as second-line therapy. TIL therapy has shown impressive clinical responses in melanoma, non-small cell lung cancer and advanced cervical cancer.
In general, TIL is prepared by mechanically treating tumor tissue and enzymatically digesting to obtain a single cell suspension, selecting cultured T lymphocytes by adding high concentration IL-2, culturing to billions of TIL cells within 2-4 weeks, and then returning to the patient. Reinfusion therapy is accompanied by high doses of IL-2 infusion to support the growth and activity of the TIL infused in vivo. However, high doses of IL-2 often cause systemic toxicity, requiring intensive monitoring and care; in addition, IL-2 may also promote the activity of suppressor T cells, thereby inhibiting the anti-tumor response of TIL; the above drawbacks greatly limit the clinical application of TIL therapy.
The cytokine IL-7 belongs to the erythropoiesis family I type short-chain cytokine. Studies have shown that expression of IL-7 is closely associated with T cell proliferation. If fresh T cells are stimulated with IL-7, the T cells can be dose-dependently expanded, including CD4+ and CD8+ subsets; knock-out of IL-7R, T cells stop growing; the transfer of IL-7 gene can promote the proliferation of CD4 +/CD 8+ T cells. However, CD4+ and CD8+ T cells have different response effects on IL-7. IL-7 had a stronger effect on the CD8+ T cell subset than on the CD4+ cell subset. Like IL-2, IL-15 is also a gamma chain cytokine. IL-15 has similar biological functions to IL-2, and IL-15 and IL-2 share a gamma c receptor subunit, and both cytokines can induce the differentiation of T cells, B cells and natural killer cells (NK cells) and promote the proliferation of the T cells, the B cells and the NK cells. However, in contrast to IL-2, IL-15 does not lead to the expansion of Tregs. At present, IL-15 is often used in combination with IL-2 for the in vitro expansion culture of T cells or NK cells.
Disclosure of Invention
The invention provides a preparation method of Tumor Infiltrating Lymphocytes (TIL), which is an infiltrating lymphocyte separated from tumor tissues and contains tumor antigen specificity CD4+ and CD8+ T cell populations, and in order to enhance the killing power and the amplification effect of the TIL on tumor cells, the preparation method of the tumor infiltrating lymphocytes provided by the invention specifically comprises the following steps:
1) preparing recombinant lentivirus containing target genes; wherein the target gene comprises an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence;
2) separating infiltrating lymphocytes from the tumor tissue, adding IL-2 to culture the cells to logarithmic phase;
3) adding the recombinant lentivirus into infiltrating lymphocytes cultured to a logarithmic phase, culturing for 2-4 days, adding a screening agent for screening culture, and culturing until all cells are transgenic positive cells;
4) and (3) using a Tet-on system, adding an inducer to induce the expression of IL-7 or/and IL-15, and maintaining the expansion culture of the infiltrating lymphocytes.
Wherein the inducer is tetracycline or a tetracycline derivative. Tetracycline derivatives behave like tetracycline and are derivatives of tetracycline, such as tetracycline hydrochloride.
Compared with controlling the expression level of IL-7 or/and IL-15 in a patient body at the protein level, the invention controls the expression of the IL-7 or/and IL-15 at the transcription level, and is easier to control and realize in the treatment process; meanwhile, the expression of IL-7 and IL-15 regulated by the Tet-on system at the transcription level has higher sensitivity; in the operation process, the expression of IL-7 or/and IL-15 can be obviously induced by a small amount of inducer, so that the effect of inducing expression is higher and more accurate and the toxicity of a patient is lower in the process of treating the tumor.
In addition, compared with the constitutive expression of IL-7 or/and IL-15, the Tet-on system adopted by the invention is an inducible expression system, has higher application flexibility, can induce or close the expression of IL-7 or/and IL-15 at any time when needed, and further avoids the potential toxicity of the continuous expression of IL-7 or/and IL-15.
In addition, the invention can improve the amplification efficiency of the TIL in vivo and simultaneously eliminate the toxicity of the IL-2 to the patient by reducing the influence of the IL-2 on the TIL activity to the maximum extent so as to fully exert the potential of the therapy. The invention adopts a transgenic method to obtain the stable transformation TIL cell containing the target gene of IL-7 or/and IL-15; and allowing the TIL cells to express IL-7 or/and IL-15 in the presence of an exogenous inducer tetracycline or a derivative thereof, instead of conventionally promoting the growth and expansion of the TIL cells using exogenously added high concentrations of IL-2. The amplification efficiency of the TIL cells obtained by the method is obviously improved and is about 5-10 times higher than that of the common TIL amplification method, and the amplified lymphocytes have higher tumor killing effect.
The present invention uses the Tet-on system to induce the expression of IL-7 and IL-15 by adding tetracycline or tetracycline derivatives.
Among them, the Tet-on system has a strong advantage in controlling the expression of target genes. Firstly, the expression level of a target gene of a Tet-on system is very low when no inducer is used, while the expression level is obviously increased when the inducer is added, and the highest induction multiple can reach 10000 times. Secondly, the induction medicine of the Tet-on system is tetracycline (Tet) or derivatives thereof such as doxycycline (Dox), Tet has a long history application record as an antibiotic, is a medicine which is safe to human bodies, and low-dose Tet in the Tet-on system can regulate the expression of genes, so that strong toxicity can not be generated to animals or cells. Finally, the tetracycline Tet-on system has reversibility, and can be closed after the inducer is removed, or the inducer can be added repeatedly to start the induction reaction for multiple times.
The invention further provides that the IL-7 and the IL-15 in the IL-7-IL-15 fusion sequence are connected by IRES. IRES enables initiation of protein translation independent of the cap structure of messenger RNA 5' and initiation of RNA translation of proteins from the middle, thus ensuring correct expression of functional proteins by both IL-7 and IL-15.
Wherein the IL-7 sequence is shown in SEQ ID No: 1 is shown in the specification;
the IL-15 sequence is shown as SEQ ID No: 2 is shown in the specification;
the IL-7-IL-15 fusion sequence is shown as SEQ ID No: 3, respectively.
In practical application, the three sequences are optimized; the CDS sequences of IL-7 and IL-15 are downloaded and then codon optimized to enhance their expression. The optimization method includes but is not limited to: human codon usage bias, modest GC content, stable mRNA secondary structure, etc., elimination of repetitive sequences, cryptic splice sites and unwanted restriction sites, while preventing depletion of tRNA pools in the cell.
The invention further provides that the recombinant lentivirus in the step 1) is prepared by adopting the following method: and obtaining and optimizing an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence, inoculating the sequences into a lentiviral vector, transfecting HEK-293T cells to carry out virus preparation and amplification, and culturing to obtain the recombinant lentivirus.
The invention further provides that the cell culture in the step 2) is carried out in a culture environment coated by the recombinant human fibrin and the CD3 antibody.
The invention further provides that in the step 3), when the recombinant lentivirus is added into infiltrating lymphocytes cultured to a logarithmic growth phase, Polybrene (Polybrene) is added at the same time; can obviously improve the infection efficiency of the retrovirus to cells.
Wherein the screening agent is Puromycin (Puromycin).
The invention provides a preferable scheme, and the preparation method comprises the following steps:
1) preparing recombinant lentivirus containing target genes; wherein the target gene comprises an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence;
the method specifically comprises the following steps: obtaining and optimizing and synthesizing an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence, inoculating the sequences into a lentiviral vector, then transfecting HEK-293T cells to carry out virus preparation and amplification, and culturing to obtain a recombinant lentivirus;
wherein the optimization includes, but is not limited to: human codon usage bias, moderate GC content, stable mRNA secondary structure, etc., elimination of repetitive sequences, cryptic splice sites and unwanted restriction sites, while preventing depletion of tRNA pools in the cell;
preferably, the IL-7 sequence is as shown in SEQ ID No: 1 is shown in the specification; the IL-15 sequence is shown as SEQ ID No: 2 is shown in the specification; the IL-7-IL-15 fusion sequence is shown as SEQ ID No: 3 is shown in the specification;
2) digesting the tumor tissue to obtain a single cell suspension containing infiltrating lymphocytes; culturing the single cell suspension by using IL-2, and separating and purifying the infiltrating lymphocytes; then in a culture environment coated by recombinant human fibrin and CD3 antibody, continuously adding IL-2 into the purified infiltrating lymphocytes, and culturing until the logarithmic phase is reached;
3) adding the recombinant lentivirus into infiltrating lymphocytes cultured to a logarithmic growth phase, and adding Polybrene at the same time, and transfecting for 2-4 days; then adding a screening agent for screening culture until all cells are transgenic positive cells;
4) using Tet-on system, tetracycline or tetracycline derivative is added to induce the expression of IL-7 and/or IL-15, and the amplification culture of infiltrating lymphocytes is maintained.
The invention further provides that the tumor tissue is selected from one of lung tumor tissue and breast tumor tissue.
The preparation method provided by the invention can be widely applied to the preparation of anti-tumor therapeutic agents.
The preparation method provided by the invention separates infiltrating lymphocytes from tumor tissues, inoculates the infiltrating lymphocytes into a culture bottle coated by recombinant human fibrin and CD3 antibody, adds IL-2, cultures to logarithmic growth phase, uses lentivirus containing IL-7, IL-15 or IL-7-IL-15 fusion gene to stably transform TIL cells, cultures for about 72h, adds screening antibiotic Puromycin (Puromycin), does not add IL-2 for culture after two weeks, uses inducer tetracycline or analogues thereof to induce the expression of IL-7 or/and IL-15, is used for maintaining the amplification culture of the TIL cells, and obtains the cells after the cells are amplified to a certain multiple. The method induces TIL cells to express transgenic IL-7 or/and IL-15 by adding tetracycline inducer, and the later culture period can be independent of the addition culture of high-concentration IL-2; and the number of returned cells can be reduced. When TIL is infused back into a patient, high-dose IL-2 does not need to be infused, and only a certain amount of tetracycline or derivatives thereof need to be injected according to needs, so that cells can be greatly amplified in vivo to maintain activity, and the systemic toxicity of high-concentration IL-2 infusion to a human body is avoided; in addition, the method of the present invention does not require concern about toxicity problems that may be caused by the constant exposure of human bodies to IL-7 and/or IL-15, since IL-7 or/and IL-15 are hardly expressed by TIL cells without the input of exogenous inducers.
Drawings
FIG. 1 is a diagram showing the results of ELISA detection of IL-7 and IL-15 secretion from non-small cell lung cancer transgenic TIL;
FIG. 2 shows the number of TIL cells obtained by different culture methods for non-small cell lung cancer;
FIG. 3 shows expansion of TIL cells obtained by different culture methods for non-small cell lung cancer;
FIG. 4 is a graph showing the results of target cell killing by TIL obtained from different culturing methods of non-small cell lung cancer;
FIG. 5 is a diagram showing the results of ELISA detection of IL-7 and IL-15 secretion from transgenic TIL of breast cancer;
FIG. 6 shows the number of TIL cells obtained by different culturing methods for breast cancer;
FIG. 7 shows the expansion fold of TIL cells obtained by different culture methods for breast cancer;
FIG. 8 is a graph showing the results of target cell killing by TIL obtained from different breast cancer culture methods.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The invention provides a preparation method of infiltrating lymphocytes, which comprises the following specific steps:
1) preparing recombinant lentivirus containing target genes;
the method specifically comprises the following steps: obtaining and optimizing and synthesizing an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence, inoculating the sequences into a lentiviral vector, then transfecting HEK-293T cells to carry out virus preparation and amplification, and culturing to obtain a recombinant lentivirus;
wherein the IL-7 sequence is shown in SEQ ID No: 1 is shown in the specification; the IL-15 sequence is shown as SEQ ID No: 2 is shown in the specification; the IL-7-IL-15 fusion sequence is shown as SEQ ID No: 3 is shown in the specification;
2) digesting the tumor tissue to obtain a single cell suspension, culturing the single cell suspension by using IL-2, and separating and purifying the infiltrated lymphocytes; then in a culture environment coated by recombinant human fibrin and CD3 antibody, continuously adding IL-2 into the purified infiltrating lymphocytes, and culturing until the logarithmic phase is reached;
3) adding the recombinant lentivirus into infiltrating lymphocytes cultured to a logarithmic growth phase, and adding Polybrene at the same time, and transfecting for 3 days; then adding puromycin for screening culture, culturing until all cells are transgenic positive cells, and then adding IL-2, but adding an inducer to culture and infiltrate the lymphocytes;
4) using Tet-on system, tetracycline or tetracycline derivative is added to induce the expression of IL-7 and/or IL-15, and the amplification culture of infiltrating lymphocytes is maintained.
Example 1 construction of lentiviral expression vectors for IL-7, IL-15, and IL-7-IL-15 genes
1. Construction of plasmid containing IL-7 or IL-15 or IL-7-IL-15 Gene
The CDS sequences of IL-7 and IL-15 are downloaded from NCBI and then codon optimized to enhance their expression, including but not limited to: human codon usage bias, modest GC content, stable mRNA secondary structure, etc., eliminates repetitive sequences and cryptic splice sites and unwanted restriction sites, while preventing depletion of the tRNA pool in the cell. The optimized sequence is directly subjected to gene synthesis, and the same double-enzyme-cut Tet-on expression plasmid pLVX-TetOne-Puro is connected into the sequence by utilizing the enzyme cutting sites of ECoRI and BamHI. The plasmids with correct enzyme digestion verification and sequencing are named pLVX-IL-7, pLVX-IL-15 and pLVX-IL-7-IL15 respectively. IL-7 and IL-15 are linked by IRES. IRES is an Internal secondary structure-rich ribosome entry site (Internal ribosome entry site) that mediates ribosome binding to RNA and initiates protein translation. IRES enables initiation of protein translation independent of the cap structure of messenger RNA 5' and initiation of RNA translation of proteins from the middle, thus ensuring correct expression of functional proteins by both IL-7 and IL-15.
2. Lentiviral preparation and titer determination
Respectively extracting and purifying pLVX-TetOne-Puro plasmids, pRSV-Rev vectors, pMD2.G vectors and pMDLg/pRRE vectors which contain IL-7, IL-15 and IL-7-1L-15 genes and are obtained in the steps, mixing and transfecting HEK-293T cells growing to about 70% of density by using Lipo2000 according to a certain proportion, replacing the cells with complete culture media after 8 hours, respectively collecting culture fluids after culturing for 48 hours and 72 hours, centrifuging and reserving supernatant, filtering the supernatant by using a 0.45 mu m filter, and obtaining filtrate which is the original solution of the recombinant lentivirus.
Recombinant lentiviruses were concentrated using a Lentivirus Concentration kit (YEASEN, 41101ES 50).
Subsequent lentivirus titer assays were performed by seeding HEK-293T cells into 24-well plates at 1X 10 per well5And cultured overnight at 37 ℃. Resuspending virus stock solution by DMEM medium, the virus stock solution amount of the first hole is 10 mu L, then carrying out gradient dilution by 10 times, adding 100 mu L of culture medium-virus mixed solution into each hole, and carrying out 3 repeated holes on each diluted concentration. Put in 5% CO at 37 DEG C2After culturing in the incubator for 24h, the culture medium containing the virus DMEM is replaced by a DMEM complete culture medium without virus liquid, and after continuous culture for 48h, the proportion of cells with positive transgene expression is detected by using a flow cytometer, and the virus titer is calculated. The sum of the total number of 3 replicate wells was calculated and averaged, assuming a (average number of fluorescent cells in the penultimate visible fluorescence well) and B (average number of fluorescent cells in the penultimate visible fluorescence well).
Lentivirus titer calculation formula: lentivirus titer (TU/ml) = (a + B10) × 1000/2/a pore virus amount.
EXAMPLE 2 preparation of non-Small cell Lung cancer TIL cells
1. Tissue pretreatment: wiping the outer surfaces of the tumor tissue specimen collecting tube, the reagent, the operating instrument and the like with 75% ethanol, putting the tumor tissue specimen collecting tube, the reagent, the operating instrument and the like into a biological safety cabinet, opening a tumor tissue specimen collecting bottle in the biological safety cabinet, cleaning and treating tumor tissues, and removing blood vessels, envelopes and other necrotic tissues.
2. Tissue digestion:
1) shearing tumor tissue in tissue enzymolysis solution to near meat foam, adding tissue enzymolysis solution for suspension, transferring suspension into a centrifugal tube, placing into an electric heating constant temperature oscillator, setting temperature at 37 deg.C, oscillation frequency at 150rpm/min, time 30-60min, and digesting at constant temperature.
2) And (3) taking 40mL of termination protection solution, placing the termination protection solution in a 50mL centrifuge tube for later use, taking a 70 mu m filter screen, placing the filter screen at the 50mL centrifuge tube opening, and preparing 15 mL disposable syringe for later use.
3) And (4) taking out the centrifuge tube from the oscillator, gradually adding the centrifuge tube into the filter screen in the previous step by using a pipette, grinding and filtering by using a grinding rod, washing while grinding, and collecting the single cell suspension.
4) Centrifuging the single cell suspension collected in 3) for 8min at 500g, and discarding the supernatant. The cell pellet was resuspended in 10mL X-VIVO-15 medium, the sample was counted, and the remaining cells were centrifuged at 500g for 8 min.
3. Plating culture
The purpose of removing residual protein in the cell suspension can be achieved by centrifugation. According to the counting result, the cell density is adjusted to 0.5-1X 106/mL, placed in 12 or 24-well culture plates, and added with 1000-3000IU/mL IL-2. Is placed in CO2Culturing in constant-temperature incubator at 37 deg.C with CO2The content was 5%.
4. Isolation and purification of TIL cells (stage S1)
1) Liquid changing and hole expanding: observing cells in B + A stage culture chamber every day under microscope, such as no obviously adherent cells, and changing liquid every other day, such as no gradual increase of lymphocytes or cancerContinuing to change the liquid if the cells are not reduced; if the density of lymphocytes is obviously increased and cell clusters appear, the cell clusters need to be expanded and then are placed at 37 ℃ under 5 percent CO2And (5) culturing in an incubator.
2) Collecting: culturing for no more than 10 days, and collecting cells. Sampling to detect the number/activity of the cells, and performing cell cryopreservation or entering the S2 stage according to the counting result. Tumor cells, fiber cells and other hybrid cells unrelated to TIL in the tissue can be removed through S1 stage culture. The growth of the cells is observed under a microscope every day to determine the subsequent process operations that need to be performed. The observed indicators include changes in the number of lymphocytes, tumor cells, and other cells and the degree of separation of lymphocytes.
5. Rapid expansion culture of TIL cells (stage S2)
1) Day 0: pre-coated, 1-5. mu.g/mL CD3 antibody + 10-15. mu.g/mL recombinant human fibrin (Retronectin abbreviated RN) was added to the flask and coated at 4 ℃. Before use, the coating solution was aspirated and washed 2-3 times with PBS. Take 2X 107TIL cell transfer flasks in CO2Culturing in a constant-temperature cell culture box, and setting the temperature: 37 ℃ CO2: 5% of the total amount of the cells, and the number of the cells in each group starting to be cultured is 2X 107And (4) respectively.
2) Day 1: adding serum substitute for CTS immunocyte into each group, adding 1000-3000IU/mL IL-2 into the first, fifth, sixth and seventh groups, adding 25 ng/mL IL-7 into the second group, adding 25 ng/mL IL-15 into the third group, adding 25 ng/mL IL-7 and IL-15 into the fourth group, and adding fluid replacement into the fourth group, observing cells under the microscope, sampling and counting, if TIL cells are gradually increased, so as to maintain the cell density at about 1 × 106/mL, after completion, placing in CO2Cell culture incubator, temperature: 37 ℃ CO2：5%。
3) Day 2: recombinant lentivirus transfection, the next day, the recombinant lentiviruses containing the IL-7, I-15 and IL-7-IL-15 gene sequences prepared in example 1 were added to five, six and seven pairs of TIL cells in the exponential growth phase, respectively, together with 8. mu.g/ml polybrene. After 3 days of transfection, 2ug/mL puromycin was added for selection and all cells were positive for the transgene after approximately 10-12 days of selection culture.
4) Day 13: the addition of IL-2 was started to be removed at the time of the five-, six-and seven-group exchanges, the culture expansion of TIL was maintained by inducing the expression of IL-7, IL-15 and IL-7-1L-15 by adding 0.5. mu.g/mL tetracycline hydrochloride (Sigma, Cat.: T3383), respectively, and the supernatants were taken the second day after the addition of the inducer to examine the secretion of cytokines IL-7 and IL-15 (IL-7 Human ELISA Kit, Invitrogen, Cat.: EH # IL7X 5; IL-15 Human ELISA Kit, Invitrogen, Cat.: BMS 2106).
As shown in FIG. 1, compared with the control group, the transgenic group was able to produce a large amount of IL-7 or/and IL-15 under the action of the inducing agent tetracycline. The transgenes IL-7 and IL-7-IL-15 TIL can respectively generate 34948.8 pg/mL and 34176.2 pg/mL IL-7; the transgenes IL-15 and IL-7-IL-15 TIL were able to produce 47615.5 pg/mL and 46209.6 pg/mL IL-15, respectively. The first group added 1000-3000IU/mL IL-2 for each liquid change, the second group added 25 ng/mL IL-7, the third group added 25 ng/mL IL-15, and the fourth group added 25 ng/mL IL-7 and IL-15 simultaneously.
5) Day 23-25: the culture is continued for 10-12 days, at which time the cell number can reach the return number. Each group was counted and the amplification fold was detected.
As shown in FIGS. 2 and 3, the transgenic method of the present invention can significantly increase the amplification factor of TIL cells, especially the IL-7-IL-15 transgenic group can reach the highest amplification factor, which reaches 2700 times.
6. TIL cell killing function assay
1) Taking a human lung adenocarcinoma cell line H1975 in an exponential growth phase for trypan blue staining, and counting; 8000 Target cells/100. mu.L per well in a 96-well plate at 37 ℃ with 5% CO2Incubating in an incubator overnight to allow the cells to adhere to the wall;
2) adding effector cells (the TIL cells harvested in the step 5 above, which are HLA-matched TIL cells that match the target cells) to target cell wells at effective-to-target ratios of 10:1 and 5:1, and making controls for separate culture of a corresponding amount of TIL; PBMC were used for negative control target cells; then transferring the mixture to 37 ℃ and 5% CO2An incubator for incubation for 24 hours;
3) after 24h, sucking 100 mu L of supernatant, and adding 20 mu L/well of MTS reagent into the well;
4）37℃，5% CO2incubating the incubator for 0.5-4 h in a dark place;
5) detecting the absorbance value at 490nm by using a microplate reader;
6) the killing rate at each effect target ratio was calculated according to the following formula.
Cytoxicity (%) =1- (OD co-incubation group-ODTIL culture alone group)/(OD tumor cell negative control group-OD blank control group) × 100%.
And (4) analyzing results: as shown in fig. 4, PBMC without specific killing of healthy people was used as a negative control group, and the group theoretically had a specific killing of 0. Whether E: T ═ 10:1 or 5:1, the seventh group of transgenic TILs (IL-7-IL-15) all showed the strongest killing effect, more than 90% killing was achieved when E: T ═ 20:1, E: T ═ 10: the killing rate is reduced to about 75% at 1 hour. These results indicate that the transgenic IL-7-IL-15 TIL cells have a greater ability to kill target cells than normal IL-2 cultured TIL cells.
EXAMPLE 3 preparation of breast cancer TIL cells
The breast cancer tissue treatment and digestion, TIL cell isolation and purification, and scale-up procedure were as in example 2. After screening transgenic positive cells and adding tetracycline inducer in the amplification culture stage, the level of IL-7 and IL-15 secretion of the transgenic TIL is detected by using an ELISA kit.
The results are shown in FIG. 5, where the breast cancer-derived transgenic TIL also secreted high levels of IL-7 and IL-15. The transgenic IL-7 and IL-7-IL-15 TIL produced 39282 pg/mL and 38746.5 pg/mL IL-7, respectively, compared to the control group; the transgenes IL-15 and IL-7-IL-15 TIL were able to produce 54114.3 pg/mL and 52106.1 pg/mL IL-15, respectively.
Cell expansion numbers and fold were measured after completion of the entire S2 phase.
The results are shown in FIGS. 6 and 7, where the IL-7-IL-15 transgenic TIL had the highest cell number, which was 2.89X 10102 to 10 from the beginning7The number was amplified 1445-fold. All groups achieved the cell therapy reinfusion dosage requirements.
The prepared breast cancer TIL cell is also subjected to killing effect detection, the breast cancer tumor cell line is MDA-MB-231, and the effector cell TIL and the target cell line are matched by HLA.
As can be seen from FIG. 8, the IL-7-IL-15 TIL cells cultured by the transgenic method of the invention have the strongest killing activity, which is significantly higher than that of the traditional method, and the method is fully demonstrated to be an effective method for culturing the TIL cells.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Beijing Biotechnology biomedical research Co., Ltd
<120> preparation method for enhancing lethality of tumor infiltrating lymphocytes
<130> P2022F0114-XS
<141> 2022-03-22
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Ala Cys Thr Cys Cys Cys Cys Cys Cys Cys Cys Thr Gly Ala Thr Cys
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Cys Thr Cys Gly Thr Cys Cys Thr Cys Cys Thr Cys Cys Cys Cys Gly
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Thr Cys Gly Cys Thr Thr Cys Gly Thr Cys Gly Gly Ala Cys Thr Gly
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Cys Gly Ala Cys Ala Thr Cys Gly Ala Ala Gly Gly Ala Ala Ala Ala
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Gly Ala Cys Gly Gly Ala Ala Ala Ala Cys Ala Ala Thr Ala Cys Gly
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Ala Ala Thr Cys Gly Gly Thr Gly Cys Thr Cys Ala Thr Gly Gly Thr
115 120 125
Gly Thr Cys Gly Ala Thr Cys Gly Ala Cys Cys Ala Gly Cys Thr Gly
130 135 140
Cys Thr Gly Gly Ala Cys Thr Cys Gly Ala Thr Gly Ala Ala Ala Gly
145 150 155 160
Ala Ala Ala Thr Cys Gly Gly Ala Thr Cys Gly Ala Ala Cys Thr Gly
165 170 175
Cys Cys Thr Cys Ala Ala Cys Ala Ala Cys Gly Ala Ala Thr Thr Thr
180 185 190
Ala Ala Cys Thr Thr Thr Thr Thr Thr Ala Ala Ala Ala Gly Ala Cys
195 200 205
Ala Thr Ala Thr Cys Thr Gly Cys Gly Ala Cys Gly Cys Thr Ala Ala
210 215 220
Cys Ala Ala Gly Gly Ala Ala Gly Gly Ala Ala Thr Gly Thr Thr Thr
225 230 235 240
Cys Thr Cys Thr Thr Thr Cys Gly Cys Gly Cys Thr Gly Cys Thr Cys
245 250 255
Gly Ala Ala Ala Ala Cys Thr Gly Cys Gly Ala Cys Ala Gly Thr Thr
260 265 270
Thr Cys Thr Cys Ala Ala Ala Ala Thr Gly Ala Ala Cys Thr Cys Gly
275 280 285
Ala Cys Gly Gly Gly Ala Gly Ala Cys Thr Thr Thr Gly Ala Cys Cys
290 295 300
Thr Cys Cys Ala Cys Cys Thr Cys Cys Thr Gly Ala Ala Gly Gly Thr
305 310 315 320
Gly Thr Cys Gly Gly Ala Ala Gly Gly Ala Ala Cys Gly Ala Cys Gly
325 330 335
Ala Thr Cys Cys Thr Gly Cys Thr Gly Ala Ala Cys Thr Gly Cys Ala
340 345 350
Cys Gly Gly Gly Ala Cys Ala Gly Gly Thr Cys Ala Ala Ala Gly Gly
355 360 365
Ala Cys Gly Ala Ala Ala Ala Cys Cys Cys Gly Cys Thr Gly Cys Thr
370 375 380
Cys Thr Cys Gly Gly Ala Gly Ala Ala Gly Cys Thr Cys Ala Gly Cys
385 390 395 400
Cys Cys Ala Cys Gly Ala Ala Gly Thr Cys Gly Cys Thr Gly Gly Ala
405 410 415
Ala Gly Ala Ala Ala Ala Cys Ala Ala Ala Thr Cys Gly Cys Thr Gly
420 425 430
Ala Ala Gly Gly Ala Ala Cys Ala Ala Ala Ala Ala Ala Ala Ala Cys
435 440 445
Thr Gly Ala Ala Cys Gly Ala Cys Cys Thr Cys Thr Gly Cys Thr Thr
450 455 460
Thr Cys Thr Cys Ala Ala Gly Cys Gly Cys Cys Thr Cys Cys Thr Cys
465 470 475 480
Cys Ala Gly Gly Ala Ala Ala Thr Cys Ala Ala Gly Ala Cys Gly Thr
485 490 495
Gly Cys Thr Gly Gly Ala Ala Cys Ala Ala Gly Ala Thr Cys Cys Thr
500 505 510
Gly Ala Thr Gly Gly Gly Ala Ala Cys Gly Ala Ala Ala Gly Ala Ala
515 520 525
Cys Ala Thr Thr Gly Ala
530
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Ala Thr Gly Ala Gly Ala Ala Thr Cys Thr Cys Gly Ala Ala Ala Cys
1 5 10 15
Cys Cys Cys Ala Cys Cys Thr Gly Ala Gly Ala Thr Cys Gly Ala Thr
20 25 30
Cys Thr Cys Gly Ala Thr Cys Cys Ala Ala Thr Gly Cys Thr Ala Cys
35 40 45
Cys Thr Gly Thr Gly Cys Cys Thr Cys Cys Thr Gly Cys Thr Gly Ala
50 55 60
Ala Cys Thr Cys Gly Cys Ala Cys Thr Thr Thr Cys Thr Cys Ala Cys
65 70 75 80
Gly Gly Ala Ala Gly Cys Thr Gly Gly Ala Ala Thr Cys Cys Ala Cys
85 90 95
Gly Thr Gly Thr Thr Thr Ala Thr Cys Cys Thr Gly Gly Gly Ala Thr
100 105 110
Gly Cys Thr Thr Thr Thr Cys Gly Gly Cys Thr Gly Gly Ala Cys Thr
115 120 125
Gly Cys Cys Cys Ala Ala Gly Ala Cys Gly Gly Ala Ala Gly Cys Thr
130 135 140
Ala Ala Cys Thr Gly Gly Gly Thr Cys Ala Ala Cys Gly Thr Cys Ala
145 150 155 160
Thr Cys Thr Cys Gly Gly Ala Cys Cys Thr Gly Ala Ala Ala Ala Ala
165 170 175
Gly Ala Thr Cys Gly Ala Ala Gly Ala Cys Cys Thr Gly Ala Thr Cys
180 185 190
Cys Ala Ala Thr Cys Gly Ala Thr Gly Cys Ala Thr Ala Thr Cys Gly
195 200 205
Ala Cys Gly Cys Thr Ala Cys Gly Cys Thr Cys Thr Ala Cys Ala Cys
210 215 220
Gly Gly Ala Ala Thr Cys Gly Gly Ala Cys Gly Thr Gly Cys Ala Thr
225 230 235 240
Cys Cys Cys Thr Cys Gly Thr Gly Cys Ala Ala Ala Gly Thr Cys Ala
245 250 255
Cys Gly Gly Cys Thr Ala Thr Gly Ala Ala Gly Thr Gly Cys Thr Thr
260 265 270
Thr Cys Thr Cys Cys Thr Gly Gly Ala Ala Cys Thr Gly Cys Ala Ala
275 280 285
Gly Thr Cys Ala Thr Cys Thr Cys Gly Cys Thr Gly Gly Ala Ala Thr
290 295 300
Cys Gly Gly Gly Ala Gly Ala Cys Gly Cys Thr Thr Cys Gly Ala Thr
305 310 315 320
Cys Cys Ala Cys Gly Ala Cys Ala Cys Gly Gly Thr Cys Gly Ala Ala
325 330 335
Ala Ala Cys Cys Thr Cys Ala Thr Cys Ala Thr Cys Cys Thr Cys Gly
340 345 350
Cys Thr Ala Ala Cys Ala Ala Cys Thr Cys Gly Cys Thr Gly Thr Cys
355 360 365
Gly Thr Cys Gly Ala Ala Cys Gly Gly Ala Ala Ala Cys Gly Thr Cys
370 375 380
Ala Cys Gly Gly Ala Ala Thr Cys Gly Gly Gly Ala Thr Gly Cys Ala
385 390 395 400
Ala Gly Gly Ala Ala Thr Gly Cys Gly Ala Ala Gly Ala Ala Cys Thr
405 410 415
Gly Gly Ala Ala Gly Ala Ala Ala Ala Ala Ala Ala Cys Ala Thr Cys
420 425 430
Ala Ala Ala Gly Ala Ala Thr Thr Thr Cys Thr Gly Cys Ala Gly Thr
435 440 445
Cys Gly Thr Thr Thr Gly Thr Gly Cys Ala Cys Ala Thr Cys Gly Thr
450 455 460
Cys Cys Ala Gly Ala Thr Gly Thr Thr Thr Ala Thr Cys Ala Ala Cys
465 470 475 480
Ala Cys Gly Thr Cys Gly Thr Gly Ala
485
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<212> PRT
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Ala Thr Gly Thr Thr Thr Cys Ala Thr Gly Thr Cys Thr Cys Gly Thr
1 5 10 15
Thr Thr Cys Gly Ala Thr Ala Cys Ala Thr Cys Thr Thr Thr Gly Gly
20 25 30
Ala Cys Thr Cys Cys Cys Cys Cys Cys Cys Cys Thr Gly Ala Thr Cys
35 40 45
Cys Thr Cys Gly Thr Cys Cys Thr Cys Cys Thr Cys Cys Cys Cys Gly
50 55 60
Thr Cys Gly Cys Thr Thr Cys Gly Thr Cys Gly Gly Ala Cys Thr Gly
65 70 75 80
Cys Gly Ala Cys Ala Thr Cys Gly Ala Ala Gly Gly Ala Ala Ala Ala
85 90 95
Gly Ala Cys Gly Gly Ala Ala Ala Ala Cys Ala Ala Thr Ala Cys Gly
100 105 110
Ala Ala Thr Cys Gly Gly Thr Gly Cys Thr Cys Ala Thr Gly Gly Thr
115 120 125
Gly Thr Cys Gly Ala Thr Cys Gly Ala Cys Cys Ala Gly Cys Thr Gly
130 135 140
Cys Thr Gly Gly Ala Cys Thr Cys Gly Ala Thr Gly Ala Ala Ala Gly
145 150 155 160
Ala Ala Ala Thr Cys Gly Gly Ala Thr Cys Gly Ala Ala Cys Thr Gly
165 170 175
Cys Cys Thr Cys Ala Ala Cys Ala Ala Cys Gly Ala Ala Thr Thr Thr
180 185 190
Ala Ala Cys Thr Thr Thr Thr Thr Thr Ala Ala Ala Ala Gly Ala Cys
195 200 205
Ala Thr Ala Thr Cys Thr Gly Cys Gly Ala Cys Gly Cys Thr Ala Ala
210 215 220
Cys Ala Ala Gly Gly Ala Ala Gly Gly Ala Ala Thr Gly Thr Thr Thr
225 230 235 240
Cys Thr Cys Thr Thr Thr Cys Gly Cys Gly Cys Thr Gly Cys Thr Cys
245 250 255
Gly Ala Ala Ala Ala Cys Thr Gly Cys Gly Ala Cys Ala Gly Thr Thr
260 265 270
Thr Cys Thr Cys Ala Ala Ala Ala Thr Gly Ala Ala Cys Thr Cys Gly
275 280 285
Ala Cys Gly Gly Gly Ala Gly Ala Cys Thr Thr Thr Gly Ala Cys Cys
290 295 300
Thr Cys Cys Ala Cys Cys Thr Cys Cys Thr Gly Ala Ala Gly Gly Thr
305 310 315 320
Gly Thr Cys Gly Gly Ala Ala Gly Gly Ala Ala Cys Gly Ala Cys Gly
325 330 335
Ala Thr Cys Cys Thr Gly Cys Thr Gly Ala Ala Cys Thr Gly Cys Ala
340 345 350
Cys Gly Gly Gly Ala Cys Ala Gly Gly Thr Cys Ala Ala Ala Gly Gly
355 360 365
Ala Cys Gly Ala Ala Ala Ala Cys Cys Cys Gly Cys Thr Gly Cys Thr
370 375 380
Cys Thr Cys Gly Gly Ala Gly Ala Ala Gly Cys Thr Cys Ala Gly Cys
385 390 395 400
Cys Cys Ala Cys Gly Ala Ala Gly Thr Cys Gly Cys Thr Gly Gly Ala
405 410 415
Ala Gly Ala Ala Ala Ala Cys Ala Ala Ala Thr Cys Gly Cys Thr Gly
420 425 430
Ala Ala Gly Gly Ala Ala Cys Ala Ala Ala Ala Ala Ala Ala Ala Cys
435 440 445
Thr Gly Ala Ala Cys Gly Ala Cys Cys Thr Cys Thr Gly Cys Thr Thr
450 455 460
Thr Cys Thr Cys Ala Ala Gly Cys Gly Cys Cys Thr Cys Cys Thr Cys
465 470 475 480
Cys Ala Gly Gly Ala Ala Ala Thr Cys Ala Ala Gly Ala Cys Gly Thr
485 490 495
Gly Cys Thr Gly Gly Ala Ala Cys Ala Ala Gly Ala Thr Cys Cys Thr
500 505 510
Gly Ala Thr Gly Gly Gly Ala Ala Cys Gly Ala Ala Ala Gly Ala Ala
515 520 525
Cys Ala Thr Thr Gly Ala Cys Cys Cys Cys Thr Cys Thr Cys Cys Cys
530 535 540
Thr Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Thr Ala Ala Cys Gly
545 550 555 560
Thr Thr Ala Cys Thr Gly Gly Cys Cys Gly Ala Ala Gly Cys Cys Gly
565 570 575
Cys Thr Thr Gly Gly Ala Ala Thr Ala Ala Gly Gly Cys Cys Gly Gly
580 585 590
Thr Gly Thr Gly Cys Gly Thr Thr Thr Gly Thr Cys Thr Ala Thr Ala
595 600 605
Thr Gly Thr Thr Ala Thr Thr Thr Thr Cys Cys Ala Cys Cys Ala Thr
610 615 620
Ala Thr Thr Gly Cys Cys Gly Thr Cys Thr Thr Thr Thr Gly Gly Cys
625 630 635 640
Ala Ala Thr Gly Thr Gly Ala Gly Gly Gly Cys Cys Cys Gly Gly Ala
645 650 655
Ala Ala Cys Cys Thr Gly Gly Cys Cys Cys Thr Gly Thr Cys Thr Thr
660 665 670
Cys Thr Thr Gly Ala Cys Gly Ala Gly Cys Ala Thr Thr Cys Cys Thr
675 680 685
Ala Gly Gly Gly Gly Thr Cys Thr Thr Thr Cys Cys Cys Cys Thr Cys
690 695 700
Thr Cys Gly Cys Cys Ala Ala Ala Gly Gly Ala Ala Thr Gly Cys Ala
705 710 715 720
Ala Gly Gly Thr Cys Thr Gly Thr Thr Gly Ala Ala Thr Gly Thr Cys
725 730 735
Gly Thr Gly Ala Ala Gly Gly Ala Ala Gly Cys Ala Gly Thr Thr Cys
740 745 750
Cys Thr Cys Thr Gly Gly Ala Ala Gly Cys Thr Thr Cys Thr Thr Gly
755 760 765
Ala Ala Gly Ala Cys Ala Ala Ala Cys Ala Ala Cys Gly Thr Cys Thr
770 775 780
Gly Thr Ala Gly Cys Gly Ala Cys Cys Cys Thr Thr Thr Gly Cys Ala
785 790 795 800
Gly Gly Cys Ala Gly Cys Gly Gly Ala Ala Cys Cys Cys Cys Cys Cys
805 810 815
Ala Cys Cys Thr Gly Gly Cys Ala Ala Cys Ala Gly Gly Thr Gly Cys
820 825 830
Cys Thr Cys Thr Gly Cys Gly Gly Cys Cys Ala Ala Ala Ala Gly Cys
835 840 845
Cys Ala Cys Gly Thr Gly Thr Ala Thr Ala Ala Gly Ala Thr Ala Cys
850 855 860
Ala Cys Cys Thr Gly Cys Ala Ala Ala Gly Gly Cys Gly Gly Cys Ala
865 870 875 880
Cys Ala Ala Cys Cys Cys Cys Ala Gly Thr Gly Cys Cys Ala Cys Gly
885 890 895
Thr Thr Gly Thr Gly Ala Gly Thr Thr Gly Gly Ala Thr Ala Gly Thr
900 905 910
Thr Gly Thr Gly Gly Ala Ala Ala Gly Ala Gly Thr Cys Ala Ala Ala
915 920 925
Thr Gly Gly Cys Thr Cys Thr Cys Cys Thr Cys Ala Ala Gly Cys Gly
930 935 940
Thr Ala Thr Thr Cys Ala Ala Cys Ala Ala Gly Gly Gly Gly Cys Thr
945 950 955 960
Gly Ala Ala Gly Gly Ala Thr Gly Cys Cys Cys Ala Gly Ala Ala Gly
965 970 975
Gly Thr Ala Cys Cys Cys Cys Ala Thr Thr Gly Thr Ala Thr Gly Gly
980 985 990
Gly Ala Thr Cys Thr Gly Ala Thr Cys Thr Gly Gly Gly Gly Cys Cys
995 1000 1005
Thr Cys Gly Gly Thr Gly Cys Ala Cys Ala Thr Gly Cys Thr Thr Thr
1010 1015 1020
Ala Cys Ala Thr Gly Thr Gly Thr Thr Thr Ala Gly Thr Cys Gly Ala
1025 1030 1035 1040
Gly Gly Thr Thr Ala Ala Ala Ala Ala Ala Ala Cys Gly Thr Cys Thr
1045 1050 1055
Ala Gly Gly Cys Cys Cys Cys Cys Cys Gly Ala Ala Cys Cys Ala Cys
1060 1065 1070
Gly Gly Gly Gly Ala Cys Gly Thr Gly Gly Thr Thr Thr Thr Cys Cys
1075 1080 1085
Thr Thr Thr Gly Ala Ala Ala Ala Ala Cys Ala Cys Gly Ala Thr Gly
1090 1095 1100
Ala Thr Ala Ala Ala Thr Gly Ala Gly Ala Ala Thr Cys Thr Cys Gly
1105 1110 1115 1120
Ala Ala Ala Cys Cys Cys Cys Ala Cys Cys Thr Gly Ala Gly Ala Thr
1125 1130 1135
Cys Gly Ala Thr Cys Thr Cys Gly Ala Thr Cys Cys Ala Ala Thr Gly
1140 1145 1150
Cys Thr Ala Cys Cys Thr Gly Thr Gly Cys Cys Thr Cys Cys Thr Gly
1155 1160 1165
Cys Thr Gly Ala Ala Cys Thr Cys Gly Cys Ala Cys Thr Thr Thr Cys
1170 1175 1180
Thr Cys Ala Cys Gly Gly Ala Ala Gly Cys Thr Gly Gly Ala Ala Thr
1185 1190 1195 1200
Cys Cys Ala Cys Gly Thr Gly Thr Thr Thr Ala Thr Cys Cys Thr Gly
1205 1210 1215
Gly Gly Ala Thr Gly Cys Thr Thr Thr Thr Cys Gly Gly Cys Thr Gly
1220 1225 1230
Gly Ala Cys Thr Gly Cys Cys Cys Ala Ala Gly Ala Cys Gly Gly Ala
1235 1240 1245
Ala Gly Cys Thr Ala Ala Cys Thr Gly Gly Gly Thr Cys Ala Ala Cys
1250 1255 1260
Gly Thr Cys Ala Thr Cys Thr Cys Gly Gly Ala Cys Cys Thr Gly Ala
1265 1270 1275 1280
Ala Ala Ala Ala Gly Ala Thr Cys Gly Ala Ala Gly Ala Cys Cys Thr
1285 1290 1295
Gly Ala Thr Cys Cys Ala Ala Thr Cys Gly Ala Thr Gly Cys Ala Thr
1300 1305 1310
Ala Thr Cys Gly Ala Cys Gly Cys Thr Ala Cys Gly Cys Thr Cys Thr
1315 1320 1325
Ala Cys Ala Cys Gly Gly Ala Ala Thr Cys Gly Gly Ala Cys Gly Thr
1330 1335 1340
Gly Cys Ala Thr Cys Cys Cys Thr Cys Gly Thr Gly Cys Ala Ala Ala
1345 1350 1355 1360
Gly Thr Cys Ala Cys Gly Gly Cys Thr Ala Thr Gly Ala Ala Gly Thr
1365 1370 1375
Gly Cys Thr Thr Thr Cys Thr Cys Cys Thr Gly Gly Ala Ala Cys Thr
1380 1385 1390
Gly Cys Ala Ala Gly Thr Cys Ala Thr Cys Thr Cys Gly Cys Thr Gly
1395 1400 1405
Gly Ala Ala Thr Cys Gly Gly Gly Ala Gly Ala Cys Gly Cys Thr Thr
1410 1415 1420
Cys Gly Ala Thr Cys Cys Ala Cys Gly Ala Cys Ala Cys Gly Gly Thr
1425 1430 1435 1440
Cys Gly Ala Ala Ala Ala Cys Cys Thr Cys Ala Thr Cys Ala Thr Cys
1445 1450 1455
Cys Thr Cys Gly Cys Thr Ala Ala Cys Ala Ala Cys Thr Cys Gly Cys
1460 1465 1470
Thr Gly Thr Cys Gly Thr Cys Gly Ala Ala Cys Gly Gly Ala Ala Ala
1475 1480 1485
Cys Gly Thr Cys Ala Cys Gly Gly Ala Ala Thr Cys Gly Gly Gly Ala
1490 1495 1500
Thr Gly Cys Ala Ala Gly Gly Ala Ala Thr Gly Cys Gly Ala Ala Gly
1505 1510 1515 1520
Ala Ala Cys Thr Gly Gly Ala Ala Gly Ala Ala Ala Ala Ala Ala Ala
1525 1530 1535
Cys Ala Thr Cys Ala Ala Ala Gly Ala Ala Thr Thr Thr Cys Thr Gly
1540 1545 1550
Cys Ala Gly Thr Cys Gly Thr Thr Thr Gly Thr Gly Cys Ala Cys Ala
1555 1560 1565
Thr Cys Gly Thr Cys Cys Ala Gly Ala Thr Gly Thr Thr Thr Ala Thr
1570 1575 1580
Cys Ala Ala Cys Ala Cys Gly Thr Cys Gly Thr Gly Ala
1585 1590 1595
Claims (10)
1. A preparation method of tumor infiltrating lymphocytes is characterized by comprising the following steps:
1) preparing recombinant lentivirus containing target genes; wherein the target gene comprises an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence;
2) separating infiltrating lymphocytes from the tumor tissue, and adding IL-2 into the infiltrating lymphocytes to perform cell culture to a logarithmic growth phase;
3) adding the recombinant lentivirus into infiltrating lymphocytes cultured to a logarithmic phase, culturing for 2-4 days, adding a screening agent for screening culture, and culturing until all cells are transgenic positive cells;
4) and (3) using a Tet-on system, adding an inducer to induce the expression of IL-7 or/and IL-15, and maintaining the expansion culture of the infiltrating lymphocytes.
2. The method of claim 1, wherein the recombinant lentivirus is produced by: and obtaining and optimizing an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence, inoculating the sequences into a lentiviral vector, transfecting HEK-293T cells to carry out virus preparation and amplification, and culturing to obtain the recombinant lentivirus.
3. The method of any one of claims 1 or 2, wherein the IL-7 and IL-15 of the IL-7-IL-15 fusion sequence are linked by an IRES.
4. The method of claim 1, wherein the IL-7-IL-15 fusion sequence is set forth in SEQ ID No: 3, respectively.
5. The method of any one of claims 1 or 4, wherein the IL-7 sequence is as set forth in SEQ ID No: 1 is shown in the specification; the IL-15 sequence is shown as SEQ ID No: 2, respectively.
6. The method according to claim 1, wherein the cell culture in step 2) is performed in a culture environment coated with recombinant human fibrin and CD3 antibody.
7. The method of claim 1, wherein the inducing agent is tetracycline or a tetracycline derivative.
8. The method of claim 1, comprising the steps of:
1) preparing recombinant lentivirus containing target genes; wherein the target gene comprises an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence;
the method specifically comprises the following steps: obtaining and optimizing and synthesizing an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence, inoculating the sequences into a lentiviral vector, then transfecting HEK-293T cells to carry out virus preparation and amplification, and culturing to obtain a recombinant lentivirus;
wherein the optimization includes, but is not limited to: human codon usage bias, moderate GC content, stable mRNA secondary structure, elimination of repetitive sequences, cryptic splice sites and unwanted restriction sites, while preventing depletion of tRNA pools in the cell;
the IL-7 sequence is shown as SEQ ID No: 1 is shown in the specification; the IL-15 sequence is shown as SEQ ID No: 2 is shown in the specification; the IL-7-IL-15 fusion sequence is shown as SEQ ID No: 3 is shown in the specification;
2) digesting the tumor tissue to obtain a single cell suspension containing infiltrating lymphocytes; culturing the single cell suspension by using IL-2, and separating and purifying the infiltrating lymphocytes; then in a culture environment coated by recombinant human fibrin and CD3 antibody, continuously adding IL-2 into the purified infiltrating lymphocytes, and culturing until the logarithmic phase is reached;
3) adding the recombinant lentivirus into infiltrating lymphocytes cultured to a logarithmic growth phase, and adding Polybrene at the same time, and transfecting for 2-4 days; then adding a screening agent for screening culture until all cells are transgenic positive cells;
4) using Tet-on system, tetracycline or tetracycline derivative is added to induce the expression of IL-7 and/or IL-15, and the amplification culture of infiltrating lymphocytes is maintained.
9. The method according to claim 1, wherein the tumor tissue is selected from one of lung tumor tissue and breast tumor tissue.
10. Use of the process according to any one of claims 1 to 9 for the preparation of an antitumor therapeutic agent.
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