Preparation method and application of epcam-car-il7-til cells with strong tumor killing activity
By preparing EpCAM-CAR-IL7-TIL cells, the problems of low expansion efficiency, insufficient targeting, and poor safety of traditional TIL therapy have been solved, achieving a highly efficient and safe tumor-killing effect, which is suitable for the treatment of EpCAM-positive tumors.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING AEONVITAL BIOMEDICINE RES CO LTD
- Filing Date
- 2026-06-26
- Publication Date
- 2026-07-24
AI Technical Summary
Traditional TIL therapy suffers from low amplification efficiency, insufficient targeting, and poor safety, resulting in limited efficacy in the treatment of solid tumors and the risk of systemic toxicity.
EpCAM-CAR-IL7-TIL cells were prepared by genetic engineering. Combined with flow cytometry screening and optimized cytokine culture strategies, TIL cells were endowed with specific targeting ability and autocrine growth signals, reducing dependence on exogenous IL-2 and enhancing tumor killing activity and survival ability.
It significantly improved the expansion efficiency and tumor targeting of TIL cells, reduced treatment toxicity, enhanced tumor killing activity and survival ability, and provided a safer adoptive cell therapy option.
Smart Images
Figure CN122445733A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of anti-tumor cell therapy technology, specifically to a method for preparing and applying EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity. Background Technology
[0002] Tumor-infiltrating lymphocytes (TILs), as a form of personalized adoptive cell transfer therapy (ACT), have shown great clinical potential in the treatment of solid tumors.
[0003] Traditional tumor-infiltrating lymphocyte (TIL) therapy suffers from numerous technical limitations, severely restricting its widespread clinical application. First, the in vitro expansion efficiency of TILs is extremely low, typically requiring 3-4 weeks to obtain the required number of cells for treatment, with limited expansion folds that fail to meet clinical needs. Second, both in vitro expansion and in vivo maintenance of TILs are highly dependent on the addition of high concentrations of exogenous IL-2. This not only leads to abnormal expansion of regulatory T cells, thus suppressing anti-tumor immune responses, but also causes life-threatening serious side effects such as capillary leakage syndrome and hypotension during reinfusion therapy due to the combined systemic administration of high doses of IL-2. Furthermore, while traditional TIL therapy exhibits tumor specificity, its recognition ability relies on the diversity of T cell receptors, making it difficult to achieve highly efficient targeting of specific tumor antigens. Additionally, the short survival time of reinfused cells in vivo hinders the formation of long-term anti-tumor immune memory.
[0004] While chimeric antigen receptor T-cell (CAR-T cell) technology endows T cells with specific targeting capabilities, it still faces challenges in the treatment of solid tumors, such as tumor microenvironment suppression and insufficient T-cell infiltration. Therefore, the persistence and therapeutic efficacy of traditional CAR-T cells in solid tumors are limited. Interleukin-7 has unique biological advantages, promoting T-cell proliferation without causing regulatory T-cell expansion, and is particularly beneficial to the activation and survival of CD8+ T cells, significantly prolonging T-cell survival time in vivo. However, systemic administration of IL-7 still raises safety concerns.
[0005] Therefore, a new approach is needed to fundamentally solve the multiple technical bottlenecks of traditional TIL therapy in terms of expansion efficiency, target specificity, safety, and durability, providing a breakthrough solution for immunotherapy of solid tumors. Summary of the Invention
[0006] Traditional TIL therapy is limited by multiple factors: on the one hand, traditional methods rely on high concentrations of IL-2 for in vitro expansion and in vivo maintenance of T cell function, which can easily cause severe systemic toxicity and may lead to T cell exhaustion and functional suppression; on the other hand, natural TILs have weak targeting and insufficient survival ability in the tumor microenvironment, making it difficult to further improve their persistence and anti-tumor effect in vivo, thus limiting their efficacy and widespread application. To address the shortcomings of traditional TIL therapy, this application provides a method for preparing and applying EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity. The EpCAM-CAR-IL7-TIL cells prepared by this method effectively improve the targeting, expansion efficiency, and anti-tumor effect of TILs, and can thus be effectively applied in the treatment of EpCAM-positive tumors.
[0007] Therefore, the first aspect of this application provides a method for preparing EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity, the method comprising the following steps: S1, Prepare a recombinant lentivirus containing a target gene, wherein the target gene includes an EpCAM-CAR sequence and a constitutively expressed IL-7 sequence; S2, Infiltrating lymphocytes were isolated from tumor tissue, and IL-2 was added to culture the cells to the logarithmic growth phase to obtain infiltrating lymphocytes in the logarithmic growth phase; S3, the recombinant lentivirus was added to the infiltrating lymphocytes in the logarithmic growth phase, and after culturing for 2-4 days, the cells were collected and subjected to double labeling staining with FITC-EpCAM recombinant protein and anti-IL-7 flow cytometry antibody; S4. EpCAM-CAR and IL-7 double-positive cell populations were sorted by flow cytometry to obtain EpCAM-CAR-IL7-TIL cells; S5, the EpCAM-CAR-IL7-TIL cells are maintained and expanded in a medium containing IL-2 and IL-10.
[0008] In this application, EpCAM-CAR-IL7-TIL cells are prepared by introducing a chimeric antigen receptor (CAR) targeting epithelial cell adhesion molecule (EpCAM) and the interleukin-7 (IL-7) gene into tumor-infiltrating lymphocytes through genetic engineering.
[0009] The core innovation of EpCAM-CAR-IL7-TIL cells lies in integrating the specific targeting ability of CARs, the natural tumor homing properties of TILs, and the survival-promoting function of IL-7 to form a synergistic therapeutic system. By introducing an EpCAM-specific CAR structure, TIL cells are not only endowed with the ability to precisely recognize and kill EpCAM-positive tumor cells, but also significantly enhanced T cell activation intensity, overcoming the deficiency of insufficient targeting in traditional TIL therapies. Simultaneously, through genetic engineering, EpCAM-CAR-IL7-TIL cells continuously express cell membrane-bound IL-7, achieving autocrine growth signal support and eliminating dependence on exogenous high-concentration IL-2. Cell membrane-bound IL-7 can exert its effects locally, avoiding the toxic reactions caused by systemic cytokine release, significantly improving treatment safety. This innovative design fundamentally solves multiple technical bottlenecks in traditional TIL therapies in terms of expansion efficiency, targeting specificity, safety, and durability, providing a breakthrough solution for immunotherapy of solid tumors.
[0010] In addition, the key innovative highlights of this application include the following two points: First, a highly efficient and precise functional positive cell screening system: Utilizing a dual staining technique of FITC-labeled EpCAM recombinant protein (FITC-EpCAM recombinant protein) and anti-IL-7 flow cytometry antibody, double-positive TIL cells simultaneously expressing functional EpCAM-CAR and secreting IL-7, i.e., EpCAM-CAR-IL7-TIL cells, are directly screened via flow cytometry. This innovative screening method not only achieves precise assessment of transduction efficiency but also ensures that the obtained cell population simultaneously possesses EpCAM target recognition ability and the function of self-secreting IL-7, fundamentally guaranteeing the quality uniformity and functional integrity of the final product. Second, an optimized cytokine combination culture strategy: After obtaining the double-positive cell population through flow cytometry sorting, a synergistic culture protocol of IL-2 and IL-10 is adopted in the subsequent expansion culture stage. Specifically, a low concentration of IL-2 is continuously added to maintain basic cell proliferation signals, while IL-10 is innovatively added to maintain cell viability. This culture protocol significantly enhances the adaptability and survival of EpCAM-CAR-IL7-TIL cells in the immunosuppressive tumor microenvironment while ensuring rapid cell proliferation.
[0011] This application introduces an EpCAM-CAR structure, enabling TILs to specifically recognize and kill various epithelial-derived tumor cells with high EpCAM expression, such as breast cancer, lung cancer, and colorectal cancer. By establishing a flow cytometry-based double-positive cell screening system, the functional consistency of the final product cells is ensured. Furthermore, by combining a synergistic culture strategy with IL-2, autocrine cell membrane-bound IL-7, and IL-10, cell viability and anti-tumor function are significantly enhanced while maintaining cell expansion efficiency.
[0012] In some embodiments, the target gene is an EpCAM-CAR-IL7 fusion sequence comprising the EpCAM-CAR sequence and a constitutively expressed IL-7 sequence, wherein the EpCAM-CAR sequence and the constitutively expressed IL-7 sequence are linked by a P2A polypeptide sequence.
[0013] In this application, the EpCAM-CAR sequence includes an anti-EpCAM single-chain antibody sequence, a transmembrane region, and an intracellular signaling domain (such as CD3ζ and co-stimulatory molecules CD28 or 4-1BB), and the IL-7 sequence is a codon-optimized sequence.
[0014] In practical applications, the constructed EpCAM-CAR-P2A-IL7 fusion sequence was systematically optimized: using the known EpCAM-CAR sequence as the molecular basis, it was linked to the codon-optimized IL-7 sequence via a P2A self-cleaving peptide linker. The codon optimization of the IL-7 sequence included, but was not limited to: adopting human codon usage preferences, adjusting the GC content to a suitable level, constructing a stable mRNA secondary structure, eliminating repetitive sequences, cryptic splicing sites, and unnecessary restriction enzyme sites, while preventing the depletion of the cellular tRNA library. Specifically, the introduction of the P2A self-cleaving peptide linker enabled efficient dissociation of EpCAM-CAR and IL-7 at the translational level, ensuring that both functional proteins could fold independently and correctly while maintaining their complete biological activity. Through this optimized design, the co-expression efficiency of EpCAM-CAR and IL-7 in TIL cells was significantly improved, laying a solid foundation for obtaining high-quality double-positive cell products.
[0015] In some preferred embodiments, the EpCAM-CAR sequence is shown in SEQ ID No:1, the constitutively expressed IL-7 sequence is shown in SEQ ID No:2, and the EpCAM-CAR-IL7 fusion sequence is shown in SEQ ID No:3.
[0016] In some embodiments, in step S1, the method for preparing the recombinant lentivirus is as follows: obtaining and synthesizing the target gene, inserting the target gene into a lentiviral vector to obtain a lentiviral vector linked with the target gene; using the lentiviral vector linked with the target gene to transfect HEK-293T cells for virus preparation and amplification, and obtaining the recombinant lentivirus after culture.
[0017] In this application, the recombinant lentivirus is prepared by cloning the target gene into a lentiviral vector and then transfecting it into HEK-293T cells.
[0018] In some embodiments, in step S2, the cell culture is carried out in a culture environment coated with recombinant human fibrin and CD3 antibody.
[0019] In this application, culturing infiltrating lymphocytes isolated from tumor tissue in a culture environment coated with recombinant human fibrin and CD3 antibody is more conducive to the growth of infiltrating lymphocytes.
[0020] In some embodiments, in step S2, the tumor tissue is selected from either lung tumor tissue or intestinal tumor tissue.
[0021] In some embodiments, in step S3, when the recombinant lentivirus is added to the infiltrating lymphocytes in the logarithmic growth phase, polybrene is added simultaneously before culturing.
[0022] In this application, the simultaneous addition of Polybrene can significantly improve the infection efficiency of recombinant lentivirus on cells and increase the yield of EpCAM-CAR and IL-7 double-positive cells.
[0023] In some embodiments, in step S5, the IL-2 content in the culture medium is 1000-3000 IU / mL, and the IL-10 content is 3000-3500 ng / mL.
[0024] In some specific embodiments, in step S5, the IL-2 content in the culture medium is 2000 IU / mL, and the IL-10 content is 3200 ng / mL.
[0025] This application achieves efficient expansion and functional maintenance of TIL cells by utilizing the synergistic effects of multiple signals, including exogenous low-concentration IL-2, exogenous high-concentration IL-10, and constitutively secreted IL-7.
[0026] In some preferred embodiments, the method for preparing EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity specifically includes the following steps: (1) Preparation of recombinant lentivirus containing a target gene: wherein the target gene is an EpCAM-CAR-IL7 fusion sequence, the EpCAM-CAR-IL7 fusion sequence includes an EpCAM-CAR sequence and a constitutively expressed IL-7 sequence, and the EpCAM-CAR sequence and the constitutively expressed IL-7 sequence are linked by a P2A polypeptide sequence. Specifically, the process involves obtaining and optimizing the synthesis of the EpCAM-CAR-IL7 fusion sequence, linking the EpCAM-CAR-IL7 fusion sequence into a lentiviral vector, transfecting HEK-293T cells for virus preparation and amplification, and then culturing to obtain recombinant lentivirus. The EpCAM-CAR sequence in the EpCAM-CAR-IL7 fusion sequence uses the known EpCAM-CAR sequence as the molecular basis; the codon optimization of the IL-7 sequence includes, but is not limited to: adopting human codon usage preferences, adjusting the appropriate GC content, constructing a stable mRNA secondary structure, eliminating repetitive sequences, hidden splicing sites and unnecessary restriction enzyme sites, while preventing the depletion of the tRNA library in the cell; Preferably, the EpCAM-CAR sequence in the EpCAM-CAR-IL7 fusion sequence is shown in SEQ ID No:1, the IL7 sequence is shown in SEQ ID No:2, and the EpCAM-CAR-IL7 fusion sequence is shown in SEQ ID No:3; (2) After digesting the tumor tissue, a single-cell suspension containing infiltrating lymphocytes was obtained; the single-cell suspension containing infiltrating lymphocytes was cultured with IL-2 to separate and purify the tumor infiltrating lymphocytes; then the purified tumor infiltrating lymphocytes were introduced into a culture environment coated with recombinant human fibrin and CD3 antibody, and IL-2 was added to the purified infiltrating lymphocytes. The cells were cultured until the logarithmic growth phase was reached to obtain infiltrating lymphocytes in the logarithmic growth phase. (3) The recombinant lentivirus was added to the infiltrating lymphocytes in the logarithmic growth phase, and Polybrene was added at the same time. After culturing for 2 to 4 days, the cells were collected and double-labeled with FITC-EpCAM recombinant protein and anti-IL-7 flow cytometry antibody. (4) EpCAM-CAR and IL-7 double-positive cell populations were sorted by flow cytometry to obtain EpCAM-CAR-IL7-TIL cells; (5) The sorted double-positive EpCAM-CAR-IL7-TIL cells were maintained and expanded under culture conditions containing 1000-3000 IU / mL IL-2 and 3000-3500 ng / mL IL-10.
[0027] The preparation method provided in this application involves isolating infiltrating lymphocytes from tumor tissue, seeding them into culture dishes coated with recombinant human fibrin and CD3 antibodies, adding IL-2, culturing to the logarithmic growth phase, using recombinant lentivirus containing the EpCAM-CAR-IL7 fusion sequence to stably transfect TIL cells, culturing for about 72 hours, and then using FITC-EpCAM recombinant protein and anti-IL-7 flow cytometry antibody to perform double-positive cell sorting to obtain fully functional EpCAM-CAR-IL7-TIL cells; the sorted cells are further expanded and cultured under culture conditions containing IL-2 and IL-10, and after expansion to a certain multiple, the cells are harvested and can be used for tumor treatment.
[0028] The second aspect of this application provides EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity prepared by the method described in the first aspect of this application.
[0029] The EpCAM-CAR-IL7-TIL cells obtained by the method described in this application exhibit a 5-10 fold increase in in vitro expansion efficiency compared to traditional methods and demonstrate stronger tumor-targeting killing activity. During reinfusion therapy, the required IL-2 infusion dose is significantly reduced, alleviating treatment-related toxicity and improving clinical safety.
[0030] The third aspect of this application provides the use of EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity prepared by the method described in the first aspect of this application in the preparation of anti-EpCAM positive tumor therapeutic drugs.
[0031] The EpCAM-CAR-IL7-TIL cells obtained by the method described in this application not only significantly enhance the targeting and killing power of TILs, but also ensure the functional uniformity of cell products through an innovative double-positive flow cytometry sorting system. Combined with a culture strategy of continuous IL-2 supply, cell membrane-bound IL-7 autocrine secretion, and IL-10 synergy, this provides a new generation of efficient, safe, and highly adaptable and durable solution for adoptive cell therapy of solid tumors, and can be effectively applied in the preparation of anti-EpCAM positive tumor therapeutic drugs.
[0032] The beneficial technical effects of this application are as follows: The EpCAM-CAR-IL7-TIL cells prepared by the method described in this application contain an EpCAM-CAR-IL7 fusion sequence, which combines the EpCAM-CAR sequence with a constitutively expressed IL-7 sequence. EpCAM-CAR endows TIL cells with specific targeted killing ability, while constitutive expression of IL-7 provides autocrine growth signals, reducing dependence on exogenous high-concentration IL-2 in the later stages of culture; it also reduces the number of cells to be reinfused. When TILs are reinfused into patients, the infusion of high-dose IL-2 can also be reduced; maintaining an appropriate concentration of IL-2 is sufficient to support cell proliferation and activity in vivo, effectively avoiding the systemic toxicity of high-concentration IL-2 infusion. Furthermore, the membrane-bound IL-7 used in this application can exert its effects locally, avoiding the toxic reactions caused by systemic cytokine release, significantly improving treatment safety, and can be effectively applied in the preparation of anti-EpCAM positive tumor therapeutic drugs. Attached Figure Description
[0033] Figure 1 Image showing the ELISA results of IL-7 secreted by transgenic TILs from non-small cell lung cancer. Figure 2 Number of TIL cells obtained by different culture methods; Figure 3 The fold expansion of TIL cells obtained by different culture methods; Figure 4 Figures showing the results of TIL killing target cells obtained by different culture methods; Figure 5 ELISA results of IL-7 secreted by transgenic TILs derived from colorectal cancer; Figure 6 Number of TIL cells obtained by different culture methods; Figure 7 The fold expansion of TIL cells obtained by different culture methods; Figure 8 The results of TIL killing target cells obtained by different culture methods are shown in the figure. Detailed Implementation
[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.
[0035] This application provides a method for preparing EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity, as detailed below: 1) Preparation of recombinant lentiviruses containing the target gene: Specifically, the process involves: obtaining and optimizing the synthesis of the EpCAM-CAR-IL7 fusion sequence; ligating the EpCAM-CAR-IL7 fusion sequence into a lentiviral vector; transfecting HEK-293T cells for virus preparation and amplification; and culturing the vector to obtain a recombinant lentivirus. The EpCAM-CAR-IL7 fusion sequence comprises an EpCAM-CAR sequence and a constitutively expressed IL-7 sequence, and the EpCAM-CAR sequence and the constitutively expressed IL-7 sequence are linked by a P2A polypeptide sequence. The EpCAM-CAR sequence is shown in SEQ ID No:1; the IL7 sequence is shown in SEQ ID No:2; and the EpCAM-CAR-IL7 fusion sequence is shown in SEQ ID No:3.
[0036] 2) After digesting the tumor tissue, a single-cell suspension containing infiltrating lymphocytes was obtained; the single-cell suspension containing infiltrating lymphocytes was cultured with IL-2 to isolate and purify the tumor infiltrating lymphocytes; then the purified tumor infiltrating lymphocytes were inoculated into a culture environment coated with recombinant human fibrin and CD3 antibody, and IL-2 was added to the purified infiltrating lymphocytes. The cells were cultured until the logarithmic growth phase was reached to obtain infiltrating lymphocytes in the logarithmic growth phase. 3) The recombinant lentivirus was added to the infiltrating lymphocytes in the logarithmic growth phase, along with Polybrene. After culturing for 2-4 days, the cells were collected and subjected to double-labeling staining with FITC-EpCAM recombinant protein and anti-IL-7 flow cytometry antibody. 4) Use flow cytometry to sort the EpCAM-CAR and IL-7 double-positive cell population to ensure that functional transgenic TIL cells are obtained, that is, EpCAM-CAR-IL7-TIL cells are obtained. 5) The sorted double-positive EpCAM-CAR-IL7-TIL cells were expanded and cultured under the conditions of 1000-3000 IU / mL IL-2 and 3000-3500 ng / mL IL-10. The synergistic effect of multiple signals, including exogenous IL-2, constitutively secreted IL-7 and exogenous IL-10, was utilized to achieve efficient expansion and functional maintenance of TIL cells.
[0037] Example 1: Construction of recombinant lentivirus containing EpCAM-CAR sequence, IL7 sequence, or EpCAM-CAR-IL7 fusion sequence 1. Construction of plasmids containing EpCAM-CAR or IL7 or EpCAM-CAR-IL7 gene The CDS sequence of IL7 was downloaded from NCBI, and codon optimization was performed to enhance its expression. These optimization methods included, but were not limited to: adopting human codon usage preferences, adjusting the GC content, constructing a stable mRNA secondary structure, eliminating repetitive sequences, hidden splicing sites, and unnecessary restriction enzyme sites, while preventing the depletion of the cellular tRNA library. The EpCAM-CAR sequence was based on a known molecular sequence. After optimization, the sequence was directly used for gene synthesis, and the pLenti expression plasmid, which was also double-digested, was ligated into the XbaI and SmaI restriction sites. Plasmids that were verified by restriction enzyme digestion and sequencing were named pLenti-EpCAM-CAR, pLenti-IL7, and pLenti-EpCAM-CAR-IL7, respectively. In EpCAM-CAR-IL7, EpCAM-CAR and IL7 are linked by P2A. P2A is a self-cleaving polypeptide sequence that can mediate a skipping reaction during ribosome translation, enabling the co-expression of multiple proteins. P2A enables protein translation to be independent of the 5' cap structure of messenger RNA-mediated initiation mechanism, allowing for the continuous translation of multiple functional proteins on a single RNA strand. Therefore, it can ensure that EpCAM-CAR and IL7 in EpCAM-CAR-IL7 can be correctly expressed as independent active proteins.
[0038] 2. Preparation and titer determination of recombinant lentivirus The pLenti plasmid, pLP1 vector, pLP2 vector, and pLP-VSVG vector containing the EpCAM-CAR, IL7, or EpCAM-CAR-IL7 genes obtained in the above steps were purified by high-level extraction. They were then transfected into HEK-293T cells at a density of approximately 70% using Lipo2000. After 16 hours, the medium was replaced with complete medium. After 48 hours and 72 hours of culture, the supernatant was collected and filtered through a 0.45 μm filter. The filtrate obtained was the stock solution of recombinant lentivirus. This yielded recombinant lentivirus stock solutions containing EpCAM-CAR, IL7, and EpCAM-CAR-IL7R, respectively.
[0039] Recombinant lentivirus was concentrated using the Lentivirus Concentration Solution (YEASEN, 41101ES50) lentivirus concentration kit.
[0040] Subsequently, lentivirus titer was determined, and HEK-293T cells were seeded into 24-well plates at 1×10⁶ cells per well. 5The cells were incubated overnight at 37°C. The original solution of the recombinant lentivirus (virus stock solution) was resuspended in DMEM medium, with 10 μL of virus stock solution in the first well. Then, 10-fold serial dilutions were performed, with 100 μL of medium-virus mixture added to each well. Each dilution was used in triplicate. After incubation at 37°C and 5% CO2 for 24 h, the virus-containing DMEM medium was replaced with DMEM complete medium without virus stock solution, and incubation continued for another 48 h. Flow cytometry was used to detect the proportion of transgene-positive cells and calculate the virus titer. The sum of the total number of cells in the three replicate wells was calculated, and the mean was calculated. Let A be the mean number of fluorescent cells in the second-to-last visible fluorescent well and B be the mean number of fluorescent cells in the penultimate visible fluorescent well.
[0041] Lentiviral titer calculation formula: Lentiviral titer (TU / ml) = (A + B) 10) Virus load in 1000 / 2 / A wells.
[0042] Example 2: Preparation of EpCAM-CAR-IL7-TIL cells for non-small cell lung cancer TILs 1. Tissue pretreatment: After wiping the outer surface of the tumor tissue specimen collection tube, reagents, and operating instruments with 75% ethanol, place them in a biosafety cabinet. Inside the biosafety cabinet, open the tumor tissue specimen collection bottle, clean and process the tumor tissue, and remove blood vessels, capsules, and other necrotic tissues.
[0043] 2. Tissue digestion: 1) The tumor tissue was minced into a near-meat-like state in the tissue enzymatic hydrolysate, and then suspended in the tissue enzymatic hydrolysate. The suspension was transferred to a centrifuge tube and placed in an electric thermostatic shaker. The temperature was set at 37°C, the shaking frequency at 150 rpm / min, and the time was 45 min for constant temperature digestion.
[0044] 2) Take 40 mL of the termination protection solution and put it into a 50 mL centrifuge tube for later use. Place a 70 μm filter screen at the mouth of the 50 mL centrifuge tube and prepare a 5 mL disposable syringe for later use.
[0045] 3) Remove the centrifuge tube from the shaker and gradually add it to the filter screen from step 2) using a pipette. Grind and filter the filter with a grinding rod while grinding and rinsing, and collect the single-cell suspension.
[0046] 4) Centrifuge the single-cell suspension collected in step 3) at 500g for 8 minutes and discard the supernatant. Resuspend the cell pellet in 10mL of X-VIVO-15 medium, take a sample for counting, and centrifuge all remaining cells at 500g for 8 minutes.
[0047] 3. Plate culture Centrifugation can remove residual proteins from cell suspensions. Based on the counting results, the cells were then cultured in a CO2 incubator at 37°C with 5% CO2.
[0048] 4. Isolation and purification of TIL cells (S1 stage) 1) Medium change and well expansion: Observe the cells under a microscope in a B+A grade culture room every day. If there are no obvious adherent cells, change the medium every other day. If the number of lymphocytes does not gradually increase or the number of cancer cells does not decrease, continue to change the medium. If the lymphocyte density increases significantly and cell clusters appear, the well expansion operation is required. After the operation is completed, place the well in a 37℃, 5% CO2 incubator for culture.
[0049] 2) Collection: Cells are collected after culturing for no more than 10 days. Cell counts and viability are assessed, and cells are either cryopreserved or proceed to the S2 stage based on the count results. The S1 stage of culture removes tumor cells, fibroblasts, and other contaminating cells unrelated to TILs. Daily microscopic observation of cell growth is used to determine subsequent processing steps. Observation indicators include changes in the number of lymphocytes, tumor cells, and other cells, as well as the degree of lymphocyte segregation.
[0050] 5. Rapid expansion culture of TIL cells (S2 phase) 1) Day 0: Pre-coating: Add coating buffer containing 3 μg / mL CD3 antibody and 12 μg / mL recombinant human fibrin (RN) to the culture flask and coat at 4°C. Before use, aspirate the coating buffer from the culture flask and wash three times with PBS. Take 2 × 10⁻⁶ [units of measurement missing]. 7 The TIL cell transfer culture flasks obtained in step 4 were placed in a CO2 cell incubator at a temperature of 37°C and a CO2 concentration of 5%. The cells were divided into five groups, with each group initially containing 2 × 10⁶ cells. 7 indivual.
[0051] 2) Day 1: Add CTS immune cell serum substitute to each group. Groups 1, 3, 4, and 5 are given 2000 IU / mL IL-2, and group 2 is given 25 ng / mL IL-7. From Day 2 to Day 4, observe the cells under a microscope and take samples for counting. If the number of TIL cells gradually increases, fluid replacement is required to maintain the cell density at approximately 1×10⁻⁶. 6 / mL, and after completion, place in a CO2 cell culture incubator at 37℃ and 5% CO2.
[0052] 3) Day 2: Recombinant lentivirus transfection. On the second day, the recombinant lentivirus containing EpCAM-CAR, IL7, and EpCAM-CAR-IL7 prepared in Example 1 were added to the third, fourth, and fifth groups of TIL cells in logarithmic growth phase, respectively, along with 8 μg / mL polybrene. Three days after transfection, FITC-EpCAM recombinant protein or anti-IL7 flow cytometry antibody was added for labeling and sorting to obtain purified transgenic TIL-positive cells (EpCAM-CAR-TIL cells, IL7-TIL cells, and EpCAM-CAR-IL7-TIL cells).
[0053] 4) Day 14: Supernatant was collected to detect the secretion of IL-7 in each group of cells (IL-7 Human ELISA Kit, Invitrogen, Cat:#EHIL7X5). The results are as follows: Figure 1 As shown.
[0054] like Figure 1 As shown, compared to the control group, both IL7 transgenic experimental groups (transgenic IL-7 group and transgenic EpCAM-CAR-IL7 group) produced large amounts of IL-7. The transgenic IL-7 group and the EpCAM-CAR-IL7 group produced 37100.8 pg / mL of IL-7 and 36217.6 pg / mL of IL-7, respectively. During the medium change process, 2000 IU / mL of IL-2 was added to the first group each time the medium was changed, and 25 ng / mL of IL-7 was added to the second group.
[0055] 5) Days 23-25: Cells were cultured for another 12 days in a medium containing 1000 IU / mL IL-2 and 3200 ng / mL IL-10, at which point the cell count reached the required amount for reinfusion. Cells in each group were counted, and the fold increase was calculated. Results are as follows: Figure 2 and 3 As shown.
[0056] like Figure 2 and 3 As shown, the transgenic method of this application can significantly increase the expansion fold of TIL cells, with the transgenic IL-7 group and the transgenic EpCAM-CAR-IL7 group both reaching an expansion fold of 1800-fold.
[0057] 6. Assay of TIL cell killing function 1) Human non-small cell lung cancer HCC827 cells in the exponential growth phase were stained with trypan blue and counted; 9000 target cells / 100 μL were added to each well of a 96-well plate and incubated overnight at 37°C in a 5% CO2 incubator to allow them to adhere to the plate. 2) Add effector cells (TIL cells harvested in each group in step 5 above, which are TIL cells that have been HLA-matched with the target cells) to the target cell wells at an effector-target ratio of 10:1 and 5:1, and make a corresponding number of blank controls for TIL culture alone; the negative control group uses PBMCs as target cells; then transfer to a 37℃, 5% CO2 incubator and incubate for 24 h. 3) After 24 hours, aspirate 100 μL of supernatant and add 20 μL of MTS reagent per well; 4) Incubate at 37℃ in a 5% CO2 incubator in the dark for 2 hours; 5) Use an ELISA reader to detect the absorbance at 490 nm; 6) Calculate the kill rate at each effective target ratio using the following formula, and the results are as follows: Figure 4 As shown.
[0058] Kill rate (%) = 1 - (OD co-incubation group - ODTIL culture alone group) / (OD tumor cell negative control group - OD blank control group) × 100%.
[0059] Results analysis: such as Figure 4 As shown, PBMCs from healthy individuals, which exhibit no specific killing effect, served as the negative control group, with a theoretical specific killing effect of 0. Regardless of whether the E:T ratio was 10:1 or 5:1, the EpCAM-CAR-IL-7 group showed the strongest killing effect, achieving over 95% killing at E:T=10:1 and decreasing to approximately 80% at E:T=5:1. These results indicate that, compared to TIL cells cultured with ordinary IL-2, transgenic EpCAM-CAR-IL-7-TIL cells possess a stronger ability to kill target cells.
[0060] Example 3: Preparation of colorectal cancer TIL cells The processing and digestion of colorectal cancer tissue, the isolation and purification of TIL cells, and the procedures for the expansion culture stage were all the same as in Example 2. The sorting of transgenic positive cells during the expansion culture stage and the detection of IL-7 expression were also performed using an ELISA kit.
[0061] The results are as follows Figure 5 As shown, the TILs in the two IL7 transgenic experimental groups derived from colorectal cancer also secreted high levels of IL-7. Compared with the control group, the transgenic IL-7 group and the transgenic EpCAM-CAR-IL-7 group produced 38231 pg / mL and 37631.5 pg / mL of IL-7, respectively.
[0062] After the entire S2 phase is completed, the cell number and amplification fold are measured.
[0063] The results are as follows Figure 6 and7 As shown, the transgenic IL-7 group and the transgenic EpCAM-CAR-IL-7 group had the highest TIL cell number, both reaching 2.9 × 10⁻⁶. 10 The number of patients was more than 1,000 times greater than the initial number of inoculations. All groups met the cell therapy infusion dose requirements.
[0064] The killing effect of the prepared colorectal cancer TIL cells was also tested. The colorectal cancer tumor cell line used was HCT116. Similarly, the effector cells TIL and the target cell line were HLA matched.
[0065] Depend on Figure 8 It can be seen that the EpCAM-CAR-IL7-TIL cells cultured by the method described in this application have the strongest tumor-killing activity, which is significantly higher than that of traditional methods. This fully demonstrates that this method is an effective way to improve the killing activity of TIL cells, and the EpCAM-CAR-IL-7-TIL cells prepared by this method have a stronger ability to kill target cells.
[0066] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A method for preparing EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity, characterized in that, The method includes the following steps: S1, Prepare a recombinant lentivirus containing a target gene, wherein the target gene includes an EpCAM-CAR sequence and a constitutively expressed IL-7 sequence; S2, Infiltrating lymphocytes were isolated from tumor tissue, and IL-2 was added to culture the cells to the logarithmic growth phase to obtain infiltrating lymphocytes in the logarithmic growth phase; S3, the recombinant lentivirus was added to the infiltrating lymphocytes in the logarithmic growth phase, and after culturing for 2-4 days, the cells were collected and subjected to double labeling staining with FITC-EpCAM recombinant protein and anti-IL-7 flow cytometry antibody; S4. EpCAM-CAR and IL-7 double-positive cell populations were sorted by flow cytometry to obtain EpCAM-CAR-IL7-TIL cells; S5, the EpCAM-CAR-IL7-TIL cells are maintained and expanded in a medium containing IL-2 and IL-10.
2. The method according to claim 1, characterized in that, The target gene is an EpCAM-CAR-IL7 fusion sequence comprising the EpCAM-CAR sequence and a constitutively expressed IL-7 sequence, wherein the EpCAM-CAR sequence and the constitutively expressed IL-7 sequence are linked by a P2A polypeptide sequence.
3. The method according to claim 2, characterized in that, The EpCAM-CAR sequence is shown in SEQ ID No:1, the constitutively expressed IL-7 sequence is shown in SEQ ID No:2, and the EpCAM-CAR-IL7 fusion sequence is shown in SEQ ID No:
3.
4. The method according to any one of claims 1-3, characterized in that, In step S1, the method for preparing the recombinant lentivirus is as follows: obtaining and synthesizing the target gene, inserting the target gene into a lentiviral vector to obtain a lentiviral vector linked with the target gene; using the lentiviral vector linked with the target gene to transfect HEK-293T cells for virus preparation and amplification, and obtaining the recombinant lentivirus after culture.
5. The method according to any one of claims 1-3, characterized in that, In step S2, the cell culture is carried out in a culture environment coated with recombinant human fibrin and CD3 antibody.
6. The method according to any one of claims 1-3, characterized in that, In step S2, the tumor tissue is selected from either lung tumor tissue or intestinal tumor tissue.
7. The method according to any one of claims 1-3, characterized in that, In step S5, the IL-2 content in the culture medium is 1000-3000 IU / mL, and the IL-10 content is 3000-3500 ng / mL.
8. An EpCAM-CAR-IL7-TIL cell with strong tumor-killing activity prepared by the method of any one of claims 1-7.
9. The use of EpCAM-CAR-IL7-TIL cells with strong tumor-killing activity prepared by any one of claims 1-7 in the preparation of anti-EpCAM positive tumor therapeutic drugs.