WO2016201658A1

WO2016201658A1 - Method for preparing tumor specific ctls

Info

Publication number: WO2016201658A1
Application number: PCT/CN2015/081713
Authority: WO
Inventors: 姜维; 吕明锦; 吴庆军
Original assignee: 深圳市达科为生物工程有限公司
Priority date: 2015-06-17
Filing date: 2015-06-17
Publication date: 2016-12-22
Also published as: CN106795492A

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

Description: A method for preparing tumor-specific CTLs

[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.

Background technique

[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] 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] 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.

technical problem

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.

Problem solution Technical solution

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, 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, separation of peripheral blood mononuclear cells: separating peripheral blood mononuclear cells from the blood of the patient, suspending for use;

[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] In the method for preparing a tumor-specific CTL provided by the present invention, the step S100 further includes:

[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, processing a cell culture bottle: a TC-treated cell culture flask;

[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.

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] 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, 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] In the method for preparing a tumor-specific CTL provided by the present invention, the step S300 further includes:

[0020] 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;

[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;

[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.

Advantageous effects of the invention

Beneficial effect

[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.

Brief description of the drawing

DRAWINGS

[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.

1 is a flow chart of a method for preparing a CTL in the prior art;

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

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.

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. Acquisition of CIK cells

[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 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 After culturing a section of the sputum, the number of CIK cells reaches a certain order of magnitude and can be harvested.

[0033] 2. Tumor antigen loading DC cell acquisition

[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;

2.2 [0035]

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. Preparation of tumor specific CTL

[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] 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] 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.

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, 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

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] 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. Processing a cell culture flask: a TC-treated cell culture flask.

[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] In another preferred embodiment of the present invention, in addition to the foregoing steps, step S200 further includes:

[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.

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, 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] In another preferred embodiment of the present invention, in addition to the foregoing steps, step S300 further includes:

[3014] S301, adjusting cell concentration: adjusting serum cell concentration of peripheral blood mononuclear cells to serum by serum-free medium

10 ⁶ ~ 2xl0 ⁶ / ml, mix well and carefully spread into the tumor antigen pre-coated culture flask.

[0055] S302, Incubation Culture: Add 400 U/m to the culture flask! ~ 800U/ml GM-CSF and 300U/m! ~ 6

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, 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

/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.

Embodiment 1

[0058] 1 coating of 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, 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.

1.2 Cell Culture Bottles: TC-treated cell culture flasks.

1.3 [1.31] 1.3

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.

2 Separation of peripheral blood mononuclear cells

[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.

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.

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.

, spare.

[0066] 3 culture of tumor-specific CTL

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 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.

3.3

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.

Example 2

[0071] 1 coating of tumor antigen

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.

1.2 Cell Culture Bottles: TC-treated cell culture flasks.

1.3 [0074] 1.3

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 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 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.

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 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.

3 culture of tumor-specific CTL

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 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.

3.3 [3.32] 3.3

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.

Example 3

[0084] 1 coating of 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 μηι filter, which was recorded as a tumor antigen, and stored at -80 ° C until use.

1.2 Cell Culture Bottles: TC-treated cell culture flasks.

[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 separation of peripheral blood mononuclear cells

[0089] 2.1 Preparation of whole blood: Direct use of refrigerated anticoagulated whole blood.

2.2 Separation of peripheral blood mononuclear cells from autologous plasma: Peripheral blood single nuclear cells obtained using a blood cell separator.

2.3 Treatment of peripheral blood mononuclear cells: Washing crude peripheral blood mononuclear cells twice with physiological saline

, 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.

, spare.

3 cultured tumor-specific CTL

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 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.

3.3 [0095]

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.

Comparative Example

[0097] The separation of the preparation of tumor antigen and peripheral blood mononuclear cells is the same as in Example 3.

[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

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 On the 7th day of culture, the number of CIK cells reached ^10 ⁹ /! ^, you can harvest.

2. Tumor antigen loading DC cell acquisition

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;

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;

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.

3. Preparation of tumor specific CTL

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] Comparison of experimental results

[0109] The quality inspection of the DC-CIK cells in the above examples and comparative examples includes:

[0110] 1 total inter-turn cycle

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] 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] Experimental count results include total cell number, CD8+, and cell viability.

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 ;

[0116] In the first embodiment, the second embodiment, the third embodiment, and the comparative example, the CD8+ is 1.32× ¹⁰ ^1ΰ and 1.26× ^{10 10 respectively.}

, 1.23x10 ¹⁰ , 3.1x10 ⁹ ;

Example 1, Example 2, Example 3. The cell viability in the comparative example was 96.7% and 98.2%, respectively.

, 97.1%. 91.5<3⁄4.

[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+, and cell viability were significantly better than the comparative examples.

[0119] 3 flow cytometry was used to detect the expression of molecules such as CD8+, CD3+, CD8+IFN- _Y + on the cell surface.

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.

%, 64.3%;

[0122] Example 1, Example 2, Example 3, Comparative Example, CD3+ were 90.3%, 95.2%^95.0%, 83.2%, respectively;

Example 1, Example 2, Example 3, CD8+ in Comparative Example

IFN-Y+ is divided into 40.4<3⁄4, 45.6%, 43.8%, 19.7%;

[0124] Example 1, Example 2, Example 3, CD107a in the comparative example was 15.6%, 17.2%.

8<3⁄4, 5.5<3⁄4.

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 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):

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.

[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] 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;

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;

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 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] It was found that the indicators in the first to third embodiments of the present invention and the comparative examples were not exceeded.

Claims

Claim

[Claim 1] A method for preparing a tumor-specific CTL, comprising the steps of:

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, separation of peripheral blood mononuclear cells: separating peripheral blood mononuclear cells from the patient's blood, and suspending for use;

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.

[Claim 2] The method for preparing a tumor-specific CTL according to claim 1, wherein the step S100 further comprises:

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. Processing a cell culture flask: a TC-treated cell culture flask;

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.

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.

[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. 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. 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.

[Claim 5] The method for preparing a tumor-specific CTL according to claim 1, wherein the step S300 further comprises:

5301, 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 placing the tumor antigen in the pre-coated culture flask;

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, 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

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.