WO2016201658A1 - Method for preparing tumor specific ctls - Google Patents

Method for preparing tumor specific ctls Download PDF

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WO2016201658A1
WO2016201658A1 PCT/CN2015/081713 CN2015081713W WO2016201658A1 WO 2016201658 A1 WO2016201658 A1 WO 2016201658A1 CN 2015081713 W CN2015081713 W CN 2015081713W WO 2016201658 A1 WO2016201658 A1 WO 2016201658A1
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tumor
cells
culture
peripheral blood
cell
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PCT/CN2015/081713
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French (fr)
Chinese (zh)
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姜维
吕明锦
吴庆军
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深圳市达科为生物工程有限公司
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Priority to PCT/CN2015/081713 priority Critical patent/WO2016201658A1/en
Priority to CN201580002103.4A priority patent/CN106795492A/en
Publication of WO2016201658A1 publication Critical patent/WO2016201658A1/en

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  • the present invention relates to the field of tumor cell immunity technology, and more particularly to a method for preparing a tumor-specific CTL.
  • Malignant tumors are the most serious diseases that endanger human health.
  • the treatments of conventional surgery, chemotherapy and radiotherapy are not satisfactory, the specificity is poor, the side effects are strong, and the effect is uncertain.
  • the treatment of malignant tumors has always been a difficult problem in the medical field.
  • adoptive cellular immunotherapy has become the fourth treatment mode after traditional tumor therapy.
  • Adoptive cell immunotherapy refers to the transmission of immune cells with anti-tumor activity to tumor patients, directly killing tumors or stimulating the body's immune response to kill tumor cells to achieve the purpose of treating tumors.
  • Adoptive immunotherapy is used to reconstruct the immune system of cancer patients, eliminate microscopic tumor lesions that are not easy to find, and provide life-saving treatment for patients to prolong survival.
  • the most critical factor in the treatment process is to obtain a large number of immunotherapeutic effector cells with strong killing effect and long survival in the body.
  • Cytotoxic lymphocyte a sub-part of leukocytes, is a specific T cell that secretes various cytokines to participate in immune function. It has a killing effect on certain viruses, tumor cells and other antigens, and it is an important defense line against natural anti-virus and anti-tumor immunity.
  • the technical problem to be solved by the present invention is to provide a method for preparing a tumor-specific CTL, which solves the problems of a complicated process for obtaining a large amount of CTL in the prior art and a low immunogenicity of the obtained CTL.
  • a method for preparing a tumor-specific CTL is provided, which comprises the following steps:
  • S100 coating of tumor antigen: preparing tumor antigen using patient tumor tissue or tumor cell line, adjusting tumor antigen concentration to 5 (Vg/ml ⁇ 20 (Vg/ml, added to TC-treated cell culture flask) Covering the bottom of the bottle, performing a coating reaction to obtain a culture bottle pre-coated with the tumor antigen;
  • S300 cultivating tumor-specific CTL: carefully plating the peripheral blood mononuclear cells into the tumor antigen pre-coated culture flask, and performing incubation culture to induce mature tumor antigen-loaded DC cells and T lymphocytes Cells; then stimulating cytokines, supplemented with fresh medium for continuous culture, and extensive expansion of T lymphocytes to obtain antigen-specific CTLs.
  • the step S100 further includes:
  • S101 preparing a tumor antigen: dispersing a patient tumor tissue or a tumor cell line into a single cell suspension, and adjusting the concentration of the suspended tumor cells with physiological saline to 1> ⁇ 10 7 ⁇ 2> ⁇ 10 7 /1 ⁇ , repeated Freeze and thaw 3 ⁇ 5 times, lyse tumor cells; centrifuge the tumor cell lysate at 2000g for 8 ⁇ 12 minutes, then aspirate the supernatant, filter and sterilize by 0.22 ⁇ filter, and record it as tumor antigen;
  • S103 coating reaction: adjust the concentration of tumor antigen to 5 (Vg / ml ⁇ 2 0 (Vg / ml, take 10m! ⁇ 20ml, added to the TC treated cell culture flask) with sterile physiological saline or lxPBS The tumor antigen covers the bottom of the culture flask, and the coating reaction is carried out at 4 ° C overnight or at 37 ° C for 11! ⁇ 2 h to obtain a tumor antigen pre-coated culture flask.
  • the method for dispersing a patient's tumor tissue includes a collagenase digestion method and a physical polishing method.
  • the step S200 further includes: [0016] S201, preparing whole blood: extracting venous blood of a tumor patient, adding an anticoagulant to an anticoagulated whole blood reserve. ;
  • S202 separation of peripheral blood mononuclear cells from autologous plasma: using a blood cell separator or a lymphocyte separation solution to separate peripheral blood mononuclear cells from autologous plasma, obtaining a peripheral blood mononuclear cell crude product
  • S203 Treatment of peripheral blood mononuclear cells: The crude peripheral blood mononuclear cells are washed twice with physiological saline or serum-free medium, 20 . Centrifuge at 500g for 3 to 7 minutes, resuspend in an appropriate amount of serum-free medium to obtain peripheral blood mononuclear cells, and set aside.
  • the step S300 further includes:
  • S301 adjusting the cell concentration: adjusting the cell concentration of the peripheral blood mononuclear cells to 1 ⁇ 10 6 ⁇ 2 ⁇ 10 6 cells/ml with the serum-free culture solution, mixing uniformly and carefully plating the tumor antigen pre-coated culture bottle in;
  • S303 continuous culture: adding 500 U/ml to 1200 U/ml of IFN- ⁇ , 60 ng/ml to 150 ng/ml of anti-CD3, and 600 U/ml to 1400 U/ml of IL2 to the tumor antigen-loaded DC cells.
  • CTL cells continuous culture: adding 500 U/ml to 1200 U/ml of IFN- ⁇ , 60 ng/ml to 150 ng/ml of anti-CD3, and 600 U/ml to 1400 U/ml of IL2 to the tumor antigen-loaded DC cells.
  • the present invention has the following beneficial effects:
  • the present invention adopts a patient's autologous peripheral blood to obtain peripheral blood mononuclear cells, incubates in a tumor antigen-coated culture flask, and then adds cytokines for continuous culture, one time.
  • a large number of DC-CTL cells with strong killing effect and tumor specificity were obtained, which were used for clinical application of tumor suppression.
  • FIG. 2 is a flow chart of a preferred embodiment of a method for preparing a tumor-specific CTL of the present invention.
  • FIG. 1 shows a flow chart of a method for preparing CTL cells in the prior art.
  • peripheral blood mononuclear cells PBMCs
  • CIK cells cytokine-induced killer, a variety of cytokine-induced killer cells
  • adherent cells were induced to obtain tumor antigen-loaded DC cells (Dendritic cells, antigen-delivering cells), and then Mature tumor antigen-loaded DC cells are mixed with CIK cells to obtain CTL.
  • CIK cells cytokine-induced killer, a variety of cytokine-induced killer cells
  • tumor antigen-loaded DC cells Dendritic cells, antigen-delivering cells
  • Mature tumor antigen-loaded DC cells are mixed with CIK cells to obtain CTL.
  • PBMCs obtained from whole blood, adjusted to a suitable cell concentration with serum-free medium, placed in a culture bottle, under appropriate culture conditions to allow monocytes to adhere;
  • the suspension cells are separated from the adherent culture flask, and the suspension cells are collected.
  • the suspension cells are mainly T lymphocyte monolayers, adjusted to a suitable cell concentration with serum-free medium, and induced by various cytokines.
  • cytokines include recombinant human IFN- ⁇ (lymphocyte-type interferon), CD3 monoclonal antibody, and recombinant human IL-2 (interleukin-2), which stimulate the growth and proliferation of CIK cells;
  • step 1.1 The remaining adherent cells in step 1.1 (mainly CD14+ monocytes, or DC precursor cells), add appropriate cytokines, such as recombinant human GM-CSF (human granulocyte-macrophage) Cell colony-stimulating factor), recombinant human IL-4 (interleukin-4), etc., induces differentiation of monocytes into DC cells;
  • cytokines such as recombinant human GM-CSF (human granulocyte-macrophage) Cell colony-stimulating factor), recombinant human IL-4 (interleukin-4), etc.
  • the above cytokines are appropriately supplemented, and the tumor antigen is added after the culture is incubated, and the DC cells are subjected to antigen loading, and then cultured, and cytokines such as recombinant human TNF-ot (tumor necrosis factor-ot) can be added for induction.
  • cytokines such as recombinant human TNF-ot (tumor necrosis factor-ot) can be added for induction.
  • TNF-ot tumor necrosis factor-ot
  • the obtained CIK cells and tumor antigen-loaded DC cells are mixed and cultured in a certain ratio, for example, 10:1, the medium is appropriately changed during the culture, and the cytokine recombinant human IL-2 is added, and cultured.
  • Tumor-specific CTL cells can be collected after a period of time.
  • the preparation process of the above CTL is complicated, and the induction workload of the suspension cells and the adherent cells is relatively large, and the diurnal cycle induced respectively is difficult to be unified.
  • the CTL prepared by the above method has low or no reaction, and may It is due to the fact that DCs affect the synthesis of CTL-specific immunosuppressive substances due to the large changes in the surrounding microenvironment of the presented antigens.
  • the main innovation of the present invention is that the tumor antigen is pre-coated in the cell culture bottle, and the DC precursor cells and the T lymphocytes do not need to be separately cultured, which simplifies the experimental process, and the DC precursor cells adhere to the wall. It can be fully contacted with the tumor antigen coated on the wall of the culture bottle, and it is helpful to obtain a large number of antigen-loaded DC cells to present the antigen, and quickly obtain a large number of specific CTL immunocompetent cells with high specificity.
  • the method for preparing a tumor-specific CTL of the present embodiment includes the following steps:
  • S100, coating of tumor antigen preparing tumor antigen using patient tumor tissue or tumor cell line, adjusting tumor antigen concentration to 5 (Vg/ml ⁇ 20 (Vg/ml, added to TC-treated cell culture flask) Cover the bottom of the bottle and carry out the coating reaction to obtain the culture bottle pre-coated with the tumor antigen.
  • the patient's autologous tumor antigen, no foreign cell introduction, strong safety, high specificity, can significantly induce the specificity against autologous tumor CTL, does not have any damage to normal cells in the body, and is conducive to rebuilding the defense monitoring ability of the tumor
  • Peripheral blood mononuclear cells are isolated from the blood of the patient and suspended for use.
  • PBMCs obtained from patients' whole blood mainly include two types of cells, one is DC cells that play antigen-presenting, and the other is T lymphocytes that can synthesize related killer immunologically active substances. Characteristics of adherent growth of DC cells The above two types of cells are isolated and cultured separately.
  • the PBMCs obtained in the present application are directly cultured in a culture flask pre-coated with tumor antigens, and are not induced by conventional methods. , the complex process of mixed culture.
  • S300 cultivating tumor-specific CTL: carefully plating the peripheral blood mononuclear cells into a tumor antigen pre-coated culture flask, and performing incubation culture to induce mature tumor antigen-loaded DC cells and T lymphocytes; Then, stimulating cytokines were added, and fresh medium was continuously cultured, and T lymphocytes were expanded in a large amount to obtain antigen-specific CTLs. Since there is no traditional method for branching culture, separately inducing, and mixing culture in the present invention, the entire culture environment is relatively stable, and the effect of presenting the antigen is not affected by changes in the surrounding microenvironment. In addition, DC precursor cells are adherently grown. Its properties allow it to be in full contact with the tumor antigen coated on the wall of the culture bottle, helping to obtain a large number of antigen-loaded DC cells.
  • the step S100 further includes: [0046] S101, preparing a tumor antigen: dispersing a patient tumor tissue or a tumor cell line into a single cell suspension Adjust the concentration of suspended tumor cells with physiological saline to 1> ⁇ 10 7 ⁇ 2> ⁇ 10 7 /1 ⁇ , freeze and thaw 3 ⁇ 5 times, lyse tumor cells; centrifuge cell lysate at 2000g for 8 ⁇ 12 After a minute, the supernatant was aspirated, and 0.22 ⁇ filter was used to filter and sterilize, and it was recorded as a tumor antigen.
  • the purpose of preparing and obtaining tumor antigens is to use DC cells loaded with tumor cell antigens.
  • DC cells loaded with tumor cell antigens There are many methods for loading DC cells in tumor cell antigens in the prior art, including loading DC with tumor cell lysate, DC with apoptotic tumor cells, necrosis or The dead tumor cells are loaded with DC, the tumor living cells are loaded with DC, the tumor cells are fused with DC, and the like.
  • the method selected in the present embodiment is that the tumor cell lysate is loaded with DC, that is, the tumor cells are lysed by repeated freeze-thaw method, and the tumor cell lysate is obtained, the operation is simple and rapid, and the antigen activity can be well preserved.
  • Repeated freezing and thawing process can also choose a variety of ways, for example, a cryotube containing a single cell suspension is immersed in liquid nitrogen or frozen in a -80 ° C refrigerator, and then quickly placed in a 37 ° C water bath for thawing for 10 min, Repeat freeze-thaw 3-5 times to ensure that the tumor cells are fully lysed.
  • a collagenase digestion method is commonly used for the collagenase-digested tumor mass, the dispersion into the tissue cells, the specific process generally: Fresh tumor tissue blocks surgery removed, under sterile conditions, washed with saline least 3 After that, it was cut, and digested with 0.1% collagenase for 30 minutes at 37 ° C. The obtained tumor cells were harvested by centrifugation, and after filtration through a 100 mesh nylon mesh, a single cell suspension was collected and suspended in an appropriate amount of physiological saline.
  • Tumor cells are filled into sterile cryotubes for freezing and thawing.
  • the physical grinding method usually uses the grinding action to disperse the tumor tissue block into cells.
  • the specific process is as follows: After the tumor tissue is cut, the R is added. PMI 1640 medium, fully ground, filtered through a 200 mesh sterile mesh and collected for single cell suspension.
  • S103 coating reaction: adjust the tumor antigen concentration to 5 (Vg / ml ⁇ 20 (Vg / ml, take 10ml ⁇ 20ml) with sterile physiological saline or lxPBS (phosphate buffer, pH 7.0 ⁇ 7.4) After adding the TC-treated cell culture flask, the tumor antigen covers the bottom of the culture flask, and the coating reaction is carried out at 4 ° C overnight or at 37 ° C for 11! ⁇ 2 h to obtain a tumor antigen pre-coated culture flask.
  • the tumor antigen preserved at -80 ° C above can be used after being dissolved at 4 ° C.
  • the washing is performed to remove the uncoated tumor antigen, for example, the coating is thoroughly washed with an appropriate amount of sterile physiological saline or lxPBS.
  • the flask is incubated 2 to 5 times. After the washing is completed, it is stored at 4 ° C for later use.
  • step S200 further includes:
  • S201 preparing whole blood: venous blood is taken from a tumor patient, and an anticoagulant is added to form anticoagulated whole blood for use. On-site extraction to prepare anticoagulated whole blood, or directly frozen anticoagulated whole blood, the refrigeration process has no significant effect on the results.
  • Peripheral blood mononuclear cells are obtained by separating a peripheral blood mononuclear cell from autologous plasma using a blood cell separator or a lymphocyte separation solution.
  • the blood cell separator can directly obtain the crude blood mononuclear cells of the peripheral blood.
  • the separation of the peripheral blood mononuclear cells by the lymphocyte separation solution may be slightly different due to the different manufacturers of the lymphocyte separation liquid.
  • the general process is: adding three in the centrifuge tube One-quarter of human peripheral blood lymphocyte separation solution, carefully spread anti-coagulated whole blood of tumor patients to the upper layer of lymphocyte separation solution, centrifuge (for example, 20 ° C, 800 g for 20 minutes), then use a Pasteur pipette or transfer The pipette carefully draws the PBMCs layer and obtains the crude blood of the peripheral blood mononuclear cells.
  • peripheral blood mononuclear cells are washed twice with physiological saline or serum-free medium, centrifuged at 500g for 3 to 7 minutes at 20 ° C, and then with an appropriate amount of serum-free medium Resuspend, obtain peripheral blood mononuclear cells, and reserve.
  • step S300 further includes:
  • S301 adjusting cell concentration: adjusting serum cell concentration of peripheral blood mononuclear cells to serum by serum-free medium
  • IL4 incubate in a CO 2 incubator 361! ⁇ 48h, induced mature tumor resistance Originally loaded DC cells.
  • the culture conditions in the C0 2 incubator can be selected at 37 ° C and the C 0 2 concentration is 5%.
  • S303 continuous culture: adding 500 U/ml to 1200 U/ml of IFN- ⁇ , 60 ng/ml to 150 ng/ml of anti-CD3, and 600 U/ml to 1400 U/ml of IL2 to the tumor antigen-loaded DC cells. After the culture is continued, IFN-Y, anti-CD3, and IL2 can be added at one time after the incubation. Samples were taken every other day, supplemented with fresh serum-free medium containing IL2, and the culture density of the cells was adjusted to 1x 10 6 to 2xl0 6
  • DC-CTL cells with specific antigen loading are obtained. During this period, fresh serum-free medium can also be added to supplement the nutrients required for cell synthesis, but the quantity is not frequent.
  • 1.1 Preparation of tumor antigen The fresh tumor tissue piece removed from the liver cancer patient is washed under sterile conditions with at least 3 times with physiological saline, and then cut with 0.1% collagenase at 37 ° C. After digestion for 30 minutes, the obtained tumor cells were harvested by centrifugation, and after filtration through a 100-mesh nylon mesh, a single cell suspension was collected, and the cell concentration of the tumor cells was suspended to 1 ⁇ 10 7 cells/ml with an appropriate amount of physiological saline, and the mixture was sterilized.
  • the cryotube containing the single cell suspension is immersed in liquid nitrogen for quick freezing, then quickly placed in a 37 ° C water bath for thawing for 10 min, repeated freeze-thaw three times to ensure that the tumor cells are fully lysed
  • the tumor cell lysate was centrifuged at 20 ° C, 2000 g for 8 minutes, and the supernatant was aspirated, filtered through a 0.22 ⁇ filter, and recorded as a tumor antigen, and stored at -80 ° C until use.
  • Coating reaction Adjust the concentration of tumor antigen to 5 (Vg/ml with sterile saline), take 20ml, add to the cell culture flask after TC treatment, and dissolve the tumor antigen stored at -80 °C at 4 °C to cover the culture flask. The bottom of the bottle was subjected to coating reaction overnight at 4 ° C to obtain a culture flask pre-coated with tumor antigen. The coated culture flask was thoroughly washed twice with an appropriate amount of sterile physiological saline, and stored at 4 ° C until the washing was completed.
  • Peripheral blood mononuclear cells were obtained by separating peripheral blood mononuclear cells from autologous plasma using a blood cell separator.
  • peripheral blood mononuclear cells Washing crude peripheral blood mononuclear cells twice with physiological saline , centrifuged at 500g for 3 minutes at 20 ° C, and then resuspended in an appropriate amount of serum-free medium to obtain peripheral blood mononuclear cells.
  • the cell concentration of the peripheral blood mononuclear cells was adjusted to 1 ⁇ 10 6 cells/ml with the serum-free medium, and the cells were uniformly mixed and carefully placed in the culture flask pre-coated with the tumor antigen.
  • liver tumor cell line HepG2 was taken, and under sterile conditions, washed at least 3 times with physiological saline, added to RPMI1640 medium, fully ground, and filtered through a 200 mesh sterile mesh to collect a single cell suspension.
  • the cryotube containing the single cell suspension is immersed in liquid nitrogen or -80 ° C refrigerator Quickly freeze, then quickly put into a 37 ° C water bath for thawing for 10 min, repeated freezing and thawing 5 times to ensure that the tumor cells are fully lysed; centrifuge the tumor cell lysate at 20 ° C, 2000g for 12 minutes, then extract the supernatant,
  • the bacteria were sterilized by filtration on a 0.22 ⁇ filter, and recorded as a tumor antigen, and stored at -80 ° C until use.
  • Coating reaction Adjust the concentration of tumor antigen to 20 (Vg/ml with sterile saline or lxPBS, take 10ml, add to the cell culture flask after TC treatment, and dissolve the tumor antigen stored at -80 °C after 4 °C dissolution. The bottom of the flask was cultured and subjected to coating reaction at 37 ° C for 1 h to obtain a culture flask pre-coated with tumor antigen. The coated flask was thoroughly washed 5 times with an appropriate amount of lxPBS, and stored at 4 ° C for storage. 2 Separation of peripheral blood mononuclear cells
  • the PBMCs layer was carefully aspirated with a Pasteur pipette or a pipette to obtain a crude peripheral blood mononuclear cell.
  • peripheral blood mononuclear cells were washed twice with serum-free medium, centrifuged at 500g for 7 minutes at 20 ° C, and then resuspended in an appropriate amount of serum-free medium to obtain peripheral blood. Mononuclear cells, spare.
  • the cell concentration of the peripheral blood mononuclear cells was adjusted to 1 ⁇ 10 6 cells/ml with the serum-free medium, and the cells were uniformly mixed and carefully placed in the culture flask pre-coated with the tumor antigen.
  • 1.1 Preparation of tumor antigen The fresh tumor tissue piece removed from the liver cancer patient is washed under sterile conditions with at least 3 times with physiological saline, then cut, and treated with 0.1% collagenase at 37 ° C. After digestion for 30 minutes, the obtained tumor cells were harvested by centrifugation, and after filtration through a 100 mesh nylon mesh, a single cell suspension was collected, and the cell concentration of the tumor cells was suspended in an appropriate amount of physiological saline to 2 ⁇ 10 7 cells/ml, and filled into a sterile frozen solution.
  • the tube is stored for freezing and thawing; the cryotube containing the single cell suspension is immersed in liquid nitrogen or frozen in a -80 ° C refrigerator, and then quickly placed in a 37 ° C water bath for thawing for 10 min, repeated freezing and thawing 4 times, Ensure that the tumor cells are fully lysed;
  • the tumor cell lysate was centrifuged at 2000 g for 10 minutes at 20 ° C, and the supernatant was aspirated, and sterilized by filtration through a 0.22 ⁇ filter, which was recorded as a tumor antigen, and stored at -80 ° C until use.
  • Peripheral blood single nuclear cells obtained using a blood cell separator.
  • peripheral blood mononuclear cells Washing crude peripheral blood mononuclear cells twice with physiological saline
  • peripheral blood mononuclear cells peripheral blood mononuclear cells
  • the cell concentration of the peripheral blood mononuclear cells was adjusted to 2 ⁇ 10 6 /ml with the serum-free medium, and the mixture was uniformly mixed and carefully placed in the culture bottle pre-coated with the tumor antigen.
  • Continuous culture After the incubation, the culture was continued by adding 1200 U/ml of IFN- ⁇ , 60 ng/ml of anti-CD3, and 600 U/ml of IL2 to the tumor antigen-loaded DC cells. The samples were counted every other day, supplemented with fresh serum-free medium containing IL2, and the cell culture density was adjusted to 2x10 6 /ml; after 15 days of culture, specific antigen-loaded DC-CTL cells were obtained.
  • step 1.1 adding serum-free medium containing 1000 U/ml recombinant human GM-CSF and 500 U/ml recombinant human IL-4, 37 ° C, 5% C0 2 3 ⁇ 4 incubator Medium culture, inducing monocytes to differentiate into DC cells;
  • the cytokine was appropriately supplemented during the culture, and the tumor antigen was added on the 5th day, and the DC cells were subjected to antigen loading, and the culture was continued. On the 6th day, 500 U/ml recombinant human TNF-ot was added for induction, and induction was induced.
  • DC cells
  • the number of cells in which the tumor antigen-loaded DC cells reached 1 ⁇ 10 6 /ml, and harvested.
  • the obtained CIK cells and tumor antigen-loaded DC cells were mixed and cultured in a ratio of 10:1, and the cells were changed every 2 days during the culture, and 300 U/ml of recombinant human IL-2 was added thereto, and mixed culture was carried out. On day 3, tumor-specific CTL cells were collected.
  • the quality inspection of the DC-CIK cells in the above examples and comparative examples includes:
  • the adherent cells are used to induce DC (GM-CSF and IL4), 50 ug/ml of antigen is added after 5 days, and 20 ng/ml of TNF is added after 6 days.
  • DC GM-CSF and IL4
  • 50 ug/ml of antigen is added after 5 days
  • 20 ng/ml of TNF is added after 6 days.
  • - ⁇ promotes maturation
  • ⁇ resuscitation of suspension cells induces the acquisition of CIK cells and co-culture with harvested mature DCs, usually requiring 21 days of culture
  • Experimental count results include total cell number, CD8+, and cell viability.
  • the total number of cells was 1.64 ⁇ 10 1 ⁇ .
  • the CD8+ is 1.32 ⁇ 10 1 ⁇ and 1.26 ⁇ 10 10 respectively.
  • Example 1 Example 2, Example 3.
  • the cell viability in the comparative example was 96.7% and 98.2%, respectively.
  • the comparison shows that the specific CTL obtained by the method for preparing a tumor-specific CTL is in the total number of cells.
  • Cell surface detection subjects include CD8+, CD3+, CD8+IFN-y+, CD107a.
  • Example 1 Example 2, Example 3, CD8+ in the comparative example were 80.2%, 85.4% ⁇ 81.2, respectively.
  • Example 1 Example 2, Example 3, Comparative Example, CD3+ were 90.3%, 95.2% ⁇ 95.0%, 83.2%, respectively;
  • IFN-Y+ is divided into 40.4 ⁇ 3 ⁇ 4, 45.6%, 43.8%, 19.7%;
  • Example 1 Example 2, Example 3, CD107a in the comparative example was 15.6%, 17.2%.
  • the specific CTL obtained by the method for producing a tumor-specific CTL was significantly superior to the comparative example in the expression of cell surface molecules.
  • DC-CIK cells are used as effector cells, tumor cells (which may be primary tumor cells or tumor cell lines) as target cells, and effector cells and target cells are 10:1, 20:1
  • the ratio of 40:1 (E:T is the ratio of the two) was added to a 96-well U-shaped plate. Each well contained 1 ⁇ 10 4 target cells, and the final volume was 200 ml. Three replicate wells were set. After 4 hours of culture, the culture supernatant was centrifuged, and the killing rate of the effector cells to the target cells was detected by a lactate dehydrogenase (LDH) kit, and a blank control, a target cell control, and an effector cell control were set. The value of each well was subtracted from the blank well and the average value of the duplicate wells was determined. Calculate the effector cells according to the following formula Cytotoxic activity, expressed as the kill rate ( ⁇ 3 ⁇ 4):
  • Killing rate target cell control value - (experimental well value - effector cell control value) / target cell control value xl00%.
  • the killing rate of the obtained tumor-specific CTL cells against the liver cancer cell lines HEPG2 and B cell leukemia K562 was examined according to this method. The experiment was repeated 6 times and averaged.

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Abstract

Provided is a method for preparing tumor specific CTLs. The method comprises the following steps: enveloping of a tumor antigen; separation of peripheral blood mononuclear cells; and culture of tumor specific CTLs. Peripheral blood mononuclear cells obtained from peripheral blood are incubated and cultured in a culture bottle in which the tumor antigen is enveloped, continuous culture is carried out by adding cell factors, and accordingly a large quantity of tumor specific CTLs are obtained in one time; and the tumor specific CTLs can be used for inhibiting or killing tumors.

Description

说明书 发明名称:一种肿瘤特异性 CTL的制备方法 技术领域  Description: A method for preparing tumor-specific CTLs
[0001] 本发明涉及肿瘤细胞免疫技术领域, 更具体的说, 涉及到一种肿瘤特异性 CTL 的制备方法。  [0001] The present invention relates to the field of tumor cell immunity technology, and more particularly to a method for preparing a tumor-specific CTL.
背景技术  Background technique
[0002] 恶性肿瘤是危害人类健康的最为严重的疾病, 常规的手术, 化疗, 放疗的治疗 效果并不令人满意, 特异性差, 毒副作用强, 效果不确定。 恶性肿瘤的治疗一 直是医学界的难题。 近几年来随着现代分子生物学和细胞免疫学的发展与进步 , 过继性细胞免疫疗法已成为继传统肿瘤疗法后的第四种治疗模式。 过继性细 胞免疫疗法是指向肿瘤患者传输具有抗肿瘤活性的免疫细胞, 直接杀伤肿瘤或 激发机体免疫反应来对肿瘤细胞进行杀伤, 以达到治疗肿瘤的目的。 过继免疫 治疗对肿瘤病人免疫系统进行重建, 消除不易发现的微小肿瘤病灶, 提供病人 生活治疗延长生存吋间都有十分重要的意义。 但是治疗过程中最关键的是要获 得大量杀伤作用强, 体内存活吋间长的免疫治疗效应细胞。  [0002] Malignant tumors are the most serious diseases that endanger human health. The treatments of conventional surgery, chemotherapy and radiotherapy are not satisfactory, the specificity is poor, the side effects are strong, and the effect is uncertain. The treatment of malignant tumors has always been a difficult problem in the medical field. In recent years, with the development and progress of modern molecular biology and cellular immunology, adoptive cellular immunotherapy has become the fourth treatment mode after traditional tumor therapy. Adoptive cell immunotherapy refers to the transmission of immune cells with anti-tumor activity to tumor patients, directly killing tumors or stimulating the body's immune response to kill tumor cells to achieve the purpose of treating tumors. Adoptive immunotherapy is used to reconstruct the immune system of cancer patients, eliminate microscopic tumor lesions that are not easy to find, and provide life-saving treatment for patients to prolong survival. However, the most critical factor in the treatment process is to obtain a large number of immunotherapeutic effector cells with strong killing effect and long survival in the body.
[0003] 细胞毒性 T淋巴细胞 (cytotoxic lymphocyte, CTL) , 是白细胞的亚部, 为一种 特异 T细胞, 专门分泌各种细胞因子参与免疫作用。 对某些病毒、 肿瘤细胞等抗 原物质具有杀伤作用, 与自然杀伤细胞构成机体抗病毒、 抗肿瘤免疫的重要防 线。  [0003] Cytotoxic lymphocyte (CTL), a sub-part of leukocytes, is a specific T cell that secretes various cytokines to participate in immune function. It has a killing effect on certain viruses, tumor cells and other antigens, and it is an important defense line against natural anti-virus and anti-tumor immunity.
[0004] 目前, 许多临床试验中, 即使检测到体内的肿瘤特异性 CTL反应, 但是临床疗 效仍不佳, 这主要是因为许多重要的分子免疫机制仍有待于阐明, 如肿瘤免疫 逃逸、 免疫耐受、 CTL活性低或无反应以及 DC在呈递抗原吋所受周围微环境的 影响。  [0004] At present, in many clinical trials, even if tumor-specific CTL responses in vivo are detected, the clinical efficacy is still poor, mainly because many important molecular immune mechanisms remain to be elucidated, such as tumor immune escape, immune tolerance. Subject to, low or no CTL activity and the influence of DC on the surrounding microenvironment of presenting antigens.
技术问题  technical problem
[0005] 本发明所要解决的技术问题在于: 提供一种肿瘤特异性 CTL的制备方法, 解决 现有技术中大量获得 CTL的过程复杂、 获得的 CTL免疫原性低等问题。  The technical problem to be solved by the present invention is to provide a method for preparing a tumor-specific CTL, which solves the problems of a complicated process for obtaining a large amount of CTL in the prior art and a low immunogenicity of the obtained CTL.
问题的解决方案 技术解决方案 Problem solution Technical solution
[0006] 本发明解决上述问题的技术方案为: 提供一种肿瘤特异性 CTL的制备方法, 包 括如下步骤:  The technical solution of the present invention to solve the above problems is as follows: A method for preparing a tumor-specific CTL is provided, which comprises the following steps:
[0007] S100、 肿瘤抗原的包被: 使用患者肿瘤组织或肿瘤细胞系制备肿瘤抗原, 调整 肿瘤抗原浓度至 5(Vg/ml〜20(Vg/ml, 加入到 TC处理过的细胞培养瓶中覆盖瓶底 , 进行包被反应, 得到肿瘤抗原预包被的培养瓶;  [0007] S100, coating of tumor antigen: preparing tumor antigen using patient tumor tissue or tumor cell line, adjusting tumor antigen concentration to 5 (Vg/ml~20 (Vg/ml, added to TC-treated cell culture flask) Covering the bottom of the bottle, performing a coating reaction to obtain a culture bottle pre-coated with the tumor antigen;
[0008] S200、 外周血单个核细胞的分离: 从患者血液中分离外周血单个核细胞, 悬浮 备用;  [0008] S200, separation of peripheral blood mononuclear cells: separating peripheral blood mononuclear cells from the blood of the patient, suspending for use;
[0009] S300、 培养肿瘤特异性 CTL: 将所述外周血单个核细胞小心铺入所述肿瘤抗原 预包被的培养瓶中, 进行孵育培养诱导获得成熟的肿瘤抗原负载的 DC细胞和 T 淋巴细胞; 然后加入刺激性细胞因子、 补充新鲜培养基连续培养, 大量扩增 T淋 巴细胞获得抗原特异性的 CTL。  [0009] S300, cultivating tumor-specific CTL: carefully plating the peripheral blood mononuclear cells into the tumor antigen pre-coated culture flask, and performing incubation culture to induce mature tumor antigen-loaded DC cells and T lymphocytes Cells; then stimulating cytokines, supplemented with fresh medium for continuous culture, and extensive expansion of T lymphocytes to obtain antigen-specific CTLs.
[0010] 在本发明提供的肿瘤特异性 CTL的制备方法中, 所述步骤 S100中还包括: [0010] In the method for preparing a tumor-specific CTL provided by the present invention, the step S100 further includes:
[0011] S101、 制备肿瘤抗原: 将患者肿瘤组织或肿瘤细胞系分散成单细胞悬液, 用生 理盐水调节悬浮肿瘤细胞浓度至 1><10 7〜2><10 7个/1^, 反复冻融 3〜5次, 裂解肿 瘤细胞; 将肿瘤细胞裂解物在 2000g下离心 8〜12分钟后吸取上清, 0.22μηι滤膜 过滤除菌, 记为肿瘤抗原; [0011] S101, preparing a tumor antigen: dispersing a patient tumor tissue or a tumor cell line into a single cell suspension, and adjusting the concentration of the suspended tumor cells with physiological saline to 1><10 7 〜2><10 7 /1^, repeated Freeze and thaw 3~5 times, lyse tumor cells; centrifuge the tumor cell lysate at 2000g for 8~12 minutes, then aspirate the supernatant, filter and sterilize by 0.22μηι filter, and record it as tumor antigen;
[0012] S102、 处理细胞培养瓶: TC处理的细胞培养瓶;  [0012] S102, processing a cell culture bottle: a TC-treated cell culture flask;
[0013] S103、 包被反应: 用无菌生理盐水或 lxPBS调整肿瘤抗原的浓度至 5(Vg/ml〜2 0(Vg/ml, 取 10m!〜 20ml, 加入 TC处理后的细胞培养瓶中, 肿瘤抗原覆盖培养瓶 瓶底, 4°C过夜或者 37°C进行包被反应 11!〜 2h, 得到肿瘤抗原预包被的培养瓶。  [0013] S103, coating reaction: adjust the concentration of tumor antigen to 5 (Vg / ml ~ 2 0 (Vg / ml, take 10m! ~ 20ml, added to the TC treated cell culture flask) with sterile physiological saline or lxPBS The tumor antigen covers the bottom of the culture flask, and the coating reaction is carried out at 4 ° C overnight or at 37 ° C for 11!~ 2 h to obtain a tumor antigen pre-coated culture flask.
[0014] 在本发明提供的肿瘤特异性 CTL的制备方法中, 所述步骤 S101中, 患者肿瘤组 织分散的方法包括胶原蛋白酶消化法、 物理研磨法。 In the method for preparing a tumor-specific CTL according to the present invention, in the step S101, the method for dispersing a patient's tumor tissue includes a collagenase digestion method and a physical polishing method.
[0015] 在本发明提供的肿瘤特异性 CTL的制备方法中, 所述步骤 S200中还包括: [0016] S201、 准备全血: 抽取肿瘤患者静脉血, 加入抗凝剂成抗凝全血备用; [0015] In the method for preparing a tumor-specific CTL provided by the present invention, the step S200 further includes: [0016] S201, preparing whole blood: extracting venous blood of a tumor patient, adding an anticoagulant to an anticoagulated whole blood reserve. ;
[0017] S202、 外周血单个核细胞与自体血浆的分离: 使用血细胞分离机或者淋巴细胞 分离液将外周血单个核细胞从自体血浆中分离, 获得的外周血单个核细胞粗品 [0018] S203、 外周血单个核细胞的处理: 将所述外周血单个核细胞粗品用生理盐水或 无血清培养基洗涤两次, 20。C下 500g离心 3〜7分钟, 再用适量无血清培养基重悬 , 得到外周血单个核细胞, 备用。 [0017] S202, separation of peripheral blood mononuclear cells from autologous plasma: using a blood cell separator or a lymphocyte separation solution to separate peripheral blood mononuclear cells from autologous plasma, obtaining a peripheral blood mononuclear cell crude product [0018] S203. Treatment of peripheral blood mononuclear cells: The crude peripheral blood mononuclear cells are washed twice with physiological saline or serum-free medium, 20 . Centrifuge at 500g for 3 to 7 minutes, resuspend in an appropriate amount of serum-free medium to obtain peripheral blood mononuclear cells, and set aside.
[0019] 在本发明提供的肿瘤特异性 CTL的制备方法中, 所述步骤 S300中还包括:  [0019] In the method for preparing a tumor-specific CTL provided by the present invention, the step S300 further includes:
[0020] S301、 调整细胞浓度: 用无血清培养液调整外周血单个核细胞的细胞浓度至 lx 10 6〜2xl0 6个 /ml, 混合均匀后小心铺入所述肿瘤抗原预包被的培养瓶中; [0020] S301, adjusting the cell concentration: adjusting the cell concentration of the peripheral blood mononuclear cells to 1×10 62 ×10 6 cells/ml with the serum-free culture solution, mixing uniformly and carefully plating the tumor antigen pre-coated culture bottle in;
[0021] S302、 孵育培养: 向培养瓶中加入 400U/m!〜 800U/ml的 GM-CSF和 300U/m!〜 6 OOU/ml的 IL4, 在 CO 2培养箱中进行孵育培养 361!〜 48h, 诱导获得成熟的肿瘤抗 原负载的 DC细胞; [0021] S302, Incubation Culture: Add 400 U/m to the culture flask! ~ 800U/ml GM-CSF and 300U/m! ~ 6 OOU/ml of IL4, incubate in a CO 2 incubator for 361! ~ 48h, induced to obtain mature tumor antigen-loaded DC cells;
[0022] S303、 连续培养: 向肿瘤抗原负载的 DC细胞中加入 500U/ml〜1200U/ml的 IFN- γ、 60ng/ml〜150ng/ml的 anti-CD3、 600U/ml〜1400U/ml的 IL2后继续培养, 隔天 取样计数, 补充含 IL2的新鲜无血清培养基, 将细胞的培养密度调整至 1x10 6〜2 X10 6个 /ml, 培养 12〜15天后, 获得特异性抗原负载的 DC-CTL细胞。  [3032] S303, continuous culture: adding 500 U/ml to 1200 U/ml of IFN-γ, 60 ng/ml to 150 ng/ml of anti-CD3, and 600 U/ml to 1400 U/ml of IL2 to the tumor antigen-loaded DC cells. Continue to culture, sample and count the next day, supplement the fresh serum-free medium containing IL2, adjust the culture density of the cells to 1x10 6~2 X10 6 /ml, and after 12 to 15 days of culture, obtain DC with specific antigen load. CTL cells.
发明的有益效果  Advantageous effects of the invention
有益效果  Beneficial effect
[0023] 实施本发明, 具有如下有益效果: 本发明采用病人自体外周血中获得外周血单 个核细胞, 在肿瘤抗原包被的培养瓶中进行孵育培养, 然后添加细胞因子进行 连续培养, 一次性获得大量杀伤作用强、 肿瘤特异性的 DC-CTL细胞, 用于抑瘤 杀瘤的临床应用。  [0023] The present invention has the following beneficial effects: The present invention adopts a patient's autologous peripheral blood to obtain peripheral blood mononuclear cells, incubates in a tumor antigen-coated culture flask, and then adds cytokines for continuous culture, one time. A large number of DC-CTL cells with strong killing effect and tumor specificity were obtained, which were used for clinical application of tumor suppression.
对附图的简要说明  Brief description of the drawing
附图说明  DRAWINGS
[0024] 为了更清楚地说明本发明实施例中的技术方案, 下面将对实施例中所需要使用 的附图作简单地介绍, 显而易见地, 下面描述中的附图仅仅是本发明的一些实 施例, 对于本领域普通技术人员来讲, 在不付出创造性劳动的前提下, 还可以 根据这些附图获得其他的附图。  [0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings to be used in the embodiments will be briefly described below. Obviously, the drawings in the following description are only some implementations of the present invention. For example, other drawings may be obtained from those of ordinary skill in the art in light of the inventive work.
[0025] 图 1为现有技术中 CTL的制备方法流程图; 1 is a flow chart of a method for preparing a CTL in the prior art;
[0026] 图 2为本发明肿瘤特异性 CTL制备方法较佳实施例的流程图。 本发明的实施方式 2 is a flow chart of a preferred embodiment of a method for preparing a tumor-specific CTL of the present invention. Embodiments of the invention
[0027] 下面将结合本发明实施例中的附图, 对现有技术和本发明实施例中的技术方案 进行清楚、 完整地描述, 显然, 所描述的实施例仅仅是本发明一部分实施例, 而不是全部的实施例。 基于本发明中的实施例, 本领域普通技术人员在没有做 出创造性劳动的前提下所获得的所有其他实施例, 都属于本发明保护的范围。  The technical solutions in the prior art and the embodiments of the present invention are clearly and completely described in the following with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention. Rather than all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.
[0028] 图 1示出了现有技术中 CTL细胞的制备方法流程, 如图 1所示, 分离得到外周血 单个核细胞 (PBMCs) 并进行孵育培养后, 将悬浮细胞与贴壁细胞分离并分别 进行诱导培养, 悬浮细胞经过诱导获得 CIK细胞 (cytokine-induced killer, 多种 细胞因子诱导的杀伤细胞) , 贴壁细胞经过诱导获得肿瘤抗原负载的 DC细胞 ( Dendriticcell, 抗原传递细胞) , 再将成熟的肿瘤抗原负载的 DC细胞与 CIK细胞 混合培养获得 CTL, 具体步骤大体如下:  1 shows a flow chart of a method for preparing CTL cells in the prior art. As shown in FIG. 1 , peripheral blood mononuclear cells (PBMCs) are isolated and cultured, and the suspension cells are separated from the adherent cells. Induction culture was carried out separately, and the suspension cells were induced to obtain CIK cells (cytokine-induced killer, a variety of cytokine-induced killer cells), and the adherent cells were induced to obtain tumor antigen-loaded DC cells (Dendritic cells, antigen-delivering cells), and then Mature tumor antigen-loaded DC cells are mixed with CIK cells to obtain CTL. The specific steps are as follows:
[0029] 1 . CIK细胞的获得  [0029] 1. Acquisition of CIK cells
[0030] 1.1从全血中获得的 PBMCs , 用无血清培养液调整至合适细胞浓度, 置于培养 瓶内, 适当培养条件下以使单核细胞贴壁;  [0030] 1.1 PBMCs obtained from whole blood, adjusted to a suitable cell concentration with serum-free medium, placed in a culture bottle, under appropriate culture conditions to allow monocytes to adhere;
[0031] 1.2将悬浮细胞从已经贴壁的培养瓶中分离, 收集悬浮细胞, 悬浮细胞主要是 T 淋巴细胞单, 用无血清培养液调整至合适细胞浓度, 使用多种细胞因子对其进 行诱导, 例如细胞因子包括重组人 IFN-γ (淋巴细胞型干扰素) 、 CD3单克隆抗 体、 重组人 IL-2 (白细胞介素 -2) , 刺激 CIK细胞的生长和增殖;  [0031] 1.2 The suspension cells are separated from the adherent culture flask, and the suspension cells are collected. The suspension cells are mainly T lymphocyte monolayers, adjusted to a suitable cell concentration with serum-free medium, and induced by various cytokines. For example, cytokines include recombinant human IFN-γ (lymphocyte-type interferon), CD3 monoclonal antibody, and recombinant human IL-2 (interleukin-2), which stimulate the growth and proliferation of CIK cells;
[0032] 1.3在培养一段吋间后, CIK细胞数量达到一定数量级, 即可收获。  [0032] 1.3 After culturing a section of the sputum, the number of CIK cells reaches a certain order of magnitude and can be harvested.
[0033] 2.肿瘤抗原负载 DC细胞的获得  [0033] 2. Tumor antigen loading DC cell acquisition
[0034] 2.1步骤 1.1中剩下的贴壁细胞 (主要是 CD14+的单核细胞, 或者称为 DC前体细胞 ), 加入适当的细胞因子, 例如重组人 GM-CSF (人粒细胞-巨噬细胞集落刺激因 子) 、 重组人 IL-4 (白细胞介素 -4) 等, 诱导单核细胞向 DC细胞分化;  [0034] 2.1 The remaining adherent cells in step 1.1 (mainly CD14+ monocytes, or DC precursor cells), add appropriate cytokines, such as recombinant human GM-CSF (human granulocyte-macrophage) Cell colony-stimulating factor), recombinant human IL-4 (interleukin-4), etc., induces differentiation of monocytes into DC cells;
[0035] 2.2  2.2 [0035]
培养过程中适当补足上述细胞因子, 在培养一定吋间后加入肿瘤抗原, 对 DC细 胞进行抗原负载后继续培养, 并可以加入重组人 TNF-ot (肿瘤坏死因子 -ot) 等细 胞因子进行诱导, 诱导得到成熟的 DC细胞; [0036] 2.3在培养一段吋间后, 肿瘤抗原负载 DC细胞的细胞数量达到一定数量级, 即 可收获。 During the culture process, the above cytokines are appropriately supplemented, and the tumor antigen is added after the culture is incubated, and the DC cells are subjected to antigen loading, and then cultured, and cytokines such as recombinant human TNF-ot (tumor necrosis factor-ot) can be added for induction. Inducing mature DC cells; [0036] 2.3 After culturing a section of the sputum, the number of cells of the tumor antigen-loaded DC cells reaches a certain order of magnitude, and can be harvested.
[0037] 3.肿瘤特异性 CTL的制备  [0037] 3. Preparation of tumor specific CTL
[0038] 将获得的 CIK细胞和肿瘤抗原负载的 DC细胞按照一定的比例, 例如 10:1, 混合 培养, 培养过程中适当更换培养基, 并补加细胞因子重组人 IL-2等, 在培养一段 吋间后, 即可收集肿瘤特异性 CTL细胞。  [0038] The obtained CIK cells and tumor antigen-loaded DC cells are mixed and cultured in a certain ratio, for example, 10:1, the medium is appropriately changed during the culture, and the cytokine recombinant human IL-2 is added, and cultured. Tumor-specific CTL cells can be collected after a period of time.
[0039] 上述 CTL的制备过程较为复杂, 悬浮细胞与贴壁细胞的分别诱导工作量较大、 分别诱导的吋间周期较难统一, 另外, 通过上述方法制备的 CTL活性低或无反应 , 可能是由于 DC在呈递抗原吋所受周围微环境变化较大等原因影响 CTL特异性 免疫杀伤物质合成。  [0039] The preparation process of the above CTL is complicated, and the induction workload of the suspension cells and the adherent cells is relatively large, and the diurnal cycle induced respectively is difficult to be unified. In addition, the CTL prepared by the above method has low or no reaction, and may It is due to the fact that DCs affect the synthesis of CTL-specific immunosuppressive substances due to the large changes in the surrounding microenvironment of the presented antigens.
[0040] 本发明的主要创新点在于, 在细胞培养瓶中预包被肿瘤抗原, DC前体细胞和 T 淋巴细胞不需要单独分幵培养, 简化了实验过程, DC前体细胞贴壁生长, 与培 养瓶壁上包被的肿瘤抗原可以充分接触, 有助于获得大量抗原负载的 DC细胞来 呈递抗原, 快速获得大量特异性强的 CTL免疫活性细胞。  [0040] The main innovation of the present invention is that the tumor antigen is pre-coated in the cell culture bottle, and the DC precursor cells and the T lymphocytes do not need to be separately cultured, which simplifies the experimental process, and the DC precursor cells adhere to the wall. It can be fully contacted with the tumor antigen coated on the wall of the culture bottle, and it is helpful to obtain a large number of antigen-loaded DC cells to present the antigen, and quickly obtain a large number of specific CTL immunocompetent cells with high specificity.
[0041] 图 2示出了本发明肿瘤特异性 CTL制备方法较佳实施例的流程, 如图 2所示, 本 实施例的肿瘤特异性 CTL的制备方法, 包括如下步骤:  2 shows a flow of a preferred embodiment of the method for preparing a tumor-specific CTL of the present invention. As shown in FIG. 2, the method for preparing a tumor-specific CTL of the present embodiment includes the following steps:
[0042] S100、 肿瘤抗原的包被: 使用患者肿瘤组织或肿瘤细胞系制备肿瘤抗原, 调整 肿瘤抗原浓度至 5(Vg/ml〜20(Vg/ml, 加入到 TC处理过的细胞培养瓶中覆盖瓶底 , 进行包被反应, 得到肿瘤抗原预包被的培养瓶。 采用患者的自体肿瘤抗原, 无外源细胞导入, 安全性强, 特异性高, 可明显诱导出抗自体肿瘤的特异性 CTL , 对体内正常的细胞无任何损伤作用, 并有利于重建对该肿瘤的防御监控能力  [0042] S100, coating of tumor antigen: preparing tumor antigen using patient tumor tissue or tumor cell line, adjusting tumor antigen concentration to 5 (Vg/ml~20 (Vg/ml, added to TC-treated cell culture flask) Cover the bottom of the bottle and carry out the coating reaction to obtain the culture bottle pre-coated with the tumor antigen. The patient's autologous tumor antigen, no foreign cell introduction, strong safety, high specificity, can significantly induce the specificity against autologous tumor CTL, does not have any damage to normal cells in the body, and is conducive to rebuilding the defense monitoring ability of the tumor
[0043] S200、 外周血单个核细胞的分离: 从患者血液中分离外周血单个核细胞, 悬浮 备用。 从患者全血中获得的 PBMCs主要包括两种类型的细胞, 一类是起到抗原 呈递作用的 DC细胞类, 一类是能够合成相关杀伤性免疫活性物质的 T淋巴细胞 , 传统方法中会根据 DC细胞类贴壁生长的特性将上述两类细胞分离并单独分幵 培养, 本申请中获得的 PBMCs会直接在肿瘤抗原预包被的培养瓶中培养, 没有 传统方法中分幵培养、 分别诱导、 混合培养的复杂过程。 [0044] S300、 培养肿瘤特异性 CTL: 将所述外周血单个核细胞小心铺入肿瘤抗原预包 被的培养瓶中, 进行孵育培养诱导获得成熟的肿瘤抗原负载的 DC细胞和 T淋巴 细胞; 然后加入刺激性细胞因子、 补充新鲜培养基连续培养, 大量扩增 T淋巴细 胞获得抗原特异性的 CTL。 由于本发明中没有传统方法中分幵培养、 分别诱导、 混合培养的过程, 整个培养环境相对稳定, 不会因为周围微环境变化对呈递抗 原照成影响, 另外, DC前体细胞贴壁生长的特性使得其与培养瓶壁上包被的肿 瘤抗原可以充分接触, 有助于获得大量抗原负载的 DC细胞。 S200, Separation of Peripheral Blood Mononuclear Cells: Peripheral blood mononuclear cells are isolated from the blood of the patient and suspended for use. PBMCs obtained from patients' whole blood mainly include two types of cells, one is DC cells that play antigen-presenting, and the other is T lymphocytes that can synthesize related killer immunologically active substances. Characteristics of adherent growth of DC cells The above two types of cells are isolated and cultured separately. The PBMCs obtained in the present application are directly cultured in a culture flask pre-coated with tumor antigens, and are not induced by conventional methods. , the complex process of mixed culture. [0044] S300, cultivating tumor-specific CTL: carefully plating the peripheral blood mononuclear cells into a tumor antigen pre-coated culture flask, and performing incubation culture to induce mature tumor antigen-loaded DC cells and T lymphocytes; Then, stimulating cytokines were added, and fresh medium was continuously cultured, and T lymphocytes were expanded in a large amount to obtain antigen-specific CTLs. Since there is no traditional method for branching culture, separately inducing, and mixing culture in the present invention, the entire culture environment is relatively stable, and the effect of presenting the antigen is not affected by changes in the surrounding microenvironment. In addition, DC precursor cells are adherently grown. Its properties allow it to be in full contact with the tumor antigen coated on the wall of the culture bottle, helping to obtain a large number of antigen-loaded DC cells.
[0045] 在本发明的另一较佳实施例中, 除包括上述步骤外, 步骤 S100中还包括: [0046] S101、 制备肿瘤抗原: 将患者肿瘤组织或肿瘤细胞系分散成单细胞悬液, 用生 理盐水调节悬浮肿瘤细胞浓度至 1><10 7〜2><10 7个/1^, 反复冻融 3〜5次, 裂解肿 瘤细胞; 将肿瘤细胞裂解物在 2000g下离心 8〜12分钟后吸取上清, 0.22μηι滤膜 过滤除菌, 记为肿瘤抗原。 滤膜过滤之后通常需要留样检测蛋白含量以及细菌 、 真菌和支原体, 剩余部分 -80°C保存备用。 制备并获取肿瘤抗原的目的是使用 肿瘤细胞抗原负载 DC细胞, 现有技术中肿瘤细胞抗原负载 DC细胞的方法很多, 包括用肿瘤细胞裂解液负载 DC、 用凋亡肿瘤细胞负载 DC、 用坏死或死亡的肿瘤 细胞负载 DC、 用肿瘤活细胞负载 DC、 将肿瘤细胞与 DC融合等。 本实施例中选 用的方法是肿瘤细胞裂解液负载 DC, 即使用反复冻融的方式将肿瘤细胞裂解, 获取肿瘤细胞裂解液, 操作简单、 快速, 并可较好的保存抗原活性。 反复冻融 的过程也可以选择多种方式, 例如装有单细胞悬液的冻存管浸入液氮或者 -80°C 冰箱中进行速冻, 再迅速放入 37°C水浴中进行解冻 10 min, 反复冻融 3-5次, 确 保肿瘤细胞充分裂解。 另外, 将确保肿瘤细胞充分裂解获取单细胞悬液的方式 也很多, 例如优选的可以为胶原蛋白酶消化法、 物理研磨法。 胶原蛋白酶消化 法通常是使用胶原蛋白酶对肿瘤组织块进行消化, 使组织分散成细胞, 具体过 程大体为: 将手术取下的鲜活肿瘤组织块, 在无菌条件下, 用生理盐水至少洗 涤 3次后, 剪碎, 并且用 0.1%胶原蛋白酶在 37°C条件下消化 30分钟, 将获得的肿 瘤细胞离心收获后, 经过 100目尼龙网过滤后收集单个细胞悬液, 用适量的生理 盐水悬浮肿瘤细胞, 装入无菌冻存管以备冻融。 物理研磨法通常是使用研磨作 用对肿瘤组织块进行分散成细胞, 具体过程大体为: 将肿瘤组织剪碎后, 加入 R PMI1640培养基, 充分研磨, 200目无菌网过滤后收集单细胞悬液。 [0045] In another preferred embodiment of the present invention, in addition to the above steps, the step S100 further includes: [0046] S101, preparing a tumor antigen: dispersing a patient tumor tissue or a tumor cell line into a single cell suspension Adjust the concentration of suspended tumor cells with physiological saline to 1><10 7 ~2><10 7 /1^, freeze and thaw 3~5 times, lyse tumor cells; centrifuge cell lysate at 2000g for 8~12 After a minute, the supernatant was aspirated, and 0.22 μηι filter was used to filter and sterilize, and it was recorded as a tumor antigen. After filtering the membrane, it is usually necessary to check the protein content as well as bacteria, fungi and mycoplasma, and the rest is stored at -80 °C for later use. The purpose of preparing and obtaining tumor antigens is to use DC cells loaded with tumor cell antigens. There are many methods for loading DC cells in tumor cell antigens in the prior art, including loading DC with tumor cell lysate, DC with apoptotic tumor cells, necrosis or The dead tumor cells are loaded with DC, the tumor living cells are loaded with DC, the tumor cells are fused with DC, and the like. The method selected in the present embodiment is that the tumor cell lysate is loaded with DC, that is, the tumor cells are lysed by repeated freeze-thaw method, and the tumor cell lysate is obtained, the operation is simple and rapid, and the antigen activity can be well preserved. Repeated freezing and thawing process can also choose a variety of ways, for example, a cryotube containing a single cell suspension is immersed in liquid nitrogen or frozen in a -80 ° C refrigerator, and then quickly placed in a 37 ° C water bath for thawing for 10 min, Repeat freeze-thaw 3-5 times to ensure that the tumor cells are fully lysed. In addition, there are many ways to ensure that the tumor cells are sufficiently lysed to obtain a single cell suspension, and for example, a collagenase digestion method or a physical polishing method is preferable. Collagenase digestion method is commonly used for the collagenase-digested tumor mass, the dispersion into the tissue cells, the specific process generally: Fresh tumor tissue blocks surgery removed, under sterile conditions, washed with saline least 3 After that, it was cut, and digested with 0.1% collagenase for 30 minutes at 37 ° C. The obtained tumor cells were harvested by centrifugation, and after filtration through a 100 mesh nylon mesh, a single cell suspension was collected and suspended in an appropriate amount of physiological saline. Tumor cells are filled into sterile cryotubes for freezing and thawing. The physical grinding method usually uses the grinding action to disperse the tumor tissue block into cells. The specific process is as follows: After the tumor tissue is cut, the R is added. PMI 1640 medium, fully ground, filtered through a 200 mesh sterile mesh and collected for single cell suspension.
[0047] S102、 处理细胞培养瓶: TC处理的细胞培养瓶。 [0047] S102. Processing a cell culture flask: a TC-treated cell culture flask.
[0048] S103、 包被反应: 用无菌生理盐水或 lxPBS (磷酸盐缓冲液, pH7.0〜7.4) 调 整肿瘤抗原的浓度至 5(Vg/ml〜20(Vg/ml, 取 10ml〜20ml, 加入 TC处理后的细胞 培养瓶中, 肿瘤抗原覆盖培养瓶瓶底, 4°C过夜或者 37°C进行包被反应 11!〜 2h, 得到肿瘤抗原预包被的培养瓶。 肿瘤抗原直接取用上述 -80°C保存的肿瘤抗原, 4 °C溶解后即可使用。 包被完成后, 洗涤以去除未包被的肿瘤抗原, 例如, 用适量 的无菌生理盐水或 lxPBS充分洗涤包被培养瓶, 2〜5次, 洗涤完成后放入 4°C保 存备用。  [0048] S103, coating reaction: adjust the tumor antigen concentration to 5 (Vg / ml ~ 20 (Vg / ml, take 10ml ~ 20ml) with sterile physiological saline or lxPBS (phosphate buffer, pH 7.0 ~ 7.4) After adding the TC-treated cell culture flask, the tumor antigen covers the bottom of the culture flask, and the coating reaction is carried out at 4 ° C overnight or at 37 ° C for 11!~ 2 h to obtain a tumor antigen pre-coated culture flask. The tumor antigen preserved at -80 ° C above can be used after being dissolved at 4 ° C. After the coating is completed, the washing is performed to remove the uncoated tumor antigen, for example, the coating is thoroughly washed with an appropriate amount of sterile physiological saline or lxPBS. The flask is incubated 2 to 5 times. After the washing is completed, it is stored at 4 ° C for later use.
[0049] 在本发明的另一较佳实施例中, 除包括上述步骤外, 步骤 S200中还包括:  [0049] In another preferred embodiment of the present invention, in addition to the foregoing steps, step S200 further includes:
[0050] S201、 准备全血: 抽取肿瘤患者静脉血, 加入抗凝剂成抗凝全血备用。 现场抽 取以制备抗凝全血, 或者直接冷藏的抗凝全血, 冷藏过程对结果影响不显著。 [0050] S201, preparing whole blood: venous blood is taken from a tumor patient, and an anticoagulant is added to form anticoagulated whole blood for use. On-site extraction to prepare anticoagulated whole blood, or directly frozen anticoagulated whole blood, the refrigeration process has no significant effect on the results.
[0051] S202、 外周血单个核细胞与自体血浆的分离: 使用血细胞分离机或者淋巴细胞 分离液将外周血单个核细胞从自体血浆中分离, 获得的外周血单个核细胞粗品 。 血细胞分离机可以直接获取外周血单个核细胞粗品, 使用淋巴细胞分离液来 分离外周血单个核细胞则会因为淋巴细胞分离液的厂家不同而略有差异, 大体 过程为: 在离心管中加入三分之一左右的人外周血淋巴细胞分离液, 将肿瘤患 者抗凝全血小心铺到淋巴细胞分离液的上层, 离心 (例如, 20°C, 800g离心 20分 钟) 后用巴氏吸管或移液器小心吸取 PBMCs层, 获得的外周血单个核细胞粗品 S202, Separation of peripheral blood mononuclear cells from autologous plasma: Peripheral blood mononuclear cells are obtained by separating a peripheral blood mononuclear cell from autologous plasma using a blood cell separator or a lymphocyte separation solution. The blood cell separator can directly obtain the crude blood mononuclear cells of the peripheral blood. The separation of the peripheral blood mononuclear cells by the lymphocyte separation solution may be slightly different due to the different manufacturers of the lymphocyte separation liquid. The general process is: adding three in the centrifuge tube One-quarter of human peripheral blood lymphocyte separation solution, carefully spread anti-coagulated whole blood of tumor patients to the upper layer of lymphocyte separation solution, centrifuge (for example, 20 ° C, 800 g for 20 minutes), then use a Pasteur pipette or transfer The pipette carefully draws the PBMCs layer and obtains the crude blood of the peripheral blood mononuclear cells.
[0052] S203、 外周血单个核细胞的处理: 将外周血单个核细胞粗品用生理盐水或无血 清培养基洗涤两次, 20°C下 500g离心 3〜7分钟, 再用适量无血清培养基重悬, 得 到外周血单个核细胞, 备用。 [0052] S203, treatment of peripheral blood mononuclear cells: the peripheral blood mononuclear cells are washed twice with physiological saline or serum-free medium, centrifuged at 500g for 3 to 7 minutes at 20 ° C, and then with an appropriate amount of serum-free medium Resuspend, obtain peripheral blood mononuclear cells, and reserve.
[0053] 在本发明的另一较佳实施例中, 除包括上述步骤外, 步骤 S300中还包括: [0053] In another preferred embodiment of the present invention, in addition to the foregoing steps, step S300 further includes:
[0054] S301、 调整细胞浓度: 用无血清培养液调整外周血单个核细胞的细胞浓度至 lx[3014] S301, adjusting cell concentration: adjusting serum cell concentration of peripheral blood mononuclear cells to serum by serum-free medium
10 6〜2xl0 6个 /ml, 混合均匀后小心铺入肿瘤抗原预包被的培养瓶中。 10 6 ~ 2xl0 6 / ml, mix well and carefully spread into the tumor antigen pre-coated culture flask.
[0055] S302、 孵育培养: 向培养瓶中加入 400U/m!〜 800U/ml的 GM-CSF和 300U/m!〜 6[0055] S302, Incubation Culture: Add 400 U/m to the culture flask! ~ 800U/ml GM-CSF and 300U/m! ~ 6
00U/ml的 IL4, 在 CO 2培养箱中进行孵育培养 361!〜 48h, 诱导获得成熟的肿瘤抗 原负载的 DC细胞。 C0 2培养箱中培养条件可以选择 37°C, C0 2浓度 5%。 00U/ml of IL4, incubate in a CO 2 incubator 361! ~ 48h, induced mature tumor resistance Originally loaded DC cells. The culture conditions in the C0 2 incubator can be selected at 37 ° C and the C 0 2 concentration is 5%.
[0056] S303、 连续培养: 向肿瘤抗原负载的 DC细胞中加入 500U/ml〜1200U/ml的 IFN- γ、 60ng/ml〜150ng/ml的 anti-CD3、 600U/ml〜1400U/ml的 IL2后继续培养, 孵育 培养完成后即可一次性加入 IFN-Y、 anti-CD3、 IL2。 隔天取样计数, 补充含 IL2 的新鲜无血清培养基, 将细胞的培养密度调整至 1x 10 6〜2xl0 6 [0056] S303, continuous culture: adding 500 U/ml to 1200 U/ml of IFN-γ, 60 ng/ml to 150 ng/ml of anti-CD3, and 600 U/ml to 1400 U/ml of IL2 to the tumor antigen-loaded DC cells. After the culture is continued, IFN-Y, anti-CD3, and IL2 can be added at one time after the incubation. Samples were taken every other day, supplemented with fresh serum-free medium containing IL2, and the culture density of the cells was adjusted to 1x 10 6 to 2xl0 6
个 /ml; 培养 12〜15天后, 获得特异性抗原负载的 DC-CTL细胞, 此期间也可适量 添加新鲜无血清培养基以补充细胞合成所需营养物质, 但是数量不宜频繁。  /ml; After 12 to 15 days of culture, DC-CTL cells with specific antigen loading are obtained. During this period, fresh serum-free medium can also be added to supplement the nutrients required for cell synthesis, but the quantity is not frequent.
[0057] 实施例 1 Embodiment 1
[0058] 1肿瘤抗原的包被 [0058] 1 coating of tumor antigen
[0059] 1.1制备肿瘤抗原: 将肝癌患者手术取下的鲜活肿瘤组织块, 在无菌条件下, 用生理盐水至少洗涤 3次后, 剪碎, 并且用 0.1%胶原蛋白酶在 37°C条件下消化 30 分钟, 将获得的肿瘤细胞离心收获后, 经过 100目尼龙网过滤后收集单个细胞悬 液, 用适量的生理盐水悬浮肿瘤细胞的细胞浓度至 1x 10 7个 /ml, 装入无菌冻存管 以备冻融; 装有单细胞悬液的冻存管浸入液氮中进行速冻, 再迅速放入 37°C水浴 中进行解冻 10 min, 反复冻融 3次, 确保肿瘤细胞充分裂解; 将肿瘤细胞裂解物 在 20°C, 2000g下离心 8分钟后吸取上清, 0.22μηι滤膜过滤除菌, 记为肿瘤抗原 , -80°C保存备用。  1.1 Preparation of tumor antigen: The fresh tumor tissue piece removed from the liver cancer patient is washed under sterile conditions with at least 3 times with physiological saline, and then cut with 0.1% collagenase at 37 ° C. After digestion for 30 minutes, the obtained tumor cells were harvested by centrifugation, and after filtration through a 100-mesh nylon mesh, a single cell suspension was collected, and the cell concentration of the tumor cells was suspended to 1×10 7 cells/ml with an appropriate amount of physiological saline, and the mixture was sterilized. Freeze the tube for freezing and thawing; the cryotube containing the single cell suspension is immersed in liquid nitrogen for quick freezing, then quickly placed in a 37 ° C water bath for thawing for 10 min, repeated freeze-thaw three times to ensure that the tumor cells are fully lysed The tumor cell lysate was centrifuged at 20 ° C, 2000 g for 8 minutes, and the supernatant was aspirated, filtered through a 0.22 μηι filter, and recorded as a tumor antigen, and stored at -80 ° C until use.
[0060] 1.2处理细胞培养瓶: TC处理的细胞培养瓶。  1.2 Cell Culture Bottles: TC-treated cell culture flasks.
[0061] 1.3  1.3 [1.31] 1.3
包被反应: 用无菌生理盐水调整肿瘤抗原的浓度至 5(Vg/ml, 取 20ml, 加入 TC处 理后的细胞培养瓶中, -80°C保存的肿瘤抗原 4°C溶解后覆盖培养瓶瓶底, 4°C过 夜进行包被反应, 得到肿瘤抗原预包被的培养瓶。 用适量的无菌生理盐水充分 洗涤包被培养瓶 2次, 洗涤完成后放入 4°C保存备用。  Coating reaction: Adjust the concentration of tumor antigen to 5 (Vg/ml with sterile saline), take 20ml, add to the cell culture flask after TC treatment, and dissolve the tumor antigen stored at -80 °C at 4 °C to cover the culture flask. The bottom of the bottle was subjected to coating reaction overnight at 4 ° C to obtain a culture flask pre-coated with tumor antigen. The coated culture flask was thoroughly washed twice with an appropriate amount of sterile physiological saline, and stored at 4 ° C until the washing was completed.
[0062] 2外周血单个核细胞的分离 2 Separation of peripheral blood mononuclear cells
[0063] 2.1准备全血: 抽取肿瘤患者静脉血, 加入抗凝剂成抗凝全血备用。  [0063] 2.1 Preparation of whole blood: The venous blood of the tumor patient is taken, and the anticoagulant is added to form anticoagulated whole blood for use.
[0064] 2.2外周血单个核细胞与自体血浆的分离: 使用血细胞分离机将外周血单个核 细胞从自体血浆中分离, 获得的外周血单个核细胞粗品。  2.2 Separation of peripheral blood mononuclear cells from autologous plasma: Peripheral blood mononuclear cells were obtained by separating peripheral blood mononuclear cells from autologous plasma using a blood cell separator.
[0065] 2.3外周血单个核细胞的处理: 将外周血单个核细胞粗品用生理盐水洗涤两次 , 20°C下 500g离心 3分钟, 再用适量无血清培养基重悬, 得到外周血单个核细胞2.3 Treatment of peripheral blood mononuclear cells: Washing crude peripheral blood mononuclear cells twice with physiological saline , centrifuged at 500g for 3 minutes at 20 ° C, and then resuspended in an appropriate amount of serum-free medium to obtain peripheral blood mononuclear cells.
, 备用。 , spare.
[0066] 3培养肿瘤特异性 CTL  [0066] 3 culture of tumor-specific CTL
[0067] 3.1调整细胞浓度: 用无血清培养液调整外周血单个核细胞的细胞浓度至 1x10 6 个 /ml, 混合均匀后小心铺入肿瘤抗原预包被的培养瓶中。  3.1 Adjusting the cell concentration: The cell concentration of the peripheral blood mononuclear cells was adjusted to 1×10 6 cells/ml with the serum-free medium, and the cells were uniformly mixed and carefully placed in the culture flask pre-coated with the tumor antigen.
[0068] 3.2孵育培养: 向培养瓶中加入 400U/ml的 GM-CSF和 300U/ml的 IL4, 在 CO 2培 养箱中进行孵育培养 48h, C0 2培养箱中培养条件可以选择 37°C, C0 2浓度 5%, 诱导获得成熟的肿瘤抗原负载的 DC细胞。 [0068] 3.2 Incubation culture: 400 U/ml GM-CSF and 300 U/ml IL4 were added to the culture flask, and the culture was carried out in a CO 2 incubator for 48 h, and the culture condition in the C0 2 incubator was 37 ° C. C0 2 concentration of 5% induced the induction of mature tumor antigen-loaded DC cells.
[0069] 3.3 3.3
连续培养: 孵育培养完成后向肿瘤抗原负载的 DC细胞中加入 500U/ml的 IFN-Y、 1 50ng/ml的 anti-CD3、 1400U/ml的 IL2后继续培养。 隔天取样计数, 补充含 IL2的 新鲜无血清培养基, 将细胞的培养密度调整至 1x10 6个 /ml; 培养 15天后, 获得特 异性抗原负载的 DC-CTL细胞。  Continuous culture: After the incubation, the culture was continued by adding 500 U/ml of IFN-Y, 150 ng/ml of anti-CD3, and 1400 U/ml of IL2 to the tumor antigen-loaded DC cells. The samples were counted every other day, supplemented with fresh serum-free medium containing IL2, and the culture density of the cells was adjusted to 1×10 6 /ml; after 15 days of culture, DC-CTL cells loaded with specific antigen were obtained.
[0070] 实施例 2  Example 2
[0071] 1肿瘤抗原的包被  [0071] 1 coating of tumor antigen
[0072] 1.1制备肿瘤抗原: 取肝脏肿瘤细胞系 HepG2, 在无菌条件下, 用生理盐水至 少洗涤 3次后, 加入 RPMI1640培养基, 充分研磨, 200目无菌网过滤后收集单细 胞悬液, 用适量的生理盐水悬浮肿瘤细胞的细胞浓度至 2x10 7个 /ml, 装入无菌冻 存管以备冻融; 装有单细胞悬液的冻存管浸入液氮或者 -80°C冰箱中进行速冻, 再迅速放入 37°C水浴中进行解冻 10 min, 反复冻融 5次, 确保肿瘤细胞充分裂解 ; 将肿瘤细胞裂解物在 20°C, 2000g下离心 12分钟后吸取上清, 0.22μηι滤膜过滤 除菌, 记为肿瘤抗原, -80°C保存备用。  1.1 Preparation of tumor antigen: The liver tumor cell line HepG2 was taken, and under sterile conditions, washed at least 3 times with physiological saline, added to RPMI1640 medium, fully ground, and filtered through a 200 mesh sterile mesh to collect a single cell suspension. , suspend the cell concentration of the tumor cells with an appropriate amount of physiological saline to 2x10 7 /ml, and put them into a sterile cryotube for freezing and thawing; the cryotube containing the single cell suspension is immersed in liquid nitrogen or -80 ° C refrigerator Quickly freeze, then quickly put into a 37 ° C water bath for thawing for 10 min, repeated freezing and thawing 5 times to ensure that the tumor cells are fully lysed; centrifuge the tumor cell lysate at 20 ° C, 2000g for 12 minutes, then extract the supernatant, The bacteria were sterilized by filtration on a 0.22 μηι filter, and recorded as a tumor antigen, and stored at -80 ° C until use.
[0073] 1.2处理细胞培养瓶: TC处理的细胞培养瓶。  1.2 Cell Culture Bottles: TC-treated cell culture flasks.
[0074] 1.3  1.3 [0074] 1.3
包被反应: 用无菌生理盐水或 lxPBS调整肿瘤抗原的浓度至 20(Vg/ml, 取 10ml, 加入 TC处理后的细胞培养瓶中, -80°C保存的肿瘤抗原 4°C溶解后覆盖培养瓶瓶 底, 37°C进行包被反应 lh, 得到肿瘤抗原预包被的培养瓶。 用适量的 lxPBS充分 洗涤包被培养瓶 5次, 洗涤完成后放入 4°C保存备用。 [0075] 2外周血单个核细胞的分离 Coating reaction: Adjust the concentration of tumor antigen to 20 (Vg/ml with sterile saline or lxPBS, take 10ml, add to the cell culture flask after TC treatment, and dissolve the tumor antigen stored at -80 °C after 4 °C dissolution. The bottom of the flask was cultured and subjected to coating reaction at 37 ° C for 1 h to obtain a culture flask pre-coated with tumor antigen. The coated flask was thoroughly washed 5 times with an appropriate amount of lxPBS, and stored at 4 ° C for storage. 2 Separation of peripheral blood mononuclear cells
[0076] 2.1准备全血: 抽取肿瘤患者静脉血, 加入抗凝剂成抗凝全血备用。  [0076] 2.1 Preparation of whole blood: The venous blood of the tumor patient is taken, and the anticoagulant is added to form anticoagulated whole blood for use.
[0077] 2.2外周血单个核细胞与自体血浆的分离: 在离心管中加入三分之一左右的人 外周血淋巴细胞分离液, 将肿瘤患者抗凝全血小心铺到淋巴细胞分离液的上层 [0077] 2.2 Separation of peripheral blood mononuclear cells from autologous plasma: Add about one-third of human peripheral blood lymphocyte separation solution in a centrifuge tube, and carefully spread the anticoagulated whole blood of the tumor patient to the upper layer of the lymphocyte separation liquid.
, 20°C, 800g离心 20分钟, 后用巴氏吸管或移液器小心吸取 PBMCs层, 获得的 外周血单个核细胞粗品。 After centrifugation at 20 ° C, 800 g for 20 minutes, the PBMCs layer was carefully aspirated with a Pasteur pipette or a pipette to obtain a crude peripheral blood mononuclear cell.
[0078] 2.3外周血单个核细胞的处理: 将外周血单个核细胞粗品用无血清培养基洗涤 两次, 20°C下 500g离心 7分钟, 再用适量无血清培养基重悬, 得到外周血单个核 细胞, 备用。 [0078] 2.3 treatment of peripheral blood mononuclear cells: the peripheral blood mononuclear cells were washed twice with serum-free medium, centrifuged at 500g for 7 minutes at 20 ° C, and then resuspended in an appropriate amount of serum-free medium to obtain peripheral blood. Mononuclear cells, spare.
[0079] 3培养肿瘤特异性 CTL 3 culture of tumor-specific CTL
[0080] 3.1调整细胞浓度: 用无血清培养液调整外周血单个核细胞的细胞浓度至 1x10 6 个 /ml, 混合均匀后小心铺入肿瘤抗原预包被的培养瓶中。 3.1 Adjusting the cell concentration: The cell concentration of the peripheral blood mononuclear cells was adjusted to 1×10 6 cells/ml with the serum-free medium, and the cells were uniformly mixed and carefully placed in the culture flask pre-coated with the tumor antigen.
[0081] 3.2孵育培养: 向培养瓶中加入 600U/ml的 GM-CSF和 400U/ml的 IL4, 在 CO 2培 养箱中进行孵育培养 40h, C0 2培养箱中培养条件可以选择 37°C, C0 2浓度 5%, 诱导获得成熟的肿瘤抗原负载的 DC细胞。 [0081] 3.2 Incubation culture: 600 U/ml GM-CSF and 400 U/ml IL4 were added to the culture flask, and the culture was carried out in a CO 2 incubator for 40 h, and the culture condition in the C0 2 incubator was 37 ° C. C0 2 concentration of 5% induced the induction of mature tumor antigen-loaded DC cells.
[0082] 3.3 3.3 [3.32] 3.3
连续培养: 孵育培养完成后向肿瘤抗原负载的 DC细胞中加入 100U/ml的 IFN-Y、 9 Ong/ml的 anti-CD3、 1000U/ml的 IL2后继续培养。 隔天取样计数, 补充含 IL2的新 鲜无血清培养基, 将细胞的培养密度调整至 1x10 6个 /ml; 培养 13天后, 获得特异 性抗原负载的 DC-CTL细胞。  Continuous culture: After the incubation, the culture was continued by adding 100 U/ml of IFN-Y, 9 Ong/ml of anti-CD3, and 1000 U/ml of IL2 to the tumor antigen-loaded DC cells. The samples were counted every other day, supplemented with fresh serum-free medium containing IL2, and the culture density of the cells was adjusted to 1×10 6 /ml; after 13 days of culture, specific antigen-loaded DC-CTL cells were obtained.
[0083] 实施例 3  Example 3
[0084] 1肿瘤抗原的包被  [0084] 1 coating of tumor antigen
[0085] 1.1制备肿瘤抗原: 将肝癌患者手术取下的鲜活肿瘤组织块, 在无菌条件下, 用生理盐水至少洗涤 3次后, 剪碎, 并且用 0.1%胶原蛋白酶在 37°C条件下消化 30 分钟, 将获得的肿瘤细胞离心收获后, 经过 100目尼龙网过滤后收集单个细胞悬 液, 用适量的生理盐水悬浮肿瘤细胞的细胞浓度至 2x10 7个 /ml, 装入无菌冻存管 以备冻融; 装有单细胞悬液的冻存管浸入液氮或者 -80°C冰箱中进行速冻, 再迅 速放入 37°C水浴中进行解冻 10 min, 反复冻融 4次, 确保肿瘤细胞充分裂解; 将 肿瘤细胞裂解物在 20°C, 2000g下离心 10分钟后吸取上清, 0.22μηι滤膜过滤除菌 , 记为肿瘤抗原, -80°C保存备用。 1.1 Preparation of tumor antigen: The fresh tumor tissue piece removed from the liver cancer patient is washed under sterile conditions with at least 3 times with physiological saline, then cut, and treated with 0.1% collagenase at 37 ° C. After digestion for 30 minutes, the obtained tumor cells were harvested by centrifugation, and after filtration through a 100 mesh nylon mesh, a single cell suspension was collected, and the cell concentration of the tumor cells was suspended in an appropriate amount of physiological saline to 2×10 7 cells/ml, and filled into a sterile frozen solution. The tube is stored for freezing and thawing; the cryotube containing the single cell suspension is immersed in liquid nitrogen or frozen in a -80 ° C refrigerator, and then quickly placed in a 37 ° C water bath for thawing for 10 min, repeated freezing and thawing 4 times, Ensure that the tumor cells are fully lysed; The tumor cell lysate was centrifuged at 2000 g for 10 minutes at 20 ° C, and the supernatant was aspirated, and sterilized by filtration through a 0.22 μηι filter, which was recorded as a tumor antigen, and stored at -80 ° C until use.
[0086] 1.2处理细胞培养瓶: TC处理的细胞培养瓶。 1.2 Cell Culture Bottles: TC-treated cell culture flasks.
[0087] 1.3包被反应: 用 lxPBS调整肿瘤抗原的浓度至 10(Vg/ml, 取 15ml, 加入 TC处 理后的细胞培养瓶中, -80°C保存的肿瘤抗原 4°C溶解后覆盖培养瓶瓶底, 4°C过 夜进行包被反应, 得到肿瘤抗原预包被的培养瓶。 用适量的 lxPBS充分洗涤包被 培养瓶, 4次, 洗涤完成后放入 4°C保存备用。  [0087] 1.3 coating reaction: adjust the concentration of tumor antigen to 10 (Vg / ml, l 15ml, add TC treated cell culture flask, -80 ° C preserved tumor antigen 4 ° C dissolved after covering culture The bottom of the bottle was subjected to coating reaction overnight at 4 ° C to obtain a culture flask pre-coated with tumor antigen. The coated culture flask was thoroughly washed with an appropriate amount of lxPBS for 4 times, and after washing, it was stored at 4 ° C for use.
[0088] 2外周血单个核细胞的分离  [0088] 2 separation of peripheral blood mononuclear cells
[0089] 2.1准备全血: 直接使用冷藏的抗凝全血。  [0089] 2.1 Preparation of whole blood: Direct use of refrigerated anticoagulated whole blood.
[0090] 2.2外周血单个核细胞与自体血浆的分离: 使用血细胞分离机获得的外周血单 个核细胞粗品。  2.2 Separation of peripheral blood mononuclear cells from autologous plasma: Peripheral blood single nuclear cells obtained using a blood cell separator.
[0091] 2.3外周血单个核细胞的处理: 将外周血单个核细胞粗品用生理盐水洗涤两次  2.3 Treatment of peripheral blood mononuclear cells: Washing crude peripheral blood mononuclear cells twice with physiological saline
, 20°C下 500g离心 5分钟, 再用适量无血清培养基重悬, 得到外周血单个核细胞 , centrifuged at 500g for 5 minutes at 20°C, and then resuspended in an appropriate amount of serum-free medium to obtain peripheral blood mononuclear cells.
, 备用。 , spare.
[0092] 3培养肿瘤特异性 CTL  3 cultured tumor-specific CTL
[0093] 3.1调整细胞浓度: 用无血清培养液调整外周血单个核细胞的细胞浓度至 2x10 6 个 /ml, 混合均匀后小心铺入肿瘤抗原预包被的培养瓶中。  3.1 Adjusting the cell concentration: The cell concentration of the peripheral blood mononuclear cells was adjusted to 2×10 6 /ml with the serum-free medium, and the mixture was uniformly mixed and carefully placed in the culture bottle pre-coated with the tumor antigen.
[0094] 3.2孵育培养: 向培养瓶中加入 800U/ml的 GM-CSF和 600U/ml的 IL4, 在 CO 2培 养箱中进行孵育培养 36h, C0 2培养箱中培养条件可以选择 37°C, C0 2浓度 5%, 诱导获得成熟的肿瘤抗原负载的 DC细胞。 [0094] 3.2 Incubation culture: 800 U/ml GM-CSF and 600 U/ml IL4 were added to the culture flask, and the culture was carried out in a CO 2 incubator for 36 h, and the culture condition in the C0 2 incubator was 37 ° C. C0 2 concentration of 5% induced the induction of mature tumor antigen-loaded DC cells.
[0095] 3.3 3.3 [0095]
连续培养: 孵育培养完成后向肿瘤抗原负载的 DC细胞中加入 1200U/ml的 IFN-γ、 60ng/ml的 anti-CD3、 600U/ml的 IL2后继续培养。 隔天取样计数, 补充含 IL2的新 鲜无血清培养基, 将细胞的培养密度调整至 2x10 6个 /ml; 培养 15天后, 获得特异 性抗原负载的 DC-CTL细胞。  Continuous culture: After the incubation, the culture was continued by adding 1200 U/ml of IFN-γ, 60 ng/ml of anti-CD3, and 600 U/ml of IL2 to the tumor antigen-loaded DC cells. The samples were counted every other day, supplemented with fresh serum-free medium containing IL2, and the cell culture density was adjusted to 2x10 6 /ml; after 15 days of culture, specific antigen-loaded DC-CTL cells were obtained.
[0096] 对比实施例 Comparative Example
[0097] 制备肿瘤抗原、 外周血单个核细胞的分离与实施例 3中相同,  [0097] The separation of the preparation of tumor antigen and peripheral blood mononuclear cells is the same as in Example 3.
[0098] 1 . CIK细胞的获得 [0099] 1.1从全血中获得的 PBMCs (同实施例 3) , 用无血清培养液调整细胞浓度至 2x 10 ^/ml, 置于培养瓶内; 37°C, 5%C0 2培养箱中孵育 2h, 以使单核细胞贴壁 [0098] 1. Acquisition of CIK cells [0099] 1.1 PBMCs obtained from whole blood (same as in Example 3), adjusted the cell concentration to 2×10 ^/ml with serum-free medium, placed in a culture flask; 37 ° C, 5% CO 2 incubator Incubate for 2h to attach monocytes to the wall
[0100] 1.2收集悬浮细胞, 用无血清培养液调整细胞浓度至 2x10 6个 /ml, ; 加入 1000 U/ml的重组人 IFN-γ进行培养; 24h后加入 50ng/ml的 CD3单克隆抗体和 300 U/ml 的重组人 IL-2, 刺激 CIK细胞的生长和增殖; 每 2天扩瓶一次, 并补加重组人 300 U/ml的重组人 IL-2。 1.2 Collecting suspended cells, adjusting the cell concentration to 2×10 6 cells/ml with serum-free medium; adding 1000 U/ml of recombinant human IFN-γ for culture; adding 50 ng/ml of CD3 monoclonal antibody after 24 hours and Recombinant human IL-2 at 300 U/ml stimulated the growth and proliferation of CIK cells; it was expanded once every 2 days and supplemented with recombinant human IL-2 at 300 U/ml.
[0101] 1.3在培养第 7天, CIK细胞数量达到 ^10 9个/!^, 即可收获。 [0101] 1.3 On the 7th day of culture, the number of CIK cells reached ^10 9 /! ^, you can harvest.
[0102] 2.肿瘤抗原负载 DC细胞的获得  2. Tumor antigen loading DC cell acquisition
[0103] 2.1步骤 1.1中剩下的贴壁细胞, 加入含 1000U/ml重组人 GM-CSF和 500U/ml重组 人 IL-4的无血清培养液, 37°C, 5%C0 2¾养箱中培养, 诱导单核细胞向 DC细胞 分化; 2.1 The remaining adherent cells in step 1.1, adding serum-free medium containing 1000 U/ml recombinant human GM-CSF and 500 U/ml recombinant human IL-4, 37 ° C, 5% C0 2 3⁄4 incubator Medium culture, inducing monocytes to differentiate into DC cells;
[0104] 2.2培养过程中适当补足上述细胞因子, 在第 5天加入肿瘤抗原, 对 DC细胞进 行抗原负载后继续培养, 在第 6天加入 500U/ml重组人 TNF-ot进行诱导, 诱导得到 成熟的 DC细胞;  2.2 The cytokine was appropriately supplemented during the culture, and the tumor antigen was added on the 5th day, and the DC cells were subjected to antigen loading, and the culture was continued. On the 6th day, 500 U/ml recombinant human TNF-ot was added for induction, and induction was induced. DC cells;
[0105] 2.3在第 8天, 肿瘤抗原负载 DC细胞的细胞数量达到 1x10 6个 /ml, 即可收获。 2.3 On the 8th day, the number of cells in which the tumor antigen-loaded DC cells reached 1×10 6 /ml, and harvested.
[0106] 3.肿瘤特异性 CTL的制备 3. Preparation of tumor specific CTL
[0107] 将获得的 CIK细胞和肿瘤抗原负载的 DC细胞按照 10:1混合培养, 培养过程中每 2天换液一次, 并补加 300 U/ml重组人 IL-2等, 在混合培养的第 3天, 即可收集肿 瘤特异性 CTL细胞。  The obtained CIK cells and tumor antigen-loaded DC cells were mixed and cultured in a ratio of 10:1, and the cells were changed every 2 days during the culture, and 300 U/ml of recombinant human IL-2 was added thereto, and mixed culture was carried out. On day 3, tumor-specific CTL cells were collected.
[0108] 对比实验结果  [0108] Comparison of experimental results
[0109] 将上述实施例、 对比实施例中 DC-CIK细胞进行质检, 包括:  [0109] The quality inspection of the DC-CIK cells in the above examples and comparative examples includes:
[0110] 1培养的总吋间周期 [0110] 1 total inter-turn cycle
[0111] 对比实施例中, 通常需要先将悬浮细胞冻存, 贴壁细胞用来诱导 DC (GM-CSF 和 IL4) , 5天吋加入 50ug/ml的抗原, 6天吋加入 20ng/ml TNF-α促成熟, 第 7天吋 复苏悬浮细胞诱导获得 CIK细胞, 并与收获成熟 DC共培养, 通常需要培养 21天  In the comparative examples, it is usually necessary to first freeze the suspended cells, the adherent cells are used to induce DC (GM-CSF and IL4), 50 ug/ml of antigen is added after 5 days, and 20 ng/ml of TNF is added after 6 days. -α promotes maturation, on day 7 吋 resuscitation of suspension cells induces the acquisition of CIK cells and co-culture with harvested mature DCs, usually requiring 21 days of culture
[0112] 本发明实施例 1〜3中, 只需要 12〜15天, 即可收获。 [0113] 2台盼蓝染色检测或细胞计数仪: 标准为活细胞在 80%以上。 [0112] In the first to third embodiments of the present invention, it takes only 12 to 15 days to harvest. [0113] 2 trypan blue staining detection or cell counter: The standard is that the living cells are above 80%.
[0114] 实验计数结果包括总细胞数、 CD8+、 细胞存活率。  [0114] Experimental count results include total cell number, CD8+, and cell viability.
[0115] 实施例 1、 实施例 2、 实施例 3、 对比实施例中总细胞数分别为 1.64x10 In the first embodiment, the second embodiment, the third embodiment, and the comparative example, the total number of cells was 1.64×10 1β.
、 1.48x10 10、 1.52x10 10、 4.8x10 9, 1.48x10 10 , 1.52x10 10 , 4.8x10 9 ;
[0116] 实施例 1、 实施例 2、 实施例 3、 对比实施例中 CD8+分别为 1.32x10 、 1.26x10 10 [0116] In the first embodiment, the second embodiment, the third embodiment, and the comparative example, the CD8+ is 1.32× 10 and 1.26× 10 10 respectively.
、 1.23x10 10、 3.1x10 9, 1.23x10 10 , 3.1x10 9 ;
[0117] 实施例 1、 实施例 2、 实施例 3、 对比实施例中细胞存活率分别为 96.7%、 98.2%Example 1, Example 2, Example 3. The cell viability in the comparative example was 96.7% and 98.2%, respectively.
、 97.1%. 91.5<¾。 , 97.1%. 91.5<3⁄4.
[0118] 对比可知, 通过发明肿瘤特异性 CTL的制备方法获得的特异性 CTL在总细胞数 [0118] The comparison shows that the specific CTL obtained by the method for preparing a tumor-specific CTL is in the total number of cells.
、 CD8+、 细胞存活率上都明显优于对比实施例。 , CD8+, and cell viability were significantly better than the comparative examples.
[0119] 3流式细胞仪检测细胞表面 CD8+,CD3+, CD8+IFN-Y+等分子的表达。 [0119] 3 flow cytometry was used to detect the expression of molecules such as CD8+, CD3+, CD8+IFN- Y + on the cell surface.
[0120] 细胞表面检测对象包括 CD8+、 CD3+、 CD8+IFN-y+、 CD107a。 Cell surface detection subjects include CD8+, CD3+, CD8+IFN-y+, CD107a.
[0121] 实施例 1、 实施例 2、 实施例 3、 对比实施例中 CD8+分别为 80.2%、 85.4% ^ 81.2Example 1, Example 2, Example 3, CD8+ in the comparative example were 80.2%, 85.4% ^ 81.2, respectively.
%、 64.3%; %, 64.3%;
[0122] 实施例 1、 实施例 2、 实施例 3、 对比实施例中 CD3+分别为 90.3%、 95.2% ^ 95.0 %、 83.2%;  [0122] Example 1, Example 2, Example 3, Comparative Example, CD3+ were 90.3%, 95.2%^95.0%, 83.2%, respectively;
[0123] 实施例 1、 实施例 2、 实施例 3、 对比实施例中 CD8+  Example 1, Example 2, Example 3, CD8+ in Comparative Example
IFN-Y+分另 IJ为 40.4<¾、 45.6%、 43.8%、 19.7%;  IFN-Y+ is divided into 40.4<3⁄4, 45.6%, 43.8%, 19.7%;
[0124] 实施例 1、 实施例 2、 实施例 3、 对比实施例中 CD107a 别为 15.6%、 17.2%. 13. [0124] Example 1, Example 2, Example 3, CD107a in the comparative example was 15.6%, 17.2%.
8<¾、 5.5<¾。  8<3⁄4, 5.5<3⁄4.
[0125] 对比可知, 通过发明肿瘤特异性 CTL的制备方法获得的特异性 CTL在细胞表面 分子的表达上都明显优于对比实施例。  As can be seen from the comparison, the specific CTL obtained by the method for producing a tumor-specific CTL was significantly superior to the comparative example in the expression of cell surface molecules.
[0126] 4细胞杀伤实验: 以 DC-CIK细胞为效应细胞, 以肿瘤细胞 (可为原代肿瘤细胞 或肿瘤细胞株)为靶细胞, 将效应细胞与靶细胞按 10:1、 20:1、 40:1 (E:T为两者数 目比)的比例加入 96孔 U型板中, 每孔含靶细胞 1x104个, 终体积为 200ml, 设 3 个复孔。 培养 4h后, 离心吸取培养上清, 用乳酸脱氢酶 (LDH)试剂盒检测效应 细胞对靶细胞的杀伤率, 同吋设置空白对照, 靶细胞对照, 效应细胞对照。 每 孔数值减去空白孔对照, 求出复孔的平均值。 按照下面公式计算效应细胞的细 胞毒活性, 以杀伤率 (<¾) 表示: [0126] 4 cell killing experiment: DC-CIK cells are used as effector cells, tumor cells (which may be primary tumor cells or tumor cell lines) as target cells, and effector cells and target cells are 10:1, 20:1 The ratio of 40:1 (E:T is the ratio of the two) was added to a 96-well U-shaped plate. Each well contained 1×10 4 target cells, and the final volume was 200 ml. Three replicate wells were set. After 4 hours of culture, the culture supernatant was centrifuged, and the killing rate of the effector cells to the target cells was detected by a lactate dehydrogenase (LDH) kit, and a blank control, a target cell control, and an effector cell control were set. The value of each well was subtracted from the blank well and the average value of the duplicate wells was determined. Calculate the effector cells according to the following formula Cytotoxic activity, expressed as the kill rate (<3⁄4):
[0127] 杀伤率 =靶细胞对照值- (实验孔值 -效应细胞对照值) /靶细胞对照值 xl00%。  Killing rate = target cell control value - (experimental well value - effector cell control value) / target cell control value xl00%.
[0128] 按照此方法分别检测获得的肿瘤特异性 CTL细胞对肝癌细胞系 HEPG2以及 B细 胞白血病 K562的杀伤率。 实验重复 6次, 取平均值。  The killing rate of the obtained tumor-specific CTL cells against the liver cancer cell lines HEPG2 and B cell leukemia K562 was examined according to this method. The experiment was repeated 6 times and averaged.
[0129] 实验结果包括 E:T=10:1、 E:T=20:1、 E:T=40:1三种情形下肝癌 HEPG2与白血病 K562为靶细胞的细胞杀伤实验结果。  [0129] The experimental results include the results of cell killing experiments of liver cancer HEPG2 and leukemia K562 as target cells in three cases: E:T=10:1, E:T=20:1, E:T=40:1.
[0130] 当 E:T=10:1吋, 实施例 1、 实施例 2、 实施例 3、 对比实施例中肝癌 HEPG2的杀 伤结果分别为 44.2%、 50.2%. 46.2%. 31.2% , 实施例 1、 实施例 2、 实施例 3、 对 比实施例中白血病 K562的杀伤结果分别为 18.7%、 23.6%、 22.2% ^ 18.7%; [0130] When E:T=10:1吋, the killing results of liver cancer HEPG2 in Example 1, Example 2, Example 3, and Comparative Example were 44.2%, 50.2%, 46.2%, 31.2%, respectively. 1. The killing results of leukemia K562 in Example 2, Example 3 and Comparative Example were 18.7%, 23.6%, 22.2% ^ 18.7%, respectively;
[0131] 当 E:T=20:1吋, 实施例 1、 实施例 2、 实施例 3、 对比实施例中肝癌 HEPG2的杀 伤结果分别为 60.8%、 63.0%、 64.2%. 35.2% , 实施例 1、 实施例 2、 实施例 3、 对 比实施例中白血病 K562的杀伤结果分别为 24.3%、 20.5%、 19.5% ^ 21.0%;When E:T=20:1吋, the killing results of liver cancer HEPG2 in Example 1, Example 2, Example 3, and Comparative Example were 60.8%, 63.0%, 64.2%, and 35.2%, respectively. 1. The killing results of leukemia K562 in Example 2, Example 3 and Comparative Example were 24.3%, 20.5%, 19.5% ^ 21.0%, respectively;
[0132] 当 E:T=40:1吋, 实施例 1、 实施例 2、 实施例 3、 对比实施例中肝癌 HEPG2的杀 伤结果分别为 81.5%、 78.5%、 86.2%. 42.0% , 实施例 1、 实施例 2、 实施例 3、 对 比实施例中白血病 K562的杀伤结果分别为 34.5%、 30.2% 29.1%^ 30.6<¾。 When E:T=40:1吋, the killing results of liver cancer HEPG2 in Example 1, Example 2, Example 3, and Comparative Example were 81.5%, 78.5%, 86.2%, and 42.0%, respectively. 1. The killing results of leukemia K562 in Example 2, Example 3 and Comparative Example were 34.5%, 30.2% 29.1%^30.6<3⁄4, respectively.
[0133] 5收获细胞前, 取少量培养物进行细菌、 真菌培养, 并检测支原体、 衣原体, 及内毒素 (标准: 病原学检测阴性, 内毒素 <5 Eu)。  [0133] 5 Before harvesting the cells, a small amount of the culture was taken for bacterial and fungal culture, and the mycoplasma, chlamydia, and endotoxin were detected (standard: negative pathogen detection, endotoxin <5 Eu).
[0134] 检测得知本发明实施例 1〜3和对比实施例中各项指标都不超标。  [0134] It was found that the indicators in the first to third embodiments of the present invention and the comparative examples were not exceeded.

Claims

权利要求书 Claim
[权利要求 1] 一种肿瘤特异性 CTL的制备方法, 其特征在于, 包括如下步骤:  [Claim 1] A method for preparing a tumor-specific CTL, comprising the steps of:
5100、 肿瘤抗原的包被: 使用患者肿瘤组织或肿瘤细胞系制备肿瘤抗 原, 调整肿瘤抗原浓度至 5(Vg/m!〜 20(Vg/ml, 加入到 TC处理过的细 胞培养瓶中覆盖瓶底, 进行包被反应, 得到肿瘤抗原预包被的培养瓶  5100, Tumor antigen coating: Prepare tumor antigen using patient tumor tissue or tumor cell line, adjust tumor antigen concentration to 5 (Vg/m!~ 20 (Vg/ml, add to TC-treated cell culture flask to cover bottle) At the bottom, the coating reaction is carried out to obtain a culture bottle pre-coated with tumor antigen.
S200、 外周血单个核细胞的分离: 从患者血液中分离外周血单个核细 胞, 悬浮备用; S200, separation of peripheral blood mononuclear cells: separating peripheral blood mononuclear cells from the patient's blood, and suspending for use;
S300、 培养肿瘤特异性 CTL: 将所述外周血单个核细胞小心铺入所述 肿瘤抗原预包被的培养瓶中, 进行孵育培养诱导获得成熟的肿瘤抗原 负载的 DC细胞和 T淋巴细胞; 然后加入刺激性细胞因子、 补充新鲜培 养基连续培养, 大量扩增 T淋巴细胞获得抗原特异性的 CTL。  S300, cultivating tumor-specific CTL: carefully plating the peripheral blood mononuclear cells into the tumor antigen pre-coated culture flask, and performing incubation culture to induce mature tumor antigen-loaded DC cells and T lymphocytes; Stimulation of cytokines, supplementation with fresh medium, continuous culture, and extensive expansion of T lymphocytes to obtain antigen-specific CTLs.
[权利要求 2] 根据权利要求 1所述的肿瘤特异性 CTL的制备方法, 其特征在于, 所 述步骤 S100中还包括: [Claim 2] The method for preparing a tumor-specific CTL according to claim 1, wherein the step S100 further comprises:
5101、 制备肿瘤抗原: 将患者肿瘤组织或肿瘤细胞系分散成单细胞悬 液, 用生理盐水调节悬浮肿瘤细胞浓度至 ^10 7〜2><10 7个/1^, 反复 冻融 3〜5次, 裂解肿瘤细胞; 将肿瘤细胞裂解物在 2000g下离心 8〜12 分钟后吸取上清, 0.22μηι滤膜过滤除菌, 记为肿瘤抗原; 5101, preparing a tumor antigen: dispersing a patient tumor tissue or a tumor cell line into a single cell suspension, and adjusting the concentration of the suspended tumor cells with physiological saline to ^10 7 〜2><10 7 /1^, repeated freezing and thawing 3~5 Next, the tumor cells are lysed; the tumor cell lysate is centrifuged at 2000 g for 8 to 12 minutes, and then the supernatant is aspirated, and 0.22 μηι filter is used for filtration and sterilization, and recorded as a tumor antigen;
5102、 处理细胞培养瓶: TC处理的细胞培养瓶;  5102. Processing a cell culture flask: a TC-treated cell culture flask;
5103、 包被反应: 用无菌生理盐水或 lxPBS调整肿瘤抗原的浓度至 50 g/m!〜 20(Vg/ml, 取 10ml〜20ml, 加入 TC处理后的细胞培养瓶中, 肿瘤抗原覆盖培养瓶瓶底, 4°C过夜或者 37°C进行包被反应 11!〜 2h, 得到肿瘤抗原预包被的培养瓶。  5103, coating reaction: Adjust the concentration of tumor antigen to 50 g/m with sterile saline or lxPBS! ~ 20 (Vg / ml, take 10ml ~ 20ml, add TC treated cell culture flask, tumor antigen cover the bottom of the flask, overnight at 4 ° C or 37 ° C coating reaction 11 ~ ~ 2h, get tumor antigen Pre-coated flasks.
[权利要求 3] 根据权利要求 2所述的肿瘤特异性 CTL的制备方法, 其特征在于, 所 述步骤 S101中, 患者肿瘤组织分散的方法包括胶原蛋白酶消化法、 物 理研磨法。  The method for producing a tumor-specific CTL according to claim 2, wherein in the step S101, the method for dispersing the tumor tissue of the patient comprises a collagenase digestion method and a physical polishing method.
[权利要求 4] 根据权利要求 1所述的肿瘤特异性 CTL的制备方法, 其特征在于, 所 述步骤 S200中还包括: S201、 准备全血: 抽取肿瘤患者静脉血, 加入抗凝剂成抗凝全血备用 [Claim 4] The method for preparing a tumor-specific CTL according to claim 1, wherein the step S200 further includes: S201, preparing whole blood: extracting venous blood from tumor patients, adding anticoagulant to anticoagulated whole blood
5202、 外周血单个核细胞与自体血浆的分离: 使用血细胞分离机或者 淋巴细胞分离液将外周血单个核细胞从自体血浆中分离, 获得的外周 血单个核细胞粗品; 5202. Separation of peripheral blood mononuclear cells from autologous plasma: Peripheral blood mononuclear cells obtained by separating a peripheral blood mononuclear cell from autologous plasma using a blood cell separator or a lymphocyte separation solution;
5203、 外周血单个核细胞的处理: 将所述外周血单个核细胞粗品用生 理盐水或无血清培养基洗涤两次, 20°C下 500g离心 3〜7分钟, 再用适 量无血清培养基重悬, 得到外周血单个核细胞, 备用。  5203. Treatment of peripheral blood mononuclear cells: The crude peripheral blood mononuclear cells are washed twice with physiological saline or serum-free medium, centrifuged at 500 g for 3 to 7 minutes at 20 ° C, and then weighed with an appropriate amount of serum-free medium. Suspended, obtained peripheral blood mononuclear cells, ready for use.
[权利要求 5] 根据权利要求 1所述的肿瘤特异性 CTL的制备方法, 其特征在于, 所 述步骤 S300中还包括:  [Claim 5] The method for preparing a tumor-specific CTL according to claim 1, wherein the step S300 further comprises:
5301、 调整细胞浓度: 用无血清培养液调整外周血单个核细胞的细胞 浓度至 1x10 6〜2xl0 6个 /ml, 混合均匀后小心铺入所述肿瘤抗原预包 被的培养瓶中; 5301, adjusting the cell concentration: adjusting the cell concentration of the peripheral blood mononuclear cells to 1×10 62 ×10 6 cells/ml with the serum-free culture solution, mixing uniformly, and carefully placing the tumor antigen in the pre-coated culture flask;
5302、 孵育培养: 向培养瓶中加入 400U/ml〜800U/ml的 GM-CSF和 30 0U/ml〜600U/ml的 IL4, 在 CO 2培养箱中进行孵育培养 361!〜 48h, 诱 导获得成熟的肿瘤抗原负载的 DC细胞; 5302, Incubation culture: Add 400 U/ml to 800 U/ml of GM-CSF and 30 0 U/ml to 600 U/ml of IL4 to the flask, and incubate in a CO 2 incubator 361! ~ 48h, induced to obtain mature tumor antigen-loaded DC cells;
5303、 连续培养: 向肿瘤抗原负载的 DC细胞中加入 500U/ml〜1200U/ ml的 IFN-Y、 60ng/m!〜 150ng/ml的 anti-CD3、 600U/ml〜1400U/ml的 IL 5303, continuous culture: Add 500U/ml~1200U/ml IFN-Y, 60ng/m to the tumor antigen-loaded DC cells! ~ 150ng/ml anti-CD3, 600U/ml~1400U/ml IL
2后继续培养, 隔天取样计数, 补充含 IL2的新鲜无血清培养基, 将细 胞的培养密度调整至 ^10 6〜2><10 6个/1^, 培养 12〜15天后, 获得特 异性抗原负载的 DC-CTL细胞。 Continue to culture after 2, sample and count the next day, supplement the fresh serum-free medium containing IL2, adjust the culture density of the cells to ^10 6 〜2><10 6 /1^, and obtain the specificity after 12-15 days of culture. Antigen-loaded DC-CTL cells.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108642005A (en) * 2018-05-22 2018-10-12 广州迪澳医疗科技有限公司 A kind of lymphocyte separation medium containing bridging agent

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113174369B (en) * 2021-04-23 2022-10-18 北京起源爱心生物科技有限公司 Establishment method of immune cell bank with pleiotropic immunoregulation function
CN113846059B (en) * 2021-10-12 2024-02-27 北京中卫医正科技有限公司 In-vitro amplification culture method for CTL (cytotoxic T lymphocyte) cells activated by induction of 3D tumor holoantigen
CN115433713B (en) * 2022-03-03 2023-10-27 中山大学孙逸仙纪念医院深汕中心医院 Preparation method and application of autologous tumor drainage lymph node lymphocyte

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002053176A2 (en) * 2001-01-08 2002-07-11 Hadasit Medical Research Services And Development Ltd. An autologous anti-cancer vaccine
RU2520091C2 (en) * 2012-08-16 2014-06-20 Общество с ограниченной ответственностью "АваксисБио" Method for stimulating cytotoxic immune response to tumour cells of mammary adenocarcinoma expressing specific antigens, with use of dendrite cells transfected by polyepitope dna construct
CN103923880A (en) * 2014-05-08 2014-07-16 成都百赛泰科生物科技有限公司 Efficient multiplication CTL preparation method killing tumors in targeted mode

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102597222B (en) * 2009-10-27 2015-07-15 因缪尼卡姆股份公司 Method for proliferation of antigen-specific T cells
CN102168066A (en) * 2011-01-31 2011-08-31 浙江大学 Method for in vitro induction of specific cytotoxic T lymphocytes of hepatitis B virus (HBV)
CN104072599A (en) * 2014-06-06 2014-10-01 深圳市阳溪生物科技有限公司 Polypeptide, dendritic cell adopting in vitro activation and application of dendritic cell
CN104651311B (en) * 2014-09-03 2018-03-13 深圳市茵冠生物科技有限公司 Prepare DC CTL kit and its application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002053176A2 (en) * 2001-01-08 2002-07-11 Hadasit Medical Research Services And Development Ltd. An autologous anti-cancer vaccine
RU2520091C2 (en) * 2012-08-16 2014-06-20 Общество с ограниченной ответственностью "АваксисБио" Method for stimulating cytotoxic immune response to tumour cells of mammary adenocarcinoma expressing specific antigens, with use of dendrite cells transfected by polyepitope dna construct
CN103923880A (en) * 2014-05-08 2014-07-16 成都百赛泰科生物科技有限公司 Efficient multiplication CTL preparation method killing tumors in targeted mode

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALBERT, M.L. ET AL.: "Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs", NATURE, vol. 392, 5 March 1998 (1998-03-05), pages 86 - 89, XP002154749, ISSN: 0028-0836 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108642005A (en) * 2018-05-22 2018-10-12 广州迪澳医疗科技有限公司 A kind of lymphocyte separation medium containing bridging agent
CN108642005B (en) * 2018-05-22 2021-07-27 广州迪澳医疗科技有限公司 Lymphocyte separation liquid containing connecting agent

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