Immune co-culture method and application of PBMC (peripheral blood mononuclear cell) and DC (dendritic cell) loaded tumor cell antigen peptide

Abstract

The invention discloses a method for co-immunoculture of patient-derived tumor cell antigen peptide loaded by PBMC (peripheral blood mononuclear cells) and autologous DC (dendritic cells) and application of the method. The method comprises the following steps: extracting tumor-associated antigen peptide from a patient-derived tumor organ (PDO), and performing IFN-gamma pretreatment and freeze-thaw cycle purification; separating CD14 < + > mononuclear cells from the PBMC, inducing mature DCs (Dendritic Cells) by adopting a two-stage culture method, and loading antigens; and co-culturing the PBMC and the antigen-loaded DC, and detecting IFN-gamma expression after culturing for 48 hours. According to the method, the sensitivity and consistency of immunoreaction are remarkably improved, the experimental period is shortened, and the method is suitable for individualized drug screening and tumor immunity research.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method and application for immunoculturing PBMC and DC loaded with tumor cell antigen peptides. Background Art

[0002] Patient-derived tumor organoids (PDOs), as an in vitro three-dimensional culture model, can highly simulate the biological characteristics of tumors in patients and have been widely used in drug screening and immunological research. Peripheral blood mononuclear cells (PBMCs) contain various immune cells, and co-culturing with PDOs can simulate the in vivo immune microenvironment for studying immune responses. However, in the prior art, the co-culture system of PBMCs and PDOs has problems such as a long experimental period, low sensitivity, and poor consistency.

[0003] Patent document CN119410741A discloses a method for verifying the function of neoantigens based on tumor organoids. By loading dendritic cells (DCs) with neoantigen polypeptides, cytotoxic T lymphocytes (CTLs) are stimulated to generate and their killing activity is verified. However, this method does not involve the specific steps of extracting antigens from tumor organoids, and the co-culture system is limited to organoids and CTLs, failing to optimize the sensitivity and consistency of the entire immune response.

[0004] In addition, although there are studies on the co-culture of PBMCs and PDOs in the existing literature, there is no systematic method for extracting antigens from patient-derived PDOs and combining them with DCs for optimization. Therefore, there is an urgent need to develop a co-culture technology to improve the sensitivity, consistency, and application value of the culture system. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method and application for immunoculturing PBMC and DC loaded with tumor cell antigen peptides. By extracting tumor-associated antigen peptides from PDOs, optimizing DC induction, and constructing a co-culture system, the sensitivity and consistency of the immune response are significantly improved, the experimental period is shortened, and it is applicable to individualized drug screening and immunological research.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for immunoculturing PBMC and DC loaded with tumor cell antigen peptides, characterized by comprising the following steps:

[0008] S1. Extracting tumor-associated antigen peptides from patient-derived PDOs;

[0009] S2. Separating CD14+ monocytes from patient-derived PBMCs, inducing the differentiation of CD14+ monocytes into mature DCs by a two-stage culture method, and loading the tumor-associated antigen peptides described in step S1 onto the mature DCs;

[0010] S3. Co-culture, including co-culturing patient-derived PBMC with mature DC loaded with tumor-associated antigen peptides, and detecting IFN-γ expression after 48 hours of culture;

[0011] The cell ratio of the co-culture of the patient-derived PBMC and the mature DC loaded with tumor-associated antigen peptides is (5 - 15):1.

[0012] Furthermore, the steps of extracting tumor-associated antigen peptides in step S1 include:

[0013] S10. Digest the PDO into single cells or small cell clusters of 1 - 3 cells with enzymes;

[0014] S11. Pretreat the digested cells in a medium containing 200 ng / ml IFN-γ for 24 hours;

[0015] S12. After washing the pretreated cells, resuspend them in PBS buffer solution containing protease and phosphatase inhibitors, and perform 1 - 3 freeze-thaw cycles, where the freezing temperature is -196°C and the thawing temperature is 37°C;

[0016] S13. After centrifugation, take the supernatant and concentrate it successively using 3KD and 1KD ultrafiltration tubes to obtain tumor-associated antigen peptides.

[0017] Furthermore, the steps of inducing CD14+ monocytes to differentiate into mature DC by the two-stage culture method in step S2 include:

[0018] S21. Extract PBMC from the patient's peripheral blood by density gradient centrifugation;

[0019] S22. Isolate CD14+ monocytes from PBMC by magnetic bead sorting;

[0020] S23. The two-stage culture method includes:

[0021] The first stage, culture the CD14+ monocytes isolated in S22 in a medium containing CellGenix GMP DC 100 ml; GM-CSF 50 - 250 ug / ml 5 ug - 25 ug and IL-4 40 - 200 ng / ml 4 ug - 20 ug for 5 days, change the medium once on the third day, and continue to culture for 2 days to obtain immature DC;

[0022] In the second stage, 60 - 200 μl of the immature DC obtained in the first stage and tumor - associated antigen peptides are added to a culture medium containing 100 ml of CellGenix GMP DC, 50 - 250 ng / ml (5 μg - 25 μg) of GM - CSF, 40 - 200 ng / ml (4 μg - 20 μg) of IL - 4, 10 - 60 ng / ml (1 μg - 6 μg) of IL - 1β, 10 - 60 ng / ml (1 μg - 6 μg) of IL - 6, 10 - 60 ng / ml (1 μg - 6 μg) of TNF - alpha, 40 - 200 ng / ml (4 μg - 20 μg) of PGE - 2, and 1 - 20 μg / ml (100 μg - 2 mg) of PolyIC, and cultured for 2 - 2.5 days to obtain mature DC.

[0023] Furthermore, the cell number ratio of the co - culture of PBMC and mature DC loaded with tumor - associated antigen peptides described in step S3 is 10:1.

[0024] Furthermore, the co - culture described in step S3 also includes PDO, and the cell ratio of PBMC, PDO, and mature DC loaded with tumor - associated antigen peptides is 10:1:1.

[0025] Another object of the present invention is to provide a method for immune co - culture of PBMC and DC loaded with tumor cell antigen peptides

[0026] method

[0027] Application in screening immune drugs.

[0028] Furthermore, the immune drugs include one or more of pembrolizumab, nivolumab, or ipilimumab.

[0029] Furthermore, the immune drug is pembrolizumab, and the concentration is 25 μg / ml.

[0030] Furthermore, the IFN - γ expression described in step S3 is detected by the ELISPOT method.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] 1. Improved sensitivity: By optimizing antigen presentation with autologous DC, T - cell activation is significantly enhanced, and the IFN - γ expression level is significantly higher than that of traditional methods.

[0033] 2. Improved consistency: Standardized antigen extraction and DC induction processes reduce individual differences and improve the stability of results.

[0034] 3. Shortened cycle: Efficient antigen presentation and the synergistic effect of the three enable the immune response to be detectable within 48 hours.

[0035] 4. Multifunctional applications: Suitable for drug screening, obtaining tumor-specific T cells, and discovering new targets, with broad application prospects. Description of the Drawings

[0036] The invention will be further described with reference to the accompanying drawings. However, the embodiments in the drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the following drawings without creative efforts.

[0037] Figure 1 It is the bright-field microscopic image of the detection result of Example 1 of the present invention;

[0038] Figure 2 It is the spot map of the ELISPOT detection result of Example 1 of the present invention;

[0039] Figure 3 It is the spot map of the ELISPOT detection result of Example 2 of the present invention;

[0040] Figure 4 It is the spot map of the ELISPOT detection result of Example 3 of the present invention;

[0041] Figure 5 It is the spot map of the ELISPOT detection result of Example 4 of the present invention. Detailed Embodiments

[0042] To better illustrate the purpose, technical solutions, and advantages of the present invention, the present invention is further described by the following embodiments. Obviously, the following embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments; it should be understood that the embodiments of the present invention are only used to illustrate the technical effects of the present invention, rather than to limit the protection scope of the present invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention.

[0044] Example 1

[0045] Using the PBMC of a lung cancer patient and patient-derived tumor organoids (PDO) as materials, co-cultured in groups according to the ratio of PBMC:PDO:DC cells of 10:1:1, and added pembrolizumab (Keytruda, K drug) at 25

[0046] μg / ml. After culturing for 48 hours, the IFN-γ expression was detected by enzyme-linked immunospot assay (ELISPOT) to verify the effectiveness and sensitivity of the method of the present invention.

[0047] The experimental procedure is as follows:

[0048] (1) Extraction and purification of tumor antigens: Digest tumor organoids with (Tryple Express (GIBCO), 1X Dissociation Reagent) until single cells or small cell clusters of 1 - 3 cells are obtained. Add IFN-γ at 200 ng / ml and culture for 24 h before protein extraction. Collect the cells, wash them twice with PBS, resuspend 600,000 tumor organoid cells in 300 μl PBS (containing 10x protease inhibitor and phosphatase inhibitor), freeze in liquid nitrogen for 1 min, incubate in a 37 °C water bath for 2 min, repeat the freeze-thaw cycle once, centrifuge at 12,000 g at 4 °C for 10 min, collect the supernatant, and lyse the remaining cell pellet with 50 μl IP lysis buffer (containing 10x protease inhibitor and phosphatase inhibitor) on ice for 30 min. Centrifuge at 12,000 g at 4 °C for 10 min, collect all the supernatants, add all the protein supernatants to a 3KD protein concentrator tube, centrifuge at 12,000 g at 4 °C for 15 min, collect the concentrated solution obtained by centrifugation, dilute the concentrated solution with 4.5 ml PBS, transfer it to a 1KD concentrator tube, centrifuge at 5,000 g at 4 °C for 1 h, collect the supernatant to obtain 180 μl of protein concentrate, and aliquot and store it at -80 °C.

[0049] (2) Magnetic bead sorting of CD14-positive cells in PBMC: After resuscitating PBMC, centrifuge at 300 g for 10 min, wash it twice with an appropriate amount of PBS, resuspend 10*7 PBMC in 80 μl of sorting buffer, add 20 μl of CD14 MicroBeads, mix gently, incubate in a 4 °C refrigerator for 15 min, add 1 ml of buffer and mix well, centrifuge at 300 g for 10 min, discard the supernatant, resuspend the cells in 500 μl of buffer for standby;

[0050] Take one MS column, insert it into the magnetic field, wash the column once with 500 μl of buffer, and while keeping the column from drying out, load 500 μl of cell suspension onto the column for filtration. Place a new 15 ml centrifuge tube below to collect the cells washed out from the column. After the cell suspension is filtered, wash the column three more times with 500 μl of buffer each time. Remove the column, add 1 ml of buffer and place it on a new 15 ml centrifuge tube, gently push the column to obtain CD14+ cells, centrifuge at 300 g for 10 min, resuspend the cell pellet in 1 ml of X-VIVO, centrifuge at 300 g for 10 min, take 1 ml of DC differentiation medium to resuspend the cells, count them, and according to the counting results, seed them into the wells of a tissue culture plate of appropriate size.

[0051] After sorting cells with CD14-positive magnetic beads, 1 million CD14-positive cells were obtained and seeded into two 24-well plates. 1.5 ml of the first-stage DC differentiation medium (containing 100 ml of CellGenix GMP DC; 160 ng / ml (16 μg) of GM-CSF and 100 ng / ml (10 μg) of IL-4) was added, and the cells were cultured for 3 days. Then, 1.5 ml of the medium was replaced, and the cells were cultured for another 2 days. Next, 1.5 ml of the second-stage DC maturation medium (containing 100 ml of CellGenix GMP DC, 160 ng / ml (16 μg) of GM-CSF, 100 ng / ml (10 μg) of IL-4, 20 ng / ml (2 μg) of IL-1β, 20 ng / ml (2 μg) of IL-6, 40 ng / ml (4 μg) of TNF-α, 100 ng / ml (10 μg) of PGE-2, and 10 μg / ml (1 mg) of PolyIC) was added. At the same time, 90 μl of tumor-purified antigen peptide was loaded into each well, and the cells were cultured for another 2 days before harvesting the mature DCs.

[0052] (3) Co-culture in groups: Digest tumor organoids into single cells or small cell clusters of 1 - 3 cells. Add 200 ng / ml of IFN-γ and culture for 24 h before protein extraction. Collect the cells, wash them twice with PBS, and resuspend the tumor PDOs with the co-culture medium (50% lung cancer organoid medium + 50% 1000 IU / ml IL-2 X-VIVO-15). After counting, adjust the cell concentration to 10,000 cells / 50 μl. Collect the DCs, resuspend them with the co-culture medium, and after counting, adjust the cell concentration to 10,000 cells / 50 μl.

[0053] Resuscitate PBMC 24 h before co-culture, culture them in the resting medium X-VIVO-15-IL-2-1000 IU / ML, wash them with an appropriate amount of PBS and then resuspend the cells with the co-culture medium. After counting, adjust the cell concentration to 100,000 cells / 50 μl. According to the requirements of the ELISPOT plate instruction manual in advance, wash the plate 4 times with PBS, incubate it with the medium containing 10% FBS for more than 30 minutes, and seed the cells into the wells of the ELISPOT plate according to the groups. Detect the expression level of INF-γ at 48H according to the ELISPOT instruction manual. The data are shown in Table 1 below:

[0054] Table 1: ELISPOT detection results

[0055]

[0056] From Table 1, Figure 1 and Figure 2It can be seen that the expression level of INF-γ in the group with DC added is significantly increased compared with that in the group without DC added, and the expression level of INF-γ in the group with K drug added and DC is also increased compared with that in the DC group without K drug added, indicating that DC not only significantly enhances the immune activation of PBMC, but also has an auxiliary effect on the action of immune drugs.

[0057] Example 2

[0058] Using PBMC from a lung cancer patient, the experimental procedure was the same as in Experimental Example 1. Different groups were co-cultured at a cell ratio of PBMC:DC of 10:1, and pembrolizumab (K drug) at 25 μg / ml was added. After culturing for 48 hours, IFN-γ expression was detected by enzyme-linked immunospot assay (ELISPOT) to verify the effectiveness and sensitivity of the method of the present invention. The data are shown in Table 2 below:

[0059] Table 2: ELISPOT detection results

[0060]

[0061]

[0062] From Table 2 and Figure 3 It can be seen that the expression level of INF-γ in the group with DC added is significantly increased compared with that in the group without DC added, and the expression level of INF-γ in the group with K drug added and DC is also significantly increased compared with that in the DC group without K drug added, indicating that DC significantly enhances the immune activation of PBMC, and DC also has an auxiliary effect on the action of immune drugs.

[0063] Example 3

[0064] Using PBMC from a colorectal cancer patient, the experimental procedure was the same as in Experimental Example 1. Different groups were co-cultured at a cell ratio of PBMC:DC of 10:1, and pembrolizumab (K drug) at 25 μg / ml was added. After culturing for 48 hours, IFN-γ expression was detected by enzyme-linked immunospot assay (ELISPOT) to verify the effectiveness and sensitivity of the method of the present invention. The data are shown in Table 3 below:

[0065] Table 3: ELISPOT detection results

[0066]

[0067] From Table 3 and Figure 4 It can be seen that the expression level of INF-γ in the group with DC added is significantly increased compared with that in the group without DC added, and the expression level of INF-γ in the group with K drug added and DC is also significantly increased compared with that in the DC group without K drug added, indicating that DC significantly enhances the immune activation of PBMC, and DC also has an auxiliary effect on the action of immune drugs.

[0068] Example 4

[0069] Using PBMC from a colorectal cancer patient as an example, the experimental procedure was the same as in Experimental Example 1. Different groups were co-cultured at different cell ratios of PBMC:DC at 10:1 and 20:1 respectively for comparison, and pembrolizumab (Keytruda, K drug) at 25 μg / ml was added. After culturing for 48 hours, the IFN-γ expression was detected by enzyme-linked immunospot assay (ELISPOT) to verify the effectiveness and sensitivity of the method of the present invention. The data is shown in Table 4 below:

[0070] Table 4: ELISPOT detection results

[0071]

[0072] As can be seen from Table 4 and Figure 5 it can be seen that the expression level of INF-γ in the group with added DC was significantly increased compared to the group without added DC. The expression level of INF-γ in the K drug group with added DC was also significantly increased compared to the DC group without added K drug. Moreover, the expression level in the high-ratio DC group was significantly enhanced compared to the low-ratio group, indicating that DC significantly enhanced the immune activation of PBMC. At the same time, DC also had an auxiliary effect on the action of immune drugs, and the addition of different amounts of DC had a significant difference in the immune response.

[0073] The above is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the equivalent embodiments with equivalent changes within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for immune co-culture of PBMC and DC loaded with tumor cell antigen peptides, characterized in that: The following steps are involved: S1. Extracting tumor-associated antigen peptides from patient-derived PDO; S2. Isolate CD14+ monocytes from patient-derived PBMCs, induce CD14+ monocytes to differentiate into mature DCs using a two-stage culture method, and load the tumor-associated antigen peptides described in step S1 onto the mature DCs; S3. Co-culture, including co-culturing patient-derived PBMCs with mature DCs loaded with tumor-associated antigen peptides, and detecting IFN-γ expression after 48 hours of culture; The cell ratio of the patient-derived PBMCs co-cultured with mature DCs loaded with tumor-associated antigen peptides is (5-15):

1.

2. The method for immune co-culture of PBMC and DC loaded with tumor cell antigen peptides according to claim 1, characterized in that: The step of extracting tumor-associated antigen peptides in step S1 includes: S10. digesting PDO with enzymes into single cells or small cell clusters of 1-3 cells; S11. The digested cells were pretreated with medium containing 200 ng / ml IFN-γ for 24 hours; S12. The pretreated cells were washed and resuspended in a PBS buffered saline solution containing protease and phosphatase inhibitors, and subjected to 1-3 freeze-thaw cycles, wherein the freezing temperature was -196°C and the thawing temperature was 37°C; S13. After centrifugation, the supernatant was collected and concentrated using 3KD and 1KD ultrafiltration tubes in sequence to obtain tumor-associated antigen peptides.

3. The method for immune co-culture of PBMC and DC loaded with tumor cell antigen peptides according to claim 1, characterized in that: The step of inducing CD14+ monocytes to differentiate into mature DCs by using a two-stage culture method in step S2 includes: S21. Extract PBMC from the patient's peripheral blood using density gradient centrifugation; S22. Isolate CD14+ monocytes from PBMCs using magnetic bead sorting; S23. The two-stage culture method comprises: In the first stage, the CD14+ monocytes isolated from S22 were cultured in a medium containing CellGenix GMP DC 100ml, GM-CSF 50-250ug / ml 5ug-25ug, and IL-440-200ng / ml 4ug-20ug for 5 days. The medium was changed once on the third day and cultured for another 2 days to obtain immature DCs. In the second stage, the immature DCs obtained in the first stage and 60-200ul of tumor-associated antigen peptides were added to the culture medium containing CellGenix GMP DC 100ml, GM-CSF 50-250ng / ml 5ug-25ug, IL-440-200ng / ml 4ug-20ug, IL-1beta 10-60ng / ml 1ug-6ug, IL-6 10-60ng / ml1ug-6ug, TNF-alpha10-60ng / ml 1ug-6ug, PGE-2 40-200ng / ml 4ug-20ug and PolyIC 1-20ug / ml 100ug-2mg, and cultured for 2-2.5 days to obtain mature DCs.

4. The method for immune co-culture of PBMC and DC loaded with tumor cell antigen peptides according to claim 1, characterized in that: In step S3, the ratio of the number of cells co-cultured with PBMC and mature DC loaded with tumor-associated antigen peptides was 10:

1.

5. The method for immune co-culture of PBMC and DC loaded with tumor cell antigen peptides according to claim 1, characterized in that: The co-culture described in step S3 also includes patient-derived PDO, and the cell ratio of PBMC, PDO and mature DC loaded with tumor-associated antigen peptide is 10:1:

1.

6. Use of the method of immune co-culture of PBMC and DC loaded with tumor cell antigen peptides according to claim 1 in screening immune drugs.

7. The use of the method of immune co-culture of PBMC and DC loaded with tumor cell antigen peptides according to claim 6 in screening immune drugs, characterized in that: The immune drug includes one or more of pembrolizumab, nivolumab or ipilimumab.

8. Application of the method of immune co-culture of PBMC and DC loaded with tumor cell antigen peptides according to claim 6 or 7 in screening immune drugs, characterized in that: The immune drug is pembrolizumab, and the concentration is 25 μg / ml.

9. The method for immune co-culture of PBMC and DC loaded with tumor cell antigen peptides according to claim 1, characterized in that: The IFN-γ expression in step S3 was detected by ELISPOT method.

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