CN114288396A - Tumor individualized DC vaccine, preparation method and application - Google Patents
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Abstract
The invention provides a personalized DC vaccine aiming at tumors, a preparation method and application in preparing anti-tumor drugs. The invention realizes the in vitro construction of primary tumor cells by using a small tumor tissue sample through the improvement of the technology, and upgrades the preparation of the DC vaccine through the tumor cell lysis, thereby creating the real tumor individualized DC vaccine.
Description
Technical Field
The invention belongs to the technical field of DC vaccines, and particularly relates to an individual tumor DC vaccine, a preparation method and application thereof.
Background
Malignant tumors have become the first killer threatening the health of people, the incidence of Chinese cancers is in a fast rising period for nearly 20 years, the number of the cancer is about 260 ten thousand and about 180 ten thousand per year according to statistics, and the cancer attack groups are increasingly younger due to fast-paced and high-pressure working environment and bad life style. Although the development of medical technology has been advanced, the development of cancer treatment mainly using surgery, radiotherapy and chemotherapy is still significant, but due to the complex pathogenesis of cancer, the search for effective treatment is always a great challenge in the field of cancer treatment today.
In recent years, with the rapid progress in molecular biology, genetic engineering techniques, and immunology, immunotherapy-based biotherapy has become a new model for cancer therapy. The CAR-T technology taking CD19 as a target point is researched and reported to be used for treating acute lymphocytic leukemia and non-Hodgkin lymphoma, even if the patients are in the later stage of treatment failure and relapse difficulty, the patients still have 50-90% of complete remission rate, and part of the patients even reach long-term survival, so that clinical cure is realized. However, CAR-T therapy has been successful in the narrow field of only a few hematologic tumors and many limitations remain in the treatment of solid tumors. Compared with CAR-T technology, T cell receptor genetically engineered T cells (TCR-T) treatment technology is safer, but is limited by two elements of target and HLA typing, so the application range is narrow. Furthermore, both CAR-T and TCR-T are directed against a single target. However, the mutation spectrum presented by the tumor can contain hundreds of somatic mutations, and the mutations are dynamically changed, new mutations are continuously generated while part of old mutations disappear, so that the treatment only aiming at a single target point is undoubtedly limited, and tumor cells carrying other mutations can continue to grow, which is also the reason for the rapid drug resistance or relapse after the current genetically engineered T cell treatment.
Tumors are the products of genetic mutations in cells, many of which are not found in normal tissue cells, and these muteins are likely to activate the immune system and induce the immune system to attack tumor cells. These abnormal proteins produced by the genetic mutation of tumor cells, which activate the immune system, are tumor neoantigens. Not only different types of tumors have different gene mutations, but even different sites within the same patient's tumor have extensive mutation heterogeneity. Since tumor mutations are highly heterogeneous, they are highly individualized. Therefore, the individual multi-target immunotherapy of the targeted tumor neoantigen brings new hopes for tumor therapy, can well solve two problems of tumor mutation inhibition and dynamic evolution, and is an accurate treatment scheme capable of truly realizing the individual multi-target.
The preparation of the individualized DC vaccine can well realize the individualized multi-target immune cell therapy of the targeted tumor neoantigen, and the DC cell can deliver a plurality of tumor-related target peptides and stimulate T cells, thereby finally realizing the elimination of tumor cells. There is increasing evidence that cancer vaccines can induce immune responses and produce clinical effects. George Coukos et al use ovarian cancer cell lines to prepare DC vaccines to achieve certain clinical efficacy in subjects with recurrent ovarian cancer. It is worth noting that the antigen provided by tumor cell line lysate is used in the clinical research, which cannot be really individualized, whether the effective anti-tumor immune effect can be activated in vivo or not, and further clinical research and potential clinical risks are provided.
Therefore, truly effective individual multi-target DC vaccines should be based on patient's own tumor antigens, and currently effective tumor antigens mainly include primary tumor cell lysates and tumor neoantigens (Neoantigen) based on secondary sequencing and HLA typing screening. The latter relies on sequencing technology for transition, and has the defects of target spot screen leakage, high cost and the like, while the primary tumor cell lysate can provide a large amount of tumor antigens without screening, thereby greatly reducing the cost.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects of the current tumor DC vaccine technology, the invention provides an individual tumor DC vaccine, a preparation method and application thereof.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:
a tumor individualized DC vaccine, the vaccine being a DC vaccine comprising a primary tumor cell lysate.
Preferably, the tumor comprises solid tumors such as prostate cancer, lung cancer, gastric cancer, colon cancer, liver cancer, breast cancer, ovarian cancer and the like.
The preparation method of the tumor individualized DC vaccine comprises the following steps:
(1) culturing primary tumor cells and DC cells in vitro;
(2) primary tumor cell lysis;
(3) and co-incubating the primary tumor cell lysate and the DC cell to obtain the tumor cell.
Preferably, in step (1), the method for culturing primary tumor cells in vitro comprises the following steps:
obtaining a fresh tumor sample, digesting, cleaning, inoculating in matrigel, culturing and incubating, taking out after the matrigel is solidified, adding a culture medium for culturing, and then performing liquid change and passage.
Preferably, in step (1), the method for culturing DC cells in vitro comprises the following steps:
collecting peripheral blood of a tumor patient, separating out leukocytes, adding a culture medium for resuspension, culturing adherent cells, and adding a DC culture factor to the adherent cells for culturing.
Preferably, in step (2), the method for lysing primary tumor cells comprises the following steps:
collecting the primary tumor cells cultured in the step (1), and lysing the primary tumor cells by using a repeated freeze-thaw method.
Further preferably, the repeated freezing and thawing method adopts liquid nitrogen for freezing and thawing, the freezing is carried out for 18-22min, then the dissolving is carried out at room temperature, and the repeated freezing and thawing method is repeated for 5-7 times.
Preferably, in step (3), the method for incubating the primary tumor cell lysate and the DC cells comprises the following steps:
and (3) mixing the primary tumor cell lysate obtained in the step (2) with the DC cells cultured in the step (1), incubating, and subsequently replacing the DC culture factors used in the DC cell culture with DC cell maturation factors to continue culturing, so as to obtain the DC vaccine.
Further preferably, the ratio of DC cells to primary tumor cell lysate is 1:1, the incubation time is 22-26h, and the DC culture factor is changed into a DC cell maturation factor to continue culturing for 46-50 h.
The invention finally provides the application of the tumor individualized DC vaccine in the preparation of a tumor treatment drug.
Preferably, the tumor comprises solid tumors such as prostate cancer, lung cancer, gastric cancer, colon cancer, liver cancer, breast cancer, ovarian cancer and the like.
The invention relates to a method for constructing an individualized primary tumor cell culture by utilizing a tumor biopsy or operation sample. Meanwhile, a series of technical methods such as tumor cell lysis, DC cell induction culture and the like are improved and upgraded, and finally, a preparation method of the individualized DC vaccine based on the primary tumor cells is established. Meanwhile, a series of in vivo and in vitro experiments verify that the vaccine has the function of activating specific T cells to kill tumor cells, and finally a tumor individualized DC vaccine preparation technical product which can be applied to clinic and commercialization is created.
Has the advantages that: compared with the prior art, the invention has the following beneficial effects:
1. the invention provides antigen after primary tumor cells are cultured by using a patient tumor sample and are cracked, and the antigen and autologous DC cells are incubated together to really realize individualized and accurate treatment.
2. The invention solves the limitation of single target point of the traditional DC vaccine through the holoantigen of primary tumor cell lysis.
3. The optimal freeze-thaw condition is explored through an early stage experiment in the preparation of the DC vaccine, primary tumor cells can be completely lysed, the clinical application risk is avoided, and the optimal DC phagocytosis effect is ensured.
4. The optimal phagocytic time of the DC cells is found out through early-stage series experiments, and more effective DC vaccines are prepared.
Drawings
FIG. 1: primary tumor cell morphology under an optical microscope;
FIG. 2: establishing a primary tumor cell nude mouse subcutaneous transplantation tumor;
FIG. 3: phenotypic characterization of mature DC cells, wherein a: DC-under-the-cell-lens morphology; b: detecting mature surface marker of DC cell;
FIG. 4: and (3) detecting the lysis effect of the primary tumor cells, wherein A: observing the lysis effect of the primary tumor under a mirror; b: flow-detecting the lysis effect of the primary tumor cells;
FIG. 5: and (3) detecting the phagocytosis efficiency of the DC cells to the primary tumor cell lysate, wherein A: detecting the effect of the DiI marked primary tumor cells; b: detecting the phagocytic effect of the DC vaccine on primary tumor cell lysate;
FIG. 6: and (3) detecting the killing effect of the reactive T cells on the primary tumor cells, wherein A: the direct killing effect of the reactive T cells on the primary tumor cells; b: expression of the cytokine IL-2; c: expression of the cytokine TNF- α; d: expression of the cytokine IFN-. gamma..
Detailed Description
The following is a general description of the embodiments of the present invention, which are the most preferred embodiments of the present invention, but the present invention is not limited to the following examples.
EXAMPLE 1 Primary tumor cell culture
1. In vitro culture of primary tumor cells
Repeatedly cutting fresh prostate tumor sample obtained by biopsy or operation to small pieces of 1-5 mm3Then, the mixture was digested overnight with collagenase type II in a centrifuge tube with gentle shaking at 37 ℃. The next day, DMEM/F12 (Gibco) was added to a total volume of 10ml for washing, centrifuged (200 g, 5 min), the supernatant was discarded, 1ml of pancreatin was added to resuspend the pellet, digested at 37 ℃ for 15 min, and pipetting up and down every 5min to ensure adequate digestion. Adding DMEM/F12 to total volume of 10ml for washing, centrifuging (200 g, 5 min), and discarding supernatant; placing the digested tissue into ice-cold Matrigel, pipetting up and down 5-10 times, counting cells using a hemacytometer plate, and seeding 40ul of Matrigel to each well of a 24-well plate, approximately 2 x 104Culturing the cells in a carbon dioxide incubator (37 deg.C, 5% CO)2) And (5) incubating for about 15 min, and taking out after the matrigel is solidified. Each well was supplemented with 500. mu.l of DMEM/F12 medium, and cultured in a carbon dioxide incubator (37 ℃ C., 5% CO)2). Changing the liquid every 2-3 days, and carrying out 1:2 passage every 1-2 weeks. Observation of cultured primary tumor cells under the mirror, as shown in FIG. 1Cell clumps were observed under 4-fold and 10-fold microscopy, respectively.
2. Tumorigenicity verification of primary tumor cell mass
Further taking out the cell mass cultured in vitro, digesting, resuspending to obtain single cell suspension, injecting into nude mice under skin, and injecting 5.0 × 10 into each nude mouse in experimental group (CR group)6The cells, control group, were injected with an equal volume of saline and the mice were observed for neoplasia, as shown in fig. 2, CR constitutively developed subcutaneous transplants on day 21 post-injection.
Example 2 DC cell culture
The DC cells are used as a peripheral blood source, 50-100ml of peripheral blood of a patient is collected, whole blood is diluted by PBS buffer according to the dilution ratio of 1:1, and the whole blood is transferred into a 50ml centrifuge tube. 15ml of human peripheral blood lymphocyte separation medium (TBD) was added to a new 50ml centrifuge tube, followed by careful addition of 25ml of diluted whole blood along the walls without disrupting the interface between the two fluids. Centrifuge at 750g speed for 20 minutes, set centrifuge up slow ramp up and slow ramp down. After centrifugation, red blood cells, a ficoll layer, a leukocyte layer and a serum layer are arranged in sequence from bottom to top. The buffy coat is sucked into a new 50ml centrifuge tube, PBS is added for washing, then the supernatant is discarded, the buffy coat is resuspended by RPMI 1640 (Gibco) culture medium, the buffy coat is cultured for 2 hours at 37 ℃, the suspension cells are sucked out for freezing, and the buffy coat cells are cultured by DC culture factors (CytoCares). The culture medium was replaced every 2-3 days and supplemented with DC culture factors. The culture was continued for 48 hours by changing to mature culture factor on day 5, and the DC cells were observed under the microscope to protrude out of the antenna, as shown in FIG. 3A. And further detecting the surface mature marker thereof, as shown in fig. 3B, the mature makerCD83 expression is higher.
Example 3 DC vaccine preparation
1. Primary tumor cell lysis
The tumor primary cells cultured in example 1 were collected and lysed by repeated freeze-thaw methods (freezing with liquid nitrogen for 20min followed by lysis at room temperature, 6 repetitions of freeze-thaw). The lysis effect was measured by under-mirror observation and flow assay, as shown in fig. 4A, intact cell morphology was not observed under the mirror after lysis, while flow cytometry assay after lysis, as shown in fig. 4B, showed essentially cell debris with no intact cell particles compared to the control.
2. Co-incubation of primary tumor cell lysate with DC cells
In order to detect the phagocytic effect of DC cells on primary tumor cell lysates, a portion of primary tumor cells were labeled with DiI, as shown in fig. 5A, which shows that the efficiency of DiI labeling of primary tumor cells was high. And further, the primary tumor cells were lysed and incubated with the above DC cells cultured up to day 4 in example 2 at a ratio of 1:1 (by cell number) in a tumor primary cell lysate. After 24 hours of incubation, the whole amount of the culture solution is changed, and the culture solution is changed into DC cell maturation factors (CytoCares) to be continuously cultured for 48 hours, so that the DC vaccine can be obtained. And further detecting the phagocytic effect of the DC cells on the primary tumor lysate, as shown in fig. 5B, the DC cells were labeled with HLA-DR, and then the DiI signal value on the DC cells was observed, and the DC cells showed higher phagocytic ability on the lysed primary tumor cells compared to the primary tumor cells that were not lysed.
EXAMPLE 4 functional verification of DC vaccines
1. Reactive T cell (Ttr) preparation
The suspension cells frozen in example 2 were recovered and cultured in a carbon dioxide incubator for 2 hours (37 ℃ C., 5% CO)2) The tumor primary cell lysate-loaded DC cells of example 3 above were collected and counted, and co-incubated with resuscitated suspension cells at a ratio of 1:10 (activated T cells). After 24 hours of incubation, IL-240 ng/ml (CytoCares) was added and culture was continued for 7 days to prepare reactive T cells.
2. Detection of killing property of reactive T cell preparation on primary tumor cells
The prepared reactive cells and primary tumor cells are co-cultured in a ratio of 0.5:1, 1:1, 2:1, 5:1, and the lysis condition of the primary tumor cells and the expression condition of cytokines are detected after the cells are cultured for 24 hours. As shown in fig. 6, the killing of the reactive T cells was higher than the killing of the primary tumor cells by control T cells that were not activated by the DC vaccine.
Claims (10)
1. An individualized DC vaccine for tumors, wherein the vaccine is a DC vaccine comprising a lysate of primary tumor cells.
2. The tumor individualized DC vaccine of claim 1, wherein the tumor comprises prostate cancer, lung cancer, gastric cancer, colon cancer, liver cancer, breast cancer or ovarian cancer.
3. The method for preparing a tumor-individualized DC vaccine according to claim 1 or 2, comprising the steps of:
(1) culturing primary tumor cells and DC cells in vitro;
(2) primary tumor cell lysis;
(3) and co-incubating the primary tumor cell lysate and the DC cell to obtain the tumor cell.
4. The method for preparing DC vaccine for individualized tumor according to claim 3, wherein the method for culturing primary tumor cells in vitro in step (1) comprises the following steps:
obtaining a fresh tumor sample, digesting, cleaning, inoculating in matrigel, culturing and incubating, taking out after the matrigel is solidified, adding a culture medium for culturing, and then performing liquid change and passage;
the method for culturing DC cells in vitro comprises the following steps:
collecting peripheral blood of a tumor patient, separating out leukocytes, adding a culture medium for resuspension, culturing adherent cells, and adding a DC culture factor to the adherent cells for culturing.
5. The method for preparing DC vaccine for individualized tumor according to claim 3, wherein in the step (2), the method for lysing primary tumor cells comprises the following steps:
collecting the primary tumor cells cultured in the step (1), and lysing the primary tumor cells by using a repeated freeze-thaw method.
6. The method for preparing the individualized DC vaccine for tumor according to claim 5, wherein the freeze-thaw method is repeated for 5-7 times, and the freeze-thaw method is performed by using liquid nitrogen for freeze-thaw, and the freeze-thaw is performed at room temperature after 18-22 min.
7. The method for preparing DC vaccine for individualized tumor according to claim 3, wherein the method for incubating the primary tumor cell lysate and the DC cells in step (3) comprises the following steps:
and (3) mixing the primary tumor cell lysate obtained in the step (2) with the DC cells cultured in the step (1), incubating, and subsequently replacing the DC culture factors used in the DC cell culture with DC cell maturation factors to continue culturing, so as to obtain the DC vaccine.
8. The method for preparing DC vaccine for tumor individualization according to claim 7, wherein the ratio of DC cells to primary tumor cell lysate is 1:1, the incubation time is 22-26h, and the DC culture factor is changed into a DC cell maturation factor to continue culturing for 46-50 h.
9. Use of the tumor individualized DC vaccine of claim 1 or 2 for the manufacture of a medicament for the treatment of tumors.
10. The use of claim 9, wherein the tumor comprises prostate cancer, lung cancer, stomach cancer, colon cancer, liver cancer, breast cancer, or ovarian cancer.
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| US20100303868A1 (en) * | 2007-09-28 | 2010-12-02 | Universidad De Chile | Ex vivo, fast and efficient process to obtain activated antigen-presenting cells that are useful for therapies against cancer and immune system-related diseases |
| CN102091327A (en) * | 2010-12-27 | 2011-06-15 | 蔡建辉 | Preparation method of dendritic cell (DC) vaccine loaded with autologous tumor associated holoantigen |
| CN111603554A (en) * | 2020-07-07 | 2020-09-01 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of anti-tumor vaccine antigen raw material |
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