Method for efficiently constructing personalized pancreatic cancer organoid to screen tumor reactive TCR (T cell receptor)

Abstract

The invention discloses a method for efficiently constructing a personalized pancreatic cancer organoid and screening a tumor reactive TCR (T cell receptor), which comprises the following steps: transferring RSPO1 and WNT3A genes into the pancreatic cancer organoid, and inducing expression and secretion of RSPO1 and WNT3A in the organoid by adding Dox (doxycycline). The pancreatic cancer antigen tumor reactive TCR can be screened from the PBMC of a pancreatic cancer patient by using the constructed transgenic organ.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunotherapy, and particularly relates to a method for efficiently constructing personalized pancreatic cancer organoids, personalized pancreatic cancer organoids, and their application in screening tumor-reactive TCRs. Background Art

[0002] As an innovative 3D in vitro model, tumor organoids can accurately simulate the microenvironment, cell composition, and functional characteristics of real tumors. Their ability to highly maintain tumor heterogeneity makes organoids show great application potential in personalized precision tumor treatment. Pancreatic cancer is a typical solid tumor with high heterogeneity. The long culture cycle and low culture success rate of its organoids limit their application in clinical research. In addition, the culture of organoids requires the activation of the Wnt signaling pathway, and the known activation methods are divided into two types. One is to prepare conditioned media containing RSPO1 or WNT3A, but the medium contains other cytokines secreted by cells and irrelevant components such as fetal bovine serum, making it difficult to achieve standardization between batches. The other is to directly add purified RSPO1 or WNT3A recombinant protein to the medium, which is expensive, degrades quickly, and needs to be frequently prepared to maintain the activity of cytokines.

[0003] Identifying and recognizing tumor-reactive TCR is an effective way to improve the cellular immune efficacy of solid tumor patients. TCR is mostly enriched in tumor-infiltrating lymphocytes (TILs). Existing identification, recognition, and functional verification of tumor-reactive TCR mostly rely on tumor-reactive TILs and PBMC based on transcriptome analysis. The traditional method of obtaining T cells from tumor-reactive TILs and PBMC based on transcriptome is difficult to obtain antigen-unknown tumor-reactive TCRs in immune-deficient tumors represented by pancreatic cancer, and the cell activity is low, and the characteristics of terminally exhausted T cells (ENTPD1, PDCD1, LAYN, TIGIT, etc.) are obvious. In addition, the tumor microenvironment of pancreatic cancer is complex and the immunosuppressive characteristics are significant, resulting in extremely low quality of pancreatic cancer TILs, and it is extremely complex to obtain personalized tumor-reactive TCRs from them. Summary of the Invention

[0004] To solve the above technical problems, the present invention first provides a method for identifying and screening tumor-reactive TCRs from PBMC of cancer (pancreatic cancer) patients by using autologous transgenic organoids combined with multi-group parallel single-cell TCR-seq sequencing.

[0005] The first aspect of the present invention provides a method for constructing tumor transgenic organoids, including introducing RSPO1 and / or WNT3A genes into tumor organoids.

[0006] In some embodiments, the tumor is pancreatic cancer.

[0007] In some embodiments, the method of introduction is selected from one or more of viral vector transfection, electroporation, liposome transfection, polymer transfection, and microinjection.

[0008] In some embodiments, viral vector transfection is used for the introduction.

[0009] In some embodiments, the viral vector is a lentivirus, an adenovirus, or a retrovirus.

[0010] In some embodiments, the lentivirus is the Tet-On system.

[0011] In some embodiments, the method further comprises adding doxycycline to the organoid culture system to induce the expression and / or secretion of RSPO1 and / or WNT3A.

[0012] The second aspect of the present invention provides a tumor autologous transgenic organoid obtained by the method according to the first aspect of the present invention.

[0013] The third aspect of the present invention provides a tumor autologous transgenic organoid, which comprises an introduced RSPO1 and / or WNT3A gene.

[0014] In some embodiments, the tumor is pancreatic cancer.

[0015] The fourth aspect of the present invention provides a method for screening tumor antigen-reactive TCRs using the tumor autologous transgenic organoid according to the second or third aspect of the present invention.

[0016] In some embodiments, the tumor is pancreatic cancer.

[0017] In some embodiments, the method comprises screening pancreatic cancer antigen tumor-reactive TCRs from the PBMCs of pancreatic cancer patients.

[0018] In some embodiments, TCRs that are double-enriched with organoids and antigen polypeptides are screened as candidate TCRs; preferably, the antigen polypeptide is a KRAS G12D polypeptide.

[0019] In some embodiments, the candidate TCRs are determined by single-cell TCR-seq sequencing.

[0020] The fifth aspect of the present invention provides a TCR obtained by the method according to the fourth aspect of the present invention.

[0021] In some embodiments, the TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β as shown in SEQ ID NOs: 1-6, respectively.

[0022] In some embodiments, the TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β as shown in SEQ ID NOs: 7-12, respectively.

[0023] In some embodiments, the TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β as shown in SEQ ID NOs: 13-18, respectively.

[0024] In some embodiments, the TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β as shown in SEQ ID NOs: 19-24, respectively.

[0025] The sixth aspect of the present invention provides the use of the TCR described in the fifth aspect of the present invention in the preparation of TCR-T cells.

[0026] The seventh aspect of the present invention provides the use of the TCR described in the sixth aspect of the present invention in the determination of pancreatic cancer neoantigens.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1) Provided a novel method for constructing pancreatic cancer organoids. Through transgenic technology, the stable introduction of RSPO1 and WNT3A components into autologous pancreatic cancer organoids was achieved. By adding Dox, the precise induction of the expression of WNT3A and RSPO1 genes was realized, and the controllable activation of the Wnt signaling pathway was achieved, which could maintain the optimal morphological structure and proliferation ability of transgenic organoids. It solved the defects of interference by irrelevant components, high price, and difficulty in standardization in the preparation of traditional organoid culture media.

[0029] 2) Provided a method for screening tumor-reactive TCRs of PDAC patients by combining autologous transgenic organoids with single-cell sequencing. The present invention combines single-cell TCR-seq and autologous transgenic organoids. In the polypeptide enrichment amplification of PBMCs and the organoid enrichment amplification, the tumor-reactive TCRs of patients with high-quality TILs and those without high-quality TILs and unknown antigens are efficiently and accurately identified through multiple conditions and dimensions, which is an effective strategy for developing TCR-T therapy for pancreatic cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objects, and advantages of the present invention will become more apparent:

[0031] Figure 1Demonstration of the stable introduction of RSPO1 and WNT3A components through the Tet-On lentiviral system. A, Schematic diagram of the insertion sequence of the Lenti-EFS-TetOn-RSPO1-P2A-WNT3A-TRE plasmid; B, Morphology of intact organoids after lentiviral infection with EGFP under a laser confocal microscope; C, Flow analysis results after digestion of organoids into single cells. ****: p < 0.0001; D, Comparison of the morphological characteristics between traditional organoids and transgenic organoids. Dox was not added to the medium of traditional organoids, and RSPO1 and WNT3A were removed from the medium of transgenic organoids, with or without Dox added. P0, D2 indicates the 2nd day after the construction of transgenic organoids, and P1, D5 indicates the 5th day after one passage; E, Concentrations of RSPO1 and WNT3A in the culture supernatant of organoids 5 days after passage. *: p < 0.05, **: p < 0.01; F, Proliferation ability of traditional organoids and transgenic organoids 1 day, 3 days, and 5 days after passage. Each group of data was compared with the passage day (the 0th day). *: p < 0.05, **: p < 0.01, ns: p > 0.05.

[0032] Figure 2 Demonstration that the histochemical characteristics of Dox-regulated transgenic organoids are highly consistent with those of the source tissues. A, Immunohistochemical results of organoids and corresponding tumor tissues. The scale bar for the organoid pictures in the figure is 50 μm, the scale bar for the unamplified tumor tissues is 500 μm, and the scale bar for the amplified tumor tissues is 100 μm; B, Pathological scoring results of organoids and corresponding tumor tissues.

[0033] Figure 3 Demonstration of screening tumor-reactive TCRs against pancreatic cancer antigens using autologous transgenic organoids. A, Screening of tumor-reactive TCRs by multiplex parallel sequencing analysis of patient PBMCs; B, Venn diagram showing TCRs enriched in autologous organoids, peptides, and anti-CD3 in PBMCs; C, ELISA detection of IFN-γ secretion after incubation of TCR-T cells with peptide-stimulated DC cells, n = 3; D, ELISA detection of IFN-γ secretion when TCR-T cells are co-cultured with organoids (with or without MHC I antibody), n = 3, *: p < 0.05, *: p < 0.005. Figure 4 Demonstration of neoantigen identification and anti-tumor function detection of TCR-3. A, Neoantigen identification procedure for TCR-3; B, ELISA detection of IFN-γ secretion after incubation of TCR-T cells with DC cells stimulated by TP53 peptides, n = 3; C, Flow representative diagram and statistical analysis of TNFRSF9 expression of TCR-T cells co-cultured with organoids, n = 3; D, A combined strategy for characterizing tumor-reactive TCRs in PDAC patients through single-cell sequencing and the application of autologous transgenic organoids. Detailed implementation methods

[0034] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions of the embodiments of the present invention in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0035] Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings as understood by those of ordinary skill in the art to which the present invention pertains.

[0036] Example 1 Experimental Materials and Methods

[0037] 1.1 Preparation of Organoid Medium

[0038] (1) Basal Medium: Take S-Reduce Serum-Free DMEM / F-12 (1:1) medium and add various additives according to Table A below. After preparation, mix well and store refrigerated at 4°C for use within 1 month.

[0039] Table A Formulation of Organoid Basal Medium

[0040] Product Name Final Concentration Penicillin-Streptomycin (100×) 1× GlutaMAX Additive (100×) 2 mM (1×) Hepes 10 mM Y-27632 10 μM

[0041] (2) Expansion Medium: Take Advanced DMEM / F-12 medium and add various additives according to Table B below.

[0042] After preparation, mix well and store refrigerated at 4°C for use within 1 month.

[0043] Table B Formulation of Organoid Expansion Medium

[0044]

[0045] 1.2 Preparation of Organoids

[0046] (3) Sample Collection: Perform sample collection on the surgical specimen within 30 minutes after it is removed from the body, place it in the organoid basal medium pre-cooled at 4°C, and transport it back to the laboratory within 15 minutes. During the sample collection process, pay attention to avoiding normal tissues, macroscopically visible blood vessels, and adipose infiltration areas;

[0047] (4) Pretreatment: Transfer the tumor specimen to PBS pre-cooled at 4°C containing 1% penicillin / streptomycin for washing.

[0048] Trim the tissue to remove vascular and adipose components. Break up the large tissue into small pieces to release the remaining red blood cells in the tissue. After washing 3 times, further cut the tumor tissue into pieces with a volume < 1 mm 3, after washing in PBS solution, centrifuge at 300×g for 5 min at 4°C, discard the supernatant, and perform enzymatic digestion;

[0049] (5) Enzymatic digestion: Use the SOLO Tissue Enzymatic Digestion Kit (JZ-SC-58201, Zhongke Puri) to prepare the enzymatic digestion solution. Add appropriate amounts of digestive enzyme A, digestive enzyme B, digestive enzyme D, digestive enzyme E, enzymatic digestion buffer R, and 1% penicillin / streptomycin according to the instructions. Resuspend the tumor tissue fragments in the enzymatic digestion solution preheated to 37°C, incubate for enzymatic digestion in a 37°C incubator, gently shake once every 10 min, and digest for 1 - 2 h according to the digestion degree of the stroma and cells;

[0050] (6) Collect cells: Add organoid basal medium to terminate digestion at a ratio of 1:5. Filter the digested cell suspension through a 70 μm cell strainer, and rinse the strainer with organoid basal medium. Collect the filtrate, centrifuge at 300×g for 5 min at room temperature, and discard the supernatant. Resuspend the cell pellet in organoid basal medium, count the cells and detect cell viability, and centrifuge again to discard the supernatant;

[0051] (7) Matrigel coating: Add Matrigel to the cell pellet from the previous step. Add 30 - 50 μL of Matrigel per 10 6 cells. Gently pipette with a pre-chilled pipette tip with a filter to mix the Matrigel and cells, avoiding the formation of air bubbles. Keep the temperature low throughout the process to prevent premature solidification of Matrigel. Drop the mixture of Matrigel and cells onto the bottom of a preheated 24-well plate, 4 - 6 drops per well, with each drop having a volume of approximately 10 μL. After completion of the operation, immediately invert the well plate and place it in a 37°C cell culture incubator for 5 - 15 min to accelerate the solidification of Matrigel.

[0052] (8) Culture: After the Matrigel has solidified, add 500 μL of organoid expansion medium preheated to 37°C, and add PBS to the side wells to slow down the evaporation of the medium. Change the medium every 2 - 3 days depending on the growth of the organoids. Organoids can generally be seen to form within 1 - 3 weeks.

[0053] 1.3 Detection of cell viability

[0054] For the detection of enzymatically digested cell suspensions and single cells after organoid digestion, use the Vi-CELL BLU automatic cell counter and viability analyzer (Beckman Coulter). Dilute the cell suspension and then perform the detection on the machine.

[0055] 1.4 Preparation of organoid paraffin sections

[0056] Collect the organoids and centrifuge; add 1 mL of 4% PFA, gently flick the centrifuge tube to resuspend the organoid pellet. Hand over the fixed organoids to Shanghai Ruiyu Biotechnology Co., Ltd. for embedding and sectioning.

[0057] 1.5 Immunohistochemical Staining

[0058] (1) Select the required blank slides, mark the information such as the required antibody, ratio, antigen retrieval solution, etc. on the side with tissue, place them on the slide rack in sequence, and bake the slides in an oven at 60 - 65 °C for 2 h for standby;

[0059] (2) After the sections are sequentially passed through the jars in the following order, gently wash them three times in ddH2O:

[0060] ① Xylene I 10 min ⑤ Absolute Ethanol II 5 min ② Xylene II 10 min ⑥ 95% Ethanol I 5 min ③ Xylene III 10 min ⑦ 95% Ethanol II 5 min ④ Absolute Ethanol I 5 min ⑧ 75% Ethanol 5 min

[0061] (3) Prepare 3% hydrogen peroxide solution, place the sections in 3% hydrogen peroxide solution, and incubate at 37 °C for 20 min to remove endogenous peroxidase;

[0062] (4) Preheat the electric pressure cooker, and heat the sodium citrate buffer (pH 6.0, containing 10 mM sodium citrate and 0.05% Tween - 20) and Tris / EDTA buffer (pH 9.0, containing 10 mM Tris, 1 mM EDTA solution and 0.05% Tween - 20) in a water bath until boiling. Select the appropriate buffer for the sections according to the recommendations in the antibody instruction manual, immerse the sections in it, pressurize the pressure cooker until no more exhaust gas is emitted, maintain for 5 min and then stop heating, manually release the pressure and take out;

[0063] (5) Incubate in an ice bath with the buffer solution for about 15 min, take out the sections after the solution cools to room temperature, gently wash them three times in ddH2O, and then immerse them in water to prevent the sections from drying;

[0064] (6) Prepare a wet box. Take out the sections one by one, gently tap them on absorbent paper to remove excess moisture, carefully observe the tissue area under light. Use an immunohistochemistry pen to circle the tissue area, and dropwise add PBS solution containing 5% goat serum, and incubate at room temperature for 1 h;

[0065] (7) Dilute the primary antibody with PBS solution containing 5% goat serum according to the recommended ratio in the antibody instruction manual. Discard the liquid on the sections, add the diluted primary antibody in the area circled by the immunohistochemistry pen, and incubate at 4 °C overnight;

[0066] (8) The next day, warm up the sections at 37 °C for 1 h. Discard the primary antibody, place the sections on a horizontal shaker, and wash them three times in PBS, 10 min each time;

[0067] (9) According to the area circled by the immunohistochemistry pen and the sample size, dropwise add reagent A

[0068] (enzyme - labeled goat anti - mouse / rabbit IgG polymer) on the tissue, and incubate at room temperature for 1 h;

[0069] (10) Discard the secondary antibody, place the sections on a horizontal shaker, wash three times in PBS for 10 minutes each time; (11) Mix reagent B2 (chromogenic buffer) and reagent B1 (DAB chromogenic agent) in the secondary antibody kit of the immunochromogenic reagent in a ratio of 50:1 for standby. Drain the liquid on the sections, add the mixed reagent dropwise to the area circled by the histochemical pen, control the chromogenic degree under a light microscope, and place it in ddH2O to terminate the chromogenesis;

[0070] (12) Take out the sections, add hematoxylin staining solution dropwise to the area circled by the histochemical pen, incubate at room temperature for 75 s, and then rinse in ddH2O. After all sections are stained, wash once more with ddH2O and immerse in PBS

[0071] solution to prepare for passing through the cylinders;

[0072] (13) After the sections are dehydrated by passing through the cylinders in the following order, seal them with neutral balsam, air-dry overnight and then take pictures:

[0073] ① 75% Ethanol 5 min ⑤ Absolute Ethanol II 5 min ② 95% Ethanol I 5 min ⑥ Xylene I 10 min ③ 95% Ethanol II 5 min ⑦ Xylene II 10 min ④ Absolute Ethanol I 5 min ⑧ Xylene III 10 min

[0074] 1.6 Lentivirus packaging and concentration

[0075] (1) Use the third-generation lentivirus packaging system. Prepare HEK293T cells in the logarithmic growth phase with good growth status in advance, and start transfection when the confluence reaches 70-90% in a 10 cm cell culture dish. The culture medium can contain serum;

[0076] (2) Operate according to the instructions of Lipofectamine 3000 transfection reagent. In the transfection system of this study, the total mass of plasmid DNA is 15 μg, and the mass ratio of various plasmids is pLP1:pLP2:pLP-VSVG:plasmid = 2:2:1:3;

[0078] (3) Add the transfection complex dropwise to the culture dish after incubating at room temperature for 10-15 minutes, and place the culture dish in a cell culture incubator for culture;

[0079] (4) Collect the cell culture supernatant 48 h after adding the transfection complex, centrifuge at 4000×g and retain the supernatant,

[0080] store at -80 °C. Add fresh growth medium to the culture dish, collect the cell culture supernatant for the second time 24 h later, centrifuge at 4000×g and retain the supernatant, store at -80 °C;

[0081] (5) The centrifuged supernatant collected in the previous step contains lentiviral particles. Add the viral-containing centrifuged supernatant to the upper layer of an Amicon Ultra-15 centrifugal filter (UFC903024, Merck). After centrifuging at 4000×g for 30 - 60 min at 4°C, take the remaining dark brown liquid in the upper layer, which is the virus concentrate, and perform virus titer determination.

[0082] 1.7 Detection of lentivirus infection efficiency by flow cytometry

[0083] (1) For HEK293T cells, refer to the cell passage method to digest the cells in a culture dish. For organoids, refer to the organoid passage method to dissociate the organoids into single cells;

[0084] (2) Centrifuge to obtain the cell pellet, resuspend it in FACS Buffer. After filtering with a 70μm filter mesh, aliquot it into flow cytometry tubes as needed and detect the fluorescence ratio on the machine.

[0085] 1.8 Lentivirus infection of organoids

[0086] (1) Harvest the normally cultured organoids into a 15 mL centrifuge tube. After centrifuging at 200×g for 5 min, discard the supernatant and place the centrifuge tube on ice for later use;

[0087] (2) According to the titer determination results, add an appropriate amount of lentivirus to the organoid expansion medium. The resuspension containing organoids and lentivirus is directly added to a 48-well plate, and the edges of the plate are sealed with sealing film. Centrifuge at 600×g for 60 min at room temperature in a plate centrifuge;

[0088] (3) Take out the plate, remove the sealing film, and transfer it to a 37°C cell culture incubator for continued culture for 4 h;

[0089] (4) Harvest the cells into a 15 mL centrifuge tube, centrifuge and take the pellet according to the organoid passage steps, resuspend it with Matrigel and plate it.

[0090] 1.9 Extraction and quantification of total protein from organoids

[0091] (1) Collect the organoids according to the organoid passage method;

[0092] (2) Add protease inhibitor to the cell lysis buffer to a final concentration of 1×. Add 200 μL of the mixture to each centrifuge tube and lyse the cells by pipetting on ice. After centrifuging at 10000×g for 5 min, take the supernatant;

[0093] (3) Use a BCA protein concentration assay kit (enhanced type) (20201ES76, Yeasen) for quantification.

[0094] The specific method refers to the instruction manual;

[0095] (4) According to the measurement results, adjust the concentrations of each protein sample to be consistent with a cell lysate containing protease inhibitors for subsequent experiments.

[0096] 1.10. PBMC Isolation

[0097] (1) Select healthy adult peripheral blood for PBMC isolation. Inclusion criteria: no history of diabetes, hyperlipidemia, infectious diseases, or malignant tumors. Collect about 2 mL of blood using a purple-top tube and reach the laboratory for processing within 3 h;

[0098] (2) Dilute the peripheral blood with an equal volume of PBS (pH 7.4). Add the same volume of Ficoll-Paque PLUS density gradient medium (17144003, Cytiva) to a 15 mL centrifuge tube and tilt the centrifuge tube at 45°; (3) Slowly add the peripheral blood dilution to the upper layer of Ficoll, keeping the interface between the peripheral blood and Ficoll stable and not disrupted. Centrifuge at 500×g for 30 min at room temperature, with the centrifuge acceleration rate of 5 and deceleration rate of 0. From bottom to top, the separated liquid layers are the red blood cell layer, granulocyte layer, separation medium layer, mononuclear cell layer (cloudy layer), and plasma layer;

[0099] (4) Carefully aspirate the cloudy layer and add it to PBS containing 2% FBS. After washing, use it for subsequent experiments.

[0100] 1.11. Human T Cell Sorting and Culture

[0101] Use the EasySep Human T Cell Isolation Kit from STEMCELL Technologies to isolate human CD8 + T cells. The specific steps are as follows:

[0102] (1) Resuspend the PBMC at a concentration of 5×10 7 cells / mL in EasySep Buffer. Take 1 mL of the cell suspension into a sterile flow cytometry tube and add 50 μL of the sorting antibody mixture. Pipette gently to mix well and incubate at room temperature for 5 min;

[0103] (2) Vortex the RapidSpheres separation magnetic beads for 30 s. Add 50 μL of the separation magnetic beads to each mL of the cell sample and mix the sample. Supplement EasySep Buffer to a total volume of up to 2.5 mL. Gently pipette 2 - 3 times to mix the liquid;

[0104] (3) Place the flow cytometry tube into the magnetic cell sorter and incubate at room temperature for 3 min. Hold at an angle and pour the cell suspension into a new centrifuge tube. The cells in the new centrifuge tube are the sorted human CD8 + T cells;

[0105] (4) The cells after primary sorting were added with human recombinant IL-2 (10 ng / mL) and human anti-CD3 / CD28 / CD2 T (25 μL / mL) cell activator to promote the growth and differentiation of CD8 + T cells. During the culture process, CD8 + T cells and the culture medium were taken out every 2 - 3 days, centrifuged at 400×g for 4 min to remove the old culture medium, and then cultured in a new culture medium. The culture medium used was ImmunoCult-XF T cell expansion medium (10981, STEMCELL), and the cell density was controlled at 1×10 6 / mL during the culture process. The methods of cryopreservation and resuscitation were the same as before;

[0106] 1.12. ELISA

[0107] (1) The culture supernatant of transgenic organoids was taken, diluted, and used with Human RSPO1 ELISA Kit (RDR-RSPO1-Hu, Reddot Biotech) and Human WNT3A ELISA Kit (RD-WNT3A-Hu, Reddot Biotech) to detect RSPO1 and WNT3A. The specific steps were referred to the instruction manual;

[0108] (2) The co-culture supernatant of organoids and CAR-T cells was taken, diluted, and used with Human IFN-γ Precoated ELISA kit (1110002, Dayou) and Human TNF-α Precoated ELISA kit (1117202, Dayou) to detect IFN-γ and TNF-α. The specific steps were referred to the instruction manual.

[0109] Experimental results of Example 2

[0110] 2.1 Stable introduction of RSPO1 and WNT3A components and endogenous controllable and continuous stable activation of the Wnt signaling pathway through the Tet-On lentiviral system

[0111] The normally cultured organoids were harvested into a 15 mL centrifuge tube, centrifuged at 200×g for 5 min, and then the supernatant was discarded. The centrifuge tube was placed on ice for standby; according to the titer determination results, an appropriate amount (MOI = 50) of lentivirus Lenti-EFS-TetOn-RSPO1-P2A-WNT3A-TRE (SEQ ID NO: 25) was added to the organoid expansion medium to achieve the co-expression of the two genes ( Figure 1A). The resuspension containing organoids and lentivirus was directly added to a 48-well plate. The edges of the plate were sealed with parafilm and centrifuged at 600×g for 60 min at room temperature in a plate centrifuge. The plate was taken out, the parafilm was removed, and it was transferred to a 37 °C cell culture incubator for continued culture for 4 h. The cells were harvested into a 15 mL centrifuge tube, centrifuged and the precipitate was taken according to the organoid passage procedure, resuspended with Matrigel and plated.

[0112] Using a laser confocal microscope, the surface structure and fluorescence expression of intact organoids were observed in a state close to in vivo ( Figure 1 B), and the infection success rate was detected by flow cytometry ( Figure 1 C). Dox (doxycycline) was added to induce the expression and secretion of RSPO1 and WNT3A, thereby maintaining the growth of transgenic organoids. Higher concentrations of RSPO1 and WNT3A cytokines could be detected in the supernatant of the incomplete medium containing doxycycline (amplification medium without RSPO1 and WNT3A added) ( Figure 1 E), and the morphological structure, average size and relative cell viability of transgenic organoids were comparable to those of traditional organoids ( Figure 1 D, F).

[0113] 2.2 Comparison of the histochemical characteristics consistency between Dox-regulated transgenic organoids and source tissues

[0114] Immunohistochemical results showed that the expression levels of E-Cadherin and EGFR in the two groups of transgenic organoids were highly consistent with the results reported by the pathology department of our hospital for the tumor tissues from which they were derived (Table 1) ( Figure 2 A, B), indicating that the transgenic organoids cultured by this method have similarity with the source tissues in terms of histochemical characteristics.

[0115] Table 1 Immunohistochemical detection results of tumor tissues corresponding to representative organoids in the pathology department of our hospital

[0116]

[0117] 2.3 Screening of pancreatic cancer antigen tumor-reactive TCRs using autologous transgenic organoids

[0118] KRAS G12D Mutation is one of the most common driver mutations in pancreatic cancer and is involved in the mechanisms of tumorigenesis, development and drug resistance. The inventors obtained PBMCs from HLA-A&KRAS G12D genotyped PDAC patients and cultured them in vitro under the following 3 conditions respectively: anti-CD28 (Biolegend, 302934), anti-CD28 + autologous transgenic organoids, anti-CD28 + KRAS G12D(SEQ ID NO: 26) Polypeptide, where the anti-CD28 group is for non-specific PBMC amplification. After 10 days, sequencing samples of the original unamplified PBMC and three PBMC amplifications (stimulating cell proliferation and enrichment by adding anti-CD3, autologous organoids, and polypeptide respectively) were prepared for scRNA and TCR-seq analysis ( Figure 3 A). The sequencing results showed that among 25,382 pairs of matched full-length TCRα / β clonotypes, the characteristic frequencies of 83 TCRs enriched by autologous organoids, 195 TCRs enriched by polypeptide, and 112 TCRs enriched by the anti-CD3 group exceeded 0.05%. Boolean operation showed that there were 4 TCRs enriched by both autologous organoids and polypeptide, and 1 TCR enriched by autologous organoids, polypeptide, and anti-CD3 ( Figure 3 B). To determine the antigen / tumor specificity of the candidate TCRs, the inventor synthesized 3 TCRs (TCR 1, 2, 3) enriched by both organoids and polypeptide and 1 non-specific reference (TCR 4), and tested their reactivity to DC cells co-incubated with polypeptide and autologous organoids. Among them, DC cells were generated using the CellXVivo human monocyte-derived DC differentiation kit (R&D systems CDK004). CD14+ monocytes were isolated from PBMC and differentiated into immature DC cells within one week using GM-CSF and IL4; the immature DC cells were incubated with 20 μg / mL peptide and induced into mature DC cells within 3 days using GM-CSF and TNFα.

[0119] The results showed that TCR 1 and 3 had a response to the mutant peptide (KRAS G12D ), but no response to the wild-type polypeptide ( Figure 3 C). TCR 2 and 3 had a response to autologous organoids and induced IFN-γ secretion ( Figure 3 D). In addition, the MHC I antibody blocked the response of TCR3 to autologous organoids, indicating that the tumor response was MHC I-dependent ( Figure 3 E).

[0120] The CDR sequence information of TCR1-4 is shown in Table 2 below.

[0121] Table 2 CDR amino acid sequences and SEQ ID NOs of TCR

[0122]

[0123] 2.4 Identification of neoantigen and detection of anti-tumor function of TCR-3

[0124] The tumor tissue (300×) and PBMCs (100×) of the patient were subjected to whole exome sequencing (WES) using the Illumine HiSeq X10 platform, and RNA sequence analysis was performed on the tumor tissue. Somatic mutations in the tumor were determined by Mutect2 in the GATK bundle. HLA haplotypes were determined by HLAcount with reference to the IMGT / HLA database. Neoantigen prediction was performed by MuPeXI, and the prediction results were sorted by priority score, which represents a combination of HLA binding affinity (with wild-type and mutant polypeptides), allele frequency, mRNA expression level, and cancer driver function. Subsequently, GenScript synthesized wild-type and mutant TP53 peptides using standard solid-phase synthetic peptide chemistry methods and confirmed them using HPLC-MS, obtaining the candidate neoantigen TP53 Mu (TSPPATATW, SEQ ID NO: 27). Peptide / autologous transgenic organoid-T cell co-culture was used to evaluate the tumor killing efficiency of tumor-reactive TCR-3-T cells ( Figure 4 A). TCR-3 was reactive to the mutant peptide TP53 Mu but not to the wild-type polypeptide TP53 WT ( Figure 4 B). In addition, the expression of TNFRSF9 (4-1BB / CD137, a known activation marker recognized by CD8 + T cells for mutant recognition) was significantly upregulated in tumor-reactive TCR-T cells under the condition of incubation with autologous transgenic organoids ( Figure 4 C). The above results indicate that in the case of insufficient TILs, using autologous transgenic organoids for multi-group parallel single-cell TCR-seq of original PBMCs and anti-CD3 / peptide / autologous organoid-enriched PBMCs, combined with MuPeXI neoantigen prediction, and efficient multi-dimensional screening based on Boolean operations, is an effective strategy for obtaining tumor-reactive TCRs for PDAC patients ( Figure 4 D)

[0125] The above shows and describes the basic principles, main features, and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claims involved.

[0126] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment contains only one independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Method for constructing tumor transgenic organoids, characterized in that, Comprising introducing the RSPO1 and / or WNT3A gene into tumor organoids.

2. The method according to claim 1, wherein the tumor is pancreatic cancer.

3. The method according to claim 1 or 2, wherein the method of introduction is selected from one or more of viral vector transfection, electroporation, liposome transfection, polymer transfection, and microinjection; preferably, the introduction is by viral vector transfection; more preferably, the viral vector is a lentivirus, adenovirus, or retrovirus; more preferably, the lentivirus is the Tet-On system.

4. The method according to any one of claims 1-3, further comprising adding doxycycline to the organoid culture system to induce the expression and / or secretion of RSPO1 and / or WNT3A.

5. Tumor autologous transgenic organoids obtained by the method according to any one of claims 1-5.

6. Autologous transgenic tumor organoids, characterized in that, Comprising the introduced RSPO1 and / or WNT3A gene; preferably, the tumor is pancreatic cancer.

7. A method for screening tumor antigen-reactive TCRs using the tumor autologous transgenic organoids according to claim 5 or 6; preferably, the tumor is pancreatic cancer.

8. The method according to claim 7, comprising screening pancreatic cancer antigen tumor-reactive TCRs from the PBMC of pancreatic cancer patients.

9. The method according to claim 7 or 8, screening TCRs that are double-enriched with organoids and antigen polypeptides as candidate TCRs; preferably, the antigen polypeptide is KRAS G12D polypeptide.

10. TCRs obtained by the method according to any one of claims 7-9.

11. Use of the TCR according to claim 10 in the preparation of TCR-T cells.

12. Use of the TCR according to claim 10 in the determination of pancreatic cancer neoantigens.

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