CN116179483B - Method for rapidly amplifying stem cell-like memory cervical cancer tumor infiltrating lymphocytes in vitro - Google Patents
Method for rapidly amplifying stem cell-like memory cervical cancer tumor infiltrating lymphocytes in vitro Download PDFInfo
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- CN116179483B CN116179483B CN202211627868.5A CN202211627868A CN116179483B CN 116179483 B CN116179483 B CN 116179483B CN 202211627868 A CN202211627868 A CN 202211627868A CN 116179483 B CN116179483 B CN 116179483B
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Abstract
The invention provides a method for rapidly amplifying stem cell-like memory cervical cancer tumor infiltrating lymphocytes in vitro, which belongs to the technical field of biological medicine, and mainly comprises the steps of culturing extracted tumor lymphocyte TILs in a first solution, collecting cells, culturing the collected cells and feeder cells in a second solution, and harvesting the stem cell-like memory cervical cancer TILs, wherein the feeder cells are obtained by processing genetically engineered Hela cells, and the genetically engineered Hela cell lines are cell lines expressing transmembrane cytokines IL-7, IL-15 and IL-21.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for rapidly expanding stem cell-like memory cervical cancer tumor infiltrating lymphocytes in vitro.
Background
Cervical cancer is a common malignant tumor in female reproductive systems, the best prevention and control strategy at present is early screening and vaccination, but due to large population base in China and unbalanced regional development, the screening and vaccination are not fully popularized, and the incidence rate of cervical cancer is still high. The immunotherapy, in particular cellular immunotherapy, can regulate the immune function of organisms, change the Tumor Microenvironment (TME), induce specific tumor immunity, achieve the purposes of treating cervical cancer and reducing recurrence and metastasis of cervical cancer, and become a fourth treatment mode after surgery, chemotherapy and radiotherapy.
Adoptive immunotherapy based on tumor-infiltrating lymphocytes (Tumor Infiltrating Lymphocytes, TILs) is also receiving increasing attention from scholars both at home and abroad. TILs are a group of tumor antigen-specific lymphocytes isolated from tumor tissue and contain T lymphocytes, B lymphocytes, NK cells. The research shows that the medicine has good curative effect in treating solid tumors such as metastatic melanoma, breast cancer, nasopharyngeal carcinoma, non-small cell lung cancer and the like, and fully shows the potential of TILs for treating the solid tumors. However, there are few clinical studies at home and abroad on TILs for the treatment of cervical cancer.
Currently, TILs extraction and culture are mainly improved based on Rosenberg professor research. In TILs extraction, the main steps are cutting tumor tissue into pieces, digesting into single cell suspensions using collagenase, and then purifying TILs by discontinuous gradient density centrifugation using lymphocyte separation fluid. In the step of obtaining the single cell suspension, the scholars use tissue mass culture (oligoclonal culture), fine puncture aspiration culture, and the like. On TILs culture, the rapid expansion protocol used by Rosenberg (Redpid explantion protocal, REP), also known as the two-step method, i.e., a first step, a dose of IL-2 is added to allow TILs to expand to a certain amount, and then a second step, cells are transferred to culture in Peripheral Blood Mononuclear Cells (PBMCs) containing anti-CD 3 monoclonal antibody (OKT 3), IL-2, and radiation inactivated healthy donors, to effect further expansion.
Tumor antigen specific memory T cells (memory T cells) are a class of T cell subtypes that have an anti-tumor effect and are capable of generating a rapid and stronger anti-tumor immune response when the same antigen is encountered again. Memory T cells can be classified into stem cell-like memory T cells (Tscm) (CCR7+CD45RA+), central memory T cells (Tcm) (CCR7+CD45RA-), effector memory T cells (Tem) (CCR 7-CD45 RA-), effector T cells (Teff) (CCR 7-CD45RA+), by whether or not CCR7 is expressed. Notably, tscm was not only able to maintain self-renewal, but after re-stimulation of antigen was differentiated into Tcm and Tem, preclinical studies found that Tscm, tcm, tem tumor-bearing mice were infused with the greatest degree of tumor shrinkage in the Tscm group, tcm times and Tem the weakest. This suggests that ex vivo amplification of a higher proportion of Tscm helps TILs to exert a more durable and efficient effect.
Therefore, a method for rapidly amplifying stem cell-like memory cervical cancer TILs in vitro is developed to obtain TILs which can be applied to clinical treatment, and has important significance for the treatment of cervical cancer.
Chinese patent document CN114686430a (application No. 202011629408.7) discloses a method for preparing TIL, which mainly comprises co-culturing an initial cell population containing TIL cells with feeder cells to obtain a first expanded cell population containing expanded TIL cells, and co-culturing the first expanded cell population with second feeder cells to obtain a second expanded cell population containing expanded TIL cells. Although this patent enables rapid culture of a large amount of TIL from a very small number of readily available tumor samples, the minimum culture period also takes 3 weeks and a long period. In addition, sources of feeders used in the present invention include, but are not limited to, PBMC and/or antigen presenting cells, which sources present not only difficulties but also heterogeneity in the quality of preparation.
Chinese patent document CN114763530a (application No. 202110054608.2) discloses a method for inducing and preparing TIL cells, the main content of which is pretreatment of tumor tissue; incubating the tumor tissue with a first medium to obtain a first population of TIL cells; finally, incubating the first TIL cell population with a second medium to obtain a second TIL cell population. Although this patent can be amplified to clinically desirable amounts, the minimum culture period also requires a longer period of 25-30 days. Furthermore, the invention increases the proportion of CD3+CD8+ cells, and does not take into account the T cell subset, which has limited antitumor activity.
At present, the expansion mode of the TILs generally needs at least 3 weeks to produce T cells which can be used for infusion, the anti-tumor activity is low, the clinical effect is not ideal, and meanwhile, the culture cost and the requirements are high, so that the popularization and the application of the TILs in cervical cancer treatment are limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for rapidly expanding stem cell-like memory cervical cancer tumor infiltrating lymphocytes in vitro.
The invention solves the technical problems of long culture period of TILs in vitro, low amplification factor and low anti-tumor activity.
The technical scheme of the invention is as follows:
a method for in vitro expansion of stem cell-like memory cervical cancer tumor infiltrating lymphocytes, comprising the following steps:
(1) Extracting tumor lymphocyte TILs from cervical cancer tumor tissue;
(2) Culturing the tumor lymphocyte TILs extracted in the step (1) in a first solution for 2-5 days, and collecting cells after culturing, wherein the formula of the first solution comprises the following components: volume fractions of 10% human AB serum, 50-150 ng/ml CD3 agonist, 50-150 ng/ml CD28 agonist, 50-150 ng/ml CD137 agonist, 5-50 ng/ml IL-2X-vivo TM 15 culture medium;
(3) Culturing the cells collected in the step (2) and feeder cells in a second solution, wherein the cell number ratio of the cells to the feeder cells is 1 (1-3), the culturing time is 10-13 days, and harvesting stem cell-like memory cervical cancer TILs;
the feeder cells are obtained by treating genetically engineered Hela cells;
the formulation of the second solution comprises: x-vivo with volume fraction of 10% human AB serum and IL-2 of 5-50 ng/ml TM 15 culture medium;
the genetically engineered Hela cell strain is a cell strain for expressing transmembrane cytokines IL-7, IL-15 and IL-21 (hereinafter referred to as Hela-rhIL-7/15/21);
the transmembrane cytokine IL-7 includes: CD8 alpha signal peptide, IL-7 and CD8 alpha transmembrane region and are connected in sequence, and the nucleotide sequence is shown as SEQ ID NO.4;
the transmembrane cytokine IL-15 includes: CD8 alpha signal peptide, IL-15 and CD8 alpha transmembrane region and connected in sequence, the nucleotide sequence is shown as SEQ ID NO.6;
the transmembrane cytokine IL-21 includes: CD8 alpha signal peptide, IL-21 and CD8 alpha transmembrane region and are connected in sequence, and the nucleotide sequence is shown as SEQ ID NO.8.
According to a preferred embodiment of the present invention, in step (1), the formulation of the first solution includes: volume fractions of 10% human AB serum, 100ng/ml CD3 agonist, 100ng/ml CD28 agonist, 100ng/ml CD137 agonist, 30ng/ml IL-2X-vivo TM 15 medium.
According to a preferred embodiment of the present invention, in the step (2), the formulation of the second solution includes: x-vivo with volume fraction of 10% human AB serum, 30ng/ml IL-2 TM 15 medium.
According to a preferred embodiment of the present invention, in step (3), the feeder cells are obtained by irradiating genetically engineered HeLa cells.
Further preferably, the genetically engineered Hela cells are irradiated to 40Gy to obtain feeder cells.
The preparation method of the genetically engineered Hela cells comprises the following steps:
(1) inserting the above-mentioned transmembrane cell factor IL-7, transmembrane cell factor IL-15 and transmembrane cell factor IL-21 into correspondent vector to construct pLV [ Exp ] -Hygro-EF1A-IL-7 plasmid, pLV [ Exp ] -Neo-EF1A-IL-15 plasmid and pLV [ Exp ] -pur-EF 1A-IL-21 plasmid, adopting slow virus packaging kit to respectively transfect 293T cell and package them into three slow viruses, namely expressing IL-7 slow virus, expressing IL-15 slow virus and expressing IL-21 slow virus;
(2) transfecting the slow virus expressing IL-7 into Hela cell strain, and screening by adopting Hygro antibiotics to obtain a stable IL-7-expressing Hela cell strain; then transfecting the slow virus expressing IL-15 into a steady expression IL-7Hela cell strain, and adopting Neo antibiotics to screen to obtain steady expression IL-7 and IL-15Hela cell strain; finally, the slow virus expressing IL-21 is transfected to stably express IL-7 and IL-15Hela cell strains, and Puro antibiotics are adopted to screen to obtain the stably expressed IL-7, IL-15 and IL-21Hela cell strains, namely the genetically engineered Hela cell strains (Hela-rhIL-7/15/21).
Advantageous effects
1. The invention solves the technical problems of long in-vitro culture period of TILs, low amplification factors and low anti-tumor activity.
2. According to the TILs amplification method provided by the invention, after 14 days of in-vitro culture, the proliferation multiple of the TILs reaches more than 2000 times, the stem cell-like memory cells account for more than 90%, the in-vitro cell killing rate and the in-vivo tumor volume inhibition rate reach more than 90%, and the TILs amplification method provided by the invention has the advantages of short culture period, strong killing power and high clinical application value.
Drawings
FIG. 1 is a map of pLV [ Exp ] -Hygro-EF1A-IL-7 plasmid.
FIG. 2 is a map of pLV [ Exp ] -Neo-EF1A-IL-15 plasmid.
FIG. 3 is a map of pLV [ Exp ] -Puro-EF1A-IL-21 plasmid.
FIG. 4 is a flow chart depicting the expression of IL-7 by Hela cells;
in the figure: the result was 99.70%.
FIG. 5 is a flow chart depicting the expression of IL-15 by Hela cells;
in the figure: the result was 99.91%.
FIG. 6 is a flow chart depicting the expression of IL-21 by Hela cells;
in the figure: the result was 98.57%.
FIG. 7 is a graph showing the effect of varying amounts of liquid change on the amplification factors of TILs.
FIG. 8 shows Hela-rhIL-7 feeder cells, hela-rhIL-7/15 feeder cells, hela-rhIL-7-
15/21 feeder cells and IL-7/15/21 factor on TILs differentiation.
Fig. 9 is a graph showing the killing effect of TILs on cervical cancer.
FIG. 10 is a graph showing the killing effect of TILs on cervical cancer in vivo;
in the figure: p <0.001 compared to stem cell-like memory high proportion TILs cell treated group.
Detailed Description
The technical scheme of the present invention is further described below with reference to examples, but the scope of the present invention is not limited thereto.
The drugs and reagents used in the examples are common products on the market unless otherwise specified, and the details not specifically described in the examples are all in accordance with the prior art.
Hela cells: purchased from the southern Beijing, bai Biotech Co.
Example 1
The genetically engineered Hela cells are genetically engineered Hela cell lines expressing transmembrane cytokines IL-7, IL-15 and IL-21 (hereinafter referred to as Hela-rhIL-7/15/21).
The preparation of the genetically engineered Hela cells comprises the following steps:
(1) Construction of pLV [ Exp ] -Hygro-EF1A-IL-7 plasmid, pLV [ Exp ] -Neo-EF1A-IL-15 plasmid, pLV [ Exp ] -Puro-EF1A-IL-21 plasmid
The transmembrane cytokine IL-7 includes:
CD8 alpha signal peptide with nucleotide sequence shown as SEQ ID NO. 1; IL-7, the nucleotide sequence of which is shown as SEQ ID NO. 2; a CD8 alpha transmembrane region, and the nucleotide sequence is shown as SEQ ID NO. 3; and are connected in sequence, and the nucleotide sequence after connection is shown as SEQ ID NO.4;
the transmembrane cytokine IL-15 includes:
CD8 alpha signal peptide with nucleotide sequence shown as SEQ ID NO. 1; IL-15, the nucleotide sequence of which is shown as SEQ ID NO. 5; the nucleotide sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO. 3; and are connected in sequence, and the nucleotide sequence after connection is shown as SEQ ID NO.6;
the transmembrane IL-21 includes:
CD8 alpha signal peptide with nucleotide sequence shown as SEQ ID NO. 1; IL-21, the nucleotide sequence of which is shown as SEQ ID NO. 7; the nucleotide sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO. 3; and are connected in sequence, and the nucleotide sequence after connection is shown as SEQ ID NO.8.
SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 were used to delegate the synthesis of the whole expression cassette from the division of the biological technology of Crohn, guangzhou, and the expression vector pLV [ Exp ] -Hygro-EF1A, pLV [ Exp ] -Neo-EF1A, pLV [ Exp ] -Puro-EF1A was inserted in sequence to construct pLV [ Exp ] -Hygro-EF1A-IL-7 (see FIG. 1), pLV [ Exp ] -Neo-EF1A-IL-15 (see FIG. 2), pLV [ Exp ] -Puro-EF1A-IL-21 (see FIG. 3); and respectively introducing the constructed vectors into escherichia coli for preservation.
(2) Extraction of pLV [ Exp ] -Hygro-EF1A-IL-7 plasmid, pLV [ Exp ] -Neo-EF1A-IL-15 plasmid, pLV [ Exp ] -Puro-EF1A-IL-21 plasmid
E.coli containing pLV [ Exp ] -Hygro-EF1A-IL-7, pLV [ Exp ] -Neo-EF1A-IL-15 and pLV [ Exp ] -Puro-EF1A-IL-21 in step (1) was cultured overnight, pLV [ Exp ] -Hygro-EF1A-IL-7, pLV [ Exp ] -Neo-EF1A-IL-15 and pLV [ Exp ] -Puro-EF1A-IL-21 were extracted by using plasmid extraction and purification kit (purchased from Qiagen Co.), and the concentrations of pLV [ Exp ] -Hygro-EF1A-IL-7 and pLV [ Exp ] -Puro-EF1A-IL-21 were all not lower than 400 ng/. Mu.l by using NanoDrop.
(3) Three lentiviruses are packaged, concentrated and titer tested
Three lentiviruses were prepared using Lentiviral Packaging Kit lentivirus packaging kit. The method comprises the following specific steps:
lentiviral packaging cell line 293T was inoculated in 10cm dishes containing DMEM+10% FBS, 37℃and 5% CO 2 Culturing under the condition, and preparing transfection when the adherence rate is 70-80%. A sterile 15ml centrifuge tube was taken and the reaction system was prepared as follows: serum-free DMEM:4ml; pLV [ Exp ]]-Hygro-EF1A-IL-7 plasmid: 10 μg; GM easyTM Lentiviral Mix:10 μl (10 μg); HG TransgeneTM Reagent:60 μl. Mixing, standing at room temperature for 20min, dripping into 293T cell culture dish, and placing into CO 2 Culturing in an incubator. After 24h transfection, the cell culture broth was aspirated and discarded in a waste liquid cup containing the disinfectant, and then 15ml of DMEM medium containing 10% by volume of human AB serum was added for further culture. After 48h, the cell supernatant was aspirated into a 50ml centrifuge tube, centrifuged at 500g for 5min at 4℃and the supernatant was filtered through a 0.45 μm filter and transferred to a new centrifuge tube. Mixing with 5 XPEG-8000+NaCl mother liquor at volume ratio of 4:1, centrifuging at 4deg.C and 12000r/min for 30min, and obtaining the final product.
Lentiviral packaging cell line 293T was inoculated in 10cm dishes containing DMEM+10% FBS, 37℃and 5% CO 2 Culturing under the condition, and preparing transfection when the adherence rate is 70-80%. A sterile 15ml centrifuge tube was taken and the reaction system was prepared as follows: serum-free DMEM:4ml; pLV [ Exp ]]Neo-EF1A-IL-15 plasmid: 10 μg; GM easyTM Lentiviral Mix:10 μl (10 μg); HG TransgeneTM Reagent:60 μl. Mixing, standing at room temperature for 20min, dripping into 293T cell culture dish, and placing into CO 2 Culturing in an incubator. After 24h transfection, the cell culture broth was aspirated and discarded in a waste liquid cup containing the disinfectant, and then 15ml of DMEM medium containing 10% by volume of human AB serum was added for further culture. After 48h, the cell supernatant was aspirated into a 50ml centrifuge tube, centrifuged at 500g for 5min at 4℃and the supernatant was filtered through a 0.45 μm filter and transferred to a new centrifuge tube. Mixing with 5 XPEG-8000+NaCl mother liquor at volume ratio of 4:1, centrifuging at 4deg.C and 12000r/min for 30min, and obtaining the final product.
Lentiviral packaging cell line 293T was inoculated in 10cm dishes containing DMEM+10% FBS, 37℃and 5% CO 2 Culturing under the condition, and preparing transfection when the adherence rate is 70-80%. A sterile 15ml centrifuge tube was taken and the reaction system was prepared as follows: serum-free DMEM:4ml; pLV [ Exp ]]-Puro-EF1A-IL-21 plasmid: 10 μg; GM easyTM Lentiviral Mix:10 μl (10 μg); HG TransgeneTM Reagent:60 μl. Mixing, standing at room temperature for 20min, dripping into 293T cell culture dish, and placing into CO 2 Culturing in an incubator. After 24h transfection, the cell culture broth was aspirated and discarded in a waste liquid cup containing the disinfectant, and then 15ml of DMEM medium containing 10% by volume of human AB serum was added for further culture. After 48h, the cell supernatant was aspirated into a 50ml centrifuge tube, centrifuged at 500g for 5min at 4℃and the supernatant was filtered through a 0.45 μm filter and transferred to a new centrifuge tube. Mixing with 5 XPEG-8000+NaCl mother liquor at volume ratio of 4:1, centrifuging at 4deg.C and 12000r/min for 30min, and obtaining the final product.
The three lentiviral pellets were dissolved in DMEM medium and stored in a-80℃refrigerator for later use. Taking 100 mu l of the three dissolved lentivirus solutions,the titer was measured using a lentiviral vector (HIV P24) rapid assay card, and the titers of all three lentiviruses were not less than 1.0X10 6 TU/ml。
(4) Construction, screening and flow identification of genetically engineered Hela cells (abbreviated as Hela-rhIL-7/15/21)
Sequentially infecting the 3 viruses in the step (3) with Hela cells to prepare Hela-rhIL-7/15/21, wherein the specific contents are as follows:
1) Will be 2X 10 5 Each HeLa cell and 100. Mu.l of the IL-7 expressing lentiviral suspension of step (3) were plated into 24 well plates, the volume was adjusted to 300. Mu.l with RPMI-1640 medium containing 10% by volume of human AB serum, and the addition of polybrene to 10. Mu.g/ml. The next day was changed and incubated for 8d at 37℃in a medium containing 600. Mu.g/ml Hygro and 10% volume fraction human AB serum RPMI-1640, 5% CO 2 Culturing under the condition to obtain a stable expression IL-7Hela cell strain, and detecting IL-7 expression by using an FC500 flow cytometer (purchased from BECKMAN Co.) FITC channel, wherein the result is 99.70%, and the result is shown in FIG. 4.
2) Will be 2X 10 5 IL-7-expressing Hela cells prepared in step 1) and 100. Mu.l of the IL-15-expressing lentiviral suspension of step (3) were plated in 24-well plates, the volume was adjusted to 300. Mu.l with RPMI-1640 medium containing 10% by volume of human AB serum, and the addition of procoagulant amine to 10. Mu.g/ml. The next day was changed and cultured in a medium containing 1500. Mu.g/ml Neo and 10% volume fraction human AB serum RPMI-1640 for 8d at 37℃with 5% CO 2 The cells were cultured under the conditions to obtain cell lines stably expressing IL-7 and IL-15, and the IL-15 expression was detected by using FC500 flow cytometry (from BECKMAN Co.) PE channel, and the result was 99.91%, and the result was shown in FIG. 5.
3) Will be 2X 10 5 IL-7 and IL-15 expressing Hela cells prepared in step 2) and 100. Mu.l of the IL-21 expressing lentiviral suspension in step (3) were plated in 24 well plates, adjusted to 300. Mu.l in volume with RPMI-1640 medium containing 10% by volume of human AB serum, and addition coagulated to 10. Mu.g/ml. The next day the liquid was changed and incubated in a medium containing 4. Mu.g/ml Puro, 10% volume fraction human AB serum RPMI-1640 for 3d at 37℃with 5% CO 2 Culturing under the condition to obtain cell lines stably expressing IL-7, IL-15 and IL-21, namely Hela-rhIL-7/15/21, and adopting FC500 flow cellThe PC5.5 channel of the apparatus (from BECKMAN) examined IL-21 expression, and the result was 98.57%, and the result was shown in FIG. 6.
(5) Preparation of Hela-rhIL-7/15/21 feeder cells
And (3) carrying out 40Gy irradiation on the Hela-rhIL-7/15/21 constructed in the step (4) by adopting a biological irradiation instrument, centrifuging 600g of the Hela-rhIL-7/15/21 cell suspension for 10min, and removing the supernatant to obtain the Hela-rhIL-7/15/21 feeder layer cells.
Example 2
Extraction of tumor lymphocyte TILs from cervical cancer tumor tissue
The cervical cancer tissue sample is placed in a 10cm culture dish containing 20ml of physiological saline, fully soaked and washed for 10 minutes, and obvious necrotic tissues, vascular tissues, adipose tissues, normal tissues and the like are removed, so that 6g of cervical cancer tissues are harvested. The tissue was transferred to a 50mL centrifuge tube, the tumor tissue was cut into paste-like tissue with medical scissors, and then 20mL of physiological saline was added thereto, followed by washing for 10 minutes with sufficient soaking, and the supernatant was removed. The tissue was transferred to a 15mL centrifuge tube with a pipette and the tissue (g): mixed enzyme (mL) =1 g:0.25mL, adding mixed enzyme (0.05% pancreatin and 0.1% collagenase type I) into pasty tissue, shaking at 37deg.C, standing for 60min, adding human AB serum X-vivo with volume equal to that of mixed enzyme and 20% of that of mixed enzyme TM Stopping digestion of the medium 15, filtering with a 40um cell filter screen, collecting filtrate, centrifuging at 600g for 10min, removing supernatant, and re-suspending cells with physiological saline; mixing the cell suspension and lymphocyte separation liquid according to the volume ratio of the cell suspension to the lymphocyte separation liquid=1:1, slowly adding the cell suspension into the upper layer of the lymphocyte separation liquid, carrying out discontinuous density gradient centrifugation, centrifuging for 30min at 800g, sucking the lymphocyte in the middle layer by using a Pasteur pipette, and centrifuging to discard the supernatant. The cells were resuspended in 45mL of physiological saline while 20ul of the cell suspension was trypan blue stained, and after counting 260g centrifuged for 10min, the supernatant was discarded for use. The extracted tumor-infiltrating tissue lymphocyte TILs viable cell count is 0.75X10 6 Individual cells.
Example 3
A method for rapidly amplifying stem cell-like memory cervical cancer tumor infiltrating lymphocytes in vitro, comprising the following steps:
(1) Activation of TILs: on day 0 (day of cell isolation was taken as day 0), TILs cells extracted in example 2 were taken at a TILs cell density of 1X 10 6 Inoculating cells/ml into the first solution, culturing in carbon dioxide incubator for 3 days at 37deg.C with 5% CO 2 Culturing under the condition to obtain activated TILs.
Wherein the first solution comprises 10% human AB serum, 100ng/ml CD3 agonist, 100ng/ml CD28 agonist, 100ng/ml CD137 agonist, 30ng/ml IL-2X-vivo TM 15 medium.
(2) Amplification of TILs: completely changing TILs after 3 days of activation in the step (1), centrifuging 600g for 10min, discarding the supernatant, and re-suspending TILs with a second solution, while adding 3 times the number of Hela-rhIL-7/15/21 feeder cells, and adjusting the cell density to 1×10 with the second solution 6 cells/ml, cultured at 37℃with 5% CO 2 Culturing under the condition;
wherein the second solution comprises 10% human AB serum, 30ng/ml IL-2X-vivo TM 15 medium.
On day 5, according to the density, the fluid infusion density is maintained at 1×10 6 cells/ml. On day 8, 3-fold numbers of HeLa-rhIL-7/15/21 feeder cells were added based on cell number while the fluid replacement density was maintained at 1X 10 based on the fluid replacement density 6 cells/ml. On day 11, according to the density, the fluid infusion density is maintained at 1×10 6 cells/ml. Cells were harvested after TILs were counted after culturing to day 14. Viable cell count was 1.65X10 9 Individual cells, and flow cytometry to detect expression rates of CCR7, CD45RA, ccr7+cd45ra+in TILs cells>90 percent, the stem cell-like memory cervical carcinoma TILs are obtained.
Experimental example 1
Effect of the amount of liquid change on the amplification factors of TILs on day 3
The TILs were amplified from 60 cervical cancer tumor tissues by the method of example 3, except that the TILs were changed on day 3, and divided into a no-change group (n=20), a half-change group (n=20), and a full-change group (n=20). The TILs culture multiples of each group were compared. The result is that on day 3, complete pipetting of activated TILs gives the highest amplification factor up to 2000, see in particular fig. 7.
Experimental results show that the preparation method provided by the invention greatly improves the proliferation activity of TILs.
Experimental example 2
Hela-rhIL-7 feeder cells, hela-rhIL-7/15 feeder cells, and effects of Hela-rhIL-7/15/21 feeder cells on TILs differentiation
80 parts of tumor tissue were subjected to TILs and amplified by the method of example 3, except that the feeder cells were added in the type of Hela-rhIL-7 group (n=20), hela-rhIL-7/15 group (n=20) and Hela-rhIL-7/15/21 group (n=20). And compared with the conventional culture mode [ (IL-7/15/21 factor group (n=20) ].
In which the culture is carried out in a conventional manner [ (IL-7/15/21 factor group (n=20))]The specific culture method comprises the following steps: extraction and activation of TILs reference is made to examples 2 and 3. The amplification of TILs was performed by pipetting the activated TILs on day 3, centrifuging 600g for 10min, discarding the supernatant and resuspending the TILs with a third solution, maintaining a density of 1X 10 6 cells/ml, wherein the third solution comprises a volume fraction of 10% human AB serum, 30ng/ml IL-2, 40ng/ml IL-7, 50ng/ml IL-15, 50ng/ml IL-21X-vivo TM 15 culture medium, and then supplementing with third solution at 5, 8, and 11 days to maintain density at 1×10 6 cells/ml, cultured to day 14, TILs were counted and cells were harvested.
The results are 26.58% of the stem cell-like memory cervical carcinoma TILs of the Hela-rhIL-7 group, 76.00% of the stem cell-like memory cervical carcinoma TILs of the Hela-rhIL-7/15 group, 94.54% of the stem cell-like memory cervical carcinoma TILs of the Hela-rhIL-7/15/21 group and 64.29% of the stem cell-like memory cervical carcinoma TILs of the IL-7/15/21 factor group. It was demonstrated that the HeLa-rhIL-7/15/21 feeder cells designed in accordance with the present invention are beneficial in promoting the production of stem cell-like memory cervical cancer TILs, see in particular FIG. 8.
Effect example 1
Killing effect of TILs on cervical cancer in vitro
The Hela cervical cancer cell line is used as a target cell, the TILs prepared in the example 3 or the TILs prepared in the Hela rhIL-7 group in the experimental example 2 are used as effector cells, the effector cells are respectively added into the target cells according to the effect/target ratio of 20:1, and corresponding target cell holes, effector cell holes and blank control holes of an equal volume of fetal bovine serum RPMI-1640 culture medium with the volume fraction of 10% are arranged. Each group had 3 parallel holes. Target cell number of 1×10 4 Respectively, inoculating target cells for 6h, adding effector cells, and respectively heating to 37deg.C and 5% CO 2 Mu.l of CCK-8 solution was added to each well and incubated at 37℃for 1 h.+ -. 10min at saturated humidity for 2, 4, 6h, and OD was measured at 450nm using an ELISA reader. The kill rate is calculated from the following formula: killing rate% = [1- (effector cell action well OD value-effector cell well OD value)/target cell well OD value]X 100%. Results after 4h of action at an effective target ratio of 20:1, the killing rate of TILs prepared in example 3 to target cells reached more than 90%, which is significantly higher than that of TILs prepared in Hela-rhIL-7 group in Experimental example 2 (t=12.20, P)<0.001). Specifically, as shown in fig. 9, the TILs prepared by the method of the invention have higher killing power.
Effect example 2
Killing effect of TILs on cervical cancer in vivo
Male BALB/c nude mice (4-6 weeks old, n=18) were kept in a sterile environment, at 24℃and 50% -70% humidity, for 12/12h of light/dark cycles, and allowed free access to food and water. All animal experiments were approved by the institutional animal ethics committee. Hela cells (6×10) 6 Per mL) was resuspended in 100 μl PBS solution and injected into the left underarm subcutaneous tissue of nude mice. After inoculation, the length and width of the tumor mass were measured daily with vernier calipers. At v=0.5×a×b 2 (a is length and b is width) the tumor volume is calculated.
When the tumor volume reaches about 500mm 3 At this time, nude mice were randomly divided into a control group, a stem cell-like memory high-proportion TILs cell-treated group, and a stem cell-like memory low-proportion TILs cell-treated group, 6/group. Cells were dissolved in 1ml PBS and injected into nude mice via tail vein as follows.
Control group: the blank was only 1ml PBS.
Stem cell-like memory high proportion TILs cell treatment group: 1X 10 7 TILs cells prepared in example 3.
Stem cell-like memory low-proportion TILs cell treatment group: 1X 10 7 TILs prepared from Hela-rhIL-7 group in Experimental example 2.
After infusion, the length and width of the tumor nodules were measured with vernier calipers, the tumor volume was assessed and observed for 60d. When the tumor volume of the control group reaches>2000mm 3 At this time, the nude mice were sacrificed. Tumor volume inhibition rate of each group of nude mice [ tumor volume inhibition rate= (V) C -V t )/V C X 100%, where V C Mean tumor volume, V, for negative control group t Mean tumor volume for each experimental group]. The results show that the stem cell-like memory high-proportion TILs cells treat the tumor volume (132.50 +/-44.20) mm 3 Is obviously smaller than that of a control group (2002.00 +/-37.78) mm 3 (P<0.05 Stem cell-like memory low proportion TILs cell treatment group (569.20 ± 72.29) mm 3 (P<0.05 See in particular fig. 10). In terms of tumor volume inhibition, the tumor volume inhibition of the stem cell-like memory low-proportion TILs cell-treated group was 71.57%, while the tumor volume inhibition of the stem cell-like memory high-proportion TILs cell-treated group was 93.38%.
According to the TILs amplification method provided by the invention, after 14 days of in-vitro culture, the proliferation multiple of the TILs reaches more than 2000 times, the stem cell-like memory cells account for more than 90%, the in-vitro cell killing rate and the in-vivo tumor volume inhibition rate reach more than 90%, and the TILs amplification method provided by the invention has the advantages of short culture period, strong killing power and high clinical application value.
Claims (6)
1. A method for in vitro expansion of stem cell-like memory cervical cancer tumor infiltrating lymphocytes, comprising the steps of:
(1) Extracting tumor lymphocyte TILs from cervical cancer tumor tissue;
(2) Culturing the tumor lymphocyte TILs extracted in the step (1) in a first solution for 2-5 days, and collecting cells after culturing, wherein the formula of the first solution comprises the following components: 10% of human AB serum, 50-150 ng/ml of CD3 agonist, 50-150 ng/ml of CD28 agonist, 50-150 ng/ml of CD137 agonist and 5-50 ng/ml of IL-2X-vivo ™ culture medium;
(3) Culturing the cells collected in the step (2) and feeder cells in a second solution, wherein the cell number ratio of the cells to the feeder cells is 1 (1-3), the culturing time is 10-13 days, and harvesting stem cell-like memory cervical cancer TILs;
the feeder cells are obtained by treating genetically engineered Hela cells;
the formula of the second solution comprises the following components: x-vivo ™ culture medium with volume fraction of 10% human AB serum and IL-2 of 5-50 ng/ml;
the genetically engineered Hela cell strain is a cell strain expressing transmembrane cytokines IL-7, IL-15 and IL-21;
the transmembrane cytokine IL-7 includes: CD8 alpha signal peptide, IL-7 and CD8 alpha transmembrane region and are connected in sequence, and the nucleotide sequence is shown as SEQ ID NO.4;
the transmembrane cytokine IL-15 includes: CD8 alpha signal peptide, IL-15 and CD8 alpha transmembrane region and connected in sequence, the nucleotide sequence is shown as SEQ ID NO.6;
the transmembrane cytokine IL-21 includes: CD8 alpha signal peptide, IL-21 and CD8 alpha transmembrane region and are connected in sequence, and the nucleotide sequence is shown as SEQ ID NO.8.
2. The method of claim 1, wherein in step (1), the formulation of the first solution comprises: volume fractions of 10% human AB serum, 100ng/ml CD3 agonist, 100ng/ml CD28 agonist, 100ng/ml CD137 agonist, 30ng/ml IL-2X-vivo ™ medium.
3. The method of claim 1, wherein in step (2), the second solution is formulated as follows: volume fraction was 10% human AB serum, 30ng/ml IL-2X-vivo ™ medium.
4. The method of claim 1, wherein in step (3), the feeder cells are obtained by irradiating genetically engineered Hela cells.
5. The method of claim 4, wherein the feeder cells are obtained by irradiating genetically engineered Hela cells to 40 Gy.
6. The method of claim 1, wherein the method of preparing genetically engineered Hela cells comprises the steps of:
(1) inserting the transmembrane cytokines IL-7, transmembrane cytokine IL-15 and transmembrane cytokine IL-21 into corresponding vectors to construct pLV [ Exp ] -Hygro-EF1A-IL-7 plasmid, pLV [ Exp ] -Neo-EF1A-IL-15 plasmid and pLV [ Exp ] -pur-EF 1A-IL-21 plasmid, and respectively transfecting 293T cells into three lentiviruses by using a lentivirus packaging kit to package the three lentiviruses, namely, expressing IL-7 lentivirus, expressing IL-15 lentivirus and expressing IL-21 lentivirus;
(2) transfecting the slow virus expressing IL-7 into Hela cell strain, and screening by adopting Hygro antibiotics to obtain a stable IL-7-expressing Hela cell strain; then transfecting the slow virus expressing IL-15 into a steady expression IL-7Hela cell strain, and adopting Neo antibiotics to screen to obtain steady expression IL-7 and IL-15Hela cell strain; finally, the slow virus expressing IL-21 is transfected to stably express IL-7 and IL-15Hela cell strains, and Puro antibiotics are adopted to screen to obtain the stably expressed IL-7, IL-15 and IL-21Hela cell strains, namely the genetically engineered Hela cell strains.
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