A car-ilc2 cell, immunotherapy method and application thereof

Abstract

The application belongs to the technical field of biological medicine and molecular biology, and particularly relates to a CAR-ILC2 cell, an immunotherapy method and application thereof. Specifically, the application takes MAdCAM-1 specifically up-regulated and expressed by intestinal inflammation lesion vascular endothelial cells as a targeted antigen, constructs a chimeric antigen receptor, and modifies ILC2 cells to obtain CAR-ILC2 cells. The CAR-ILC2 cells utilize the biological functions of ILC2 cells in naturally secreting Areg and other cytokines, promoting intestinal epithelial repair, and relieving colitis, and the specific targeting ability of the CAR structure to MAdCAM-1, so that the CAR-ILC2 cells can accurately target intestinal inflammation lesion sites and play the roles of intestinal injury repair and anti-inflammation, and therefore have good practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to a CAR-ILC2 cell immunotherapy method and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Enteritis, especially inflammatory bowel disease (IBD), is a chronic, relapsing inflammatory bowel disease, primarily including ulcerative colitis (UC) and Crohn's disease (CD). Existing treatments, such as aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and anti-TNF-α antibodies, can control inflammation and alleviate clinical symptoms to some extent, but they are insufficient to effectively repair the damaged intestinal epithelial barrier. Furthermore, some patients exhibit primary non-response or secondary drug resistance, and long-term use can lead to adverse reactions such as serious infections and malignant tumors. Therefore, there is an urgent need to develop novel treatment strategies with intestinal epithelial repair capabilities.

[0004] Type II innate lymphoid cells (ILC2) are a type of innate immune cell discovered in recent years. Upon stimulation with IL-25, IL-33, and other stimuli, they can secrete large amounts of cytokines such as areg, IL-5, and IL-13. Among these, areg, as a member of the epidermal growth factor family, can directly promote the proliferation and differentiation of intestinal epithelial stem cells by activating the EGF receptor pathway, accelerating the repair of damaged mucosal barriers. However, the inventors discovered that natural ILC2 cells lack the ability to actively target enteritis lesions, and after intravenous infusion, they are difficult to effectively accumulate at the lesion site, severely limiting their therapeutic efficacy. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides an immunotherapy strategy based on genetically modified ILC2 cells and its application. Specifically, it relates to an ILC2 cell carrying a chimeric antigen receptor (CAR) (CAR-ILC2) and its use in the preparation of drugs for treating intestinal inflammatory diseases, immune diseases, and tissue damage repair. The core of the strategy lies in using ILC2 cells as a carrier and, through genetic engineering, enabling them to express chimeric antigen receptors that specifically recognize one or more disease-related antigens in the intestinal inflammatory microenvironment. This endows ILC2 cells with the ability to actively chemotactically attract, anchor, and remain in the lesion area, while preserving and enhancing the inherent tissue repair and immune regulation functions of ILC2 cells, achieving precise and efficient local treatment.

[0006] More broadly, the intestinal inflammation-related antigens described in this invention include, but are not limited to, adhesion molecules, chemokine receptors, integrins, or other inflammatory markers that are abnormally highly expressed on the surface of vascular endothelial cells, immune cells, or stromal cells at sites of intestinal inflammation. As a typical embodiment of this invention, the target antigen of the chimeric antigen receptor is mucosal addressin cell adhesion molecule 1 (MAdCAM-1)—an adhesion molecule specifically upregulated on vascular endothelial cells in enteritis lesions. By constructing an anti-MAdCAM-1 single-chain antibody (scFv) as the extracellular antigen-binding domain, connecting the transmembrane domain and the intracellular co-stimulatory signaling domain to the ILC2 endogenous signaling adaptor element, a functional CAR structure is formed. This CAR structure is then transduced into ILC2 cells to obtain MAdCAM-1-targeted CAR-ILC2 cells. Based on the strategy of this invention, the antigen target can be replaced with other intestinal inflammation-related molecules or tumor-related antigens, and the scFv sequence in the corresponding CAR structure can be conventionally replaced according to the target antigen without departing from the core concept of this invention.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a CAR-ILC2 cell, wherein the CAR molecule is composed of four parts: A, B, C, and D, wherein A is an antigen recognition domain, B is a hinge region, C is a transmembrane region, and D is an intracellular signaling domain.

[0008] Part A can be configured to use different antigen recognition domains for different targets. Specifically, for inflammatory bowel disease, Part A can be an antigen recognition domain targeting targets such as MAdCAM-1, CD13, CD146, ICAM-1, VCAM-1, E-selectin, P-selectin, CMTM3, αvβ3, and αvβ5. For cancer treatment, Part A can be an antigen recognition domain targeting targets such as CD19, CD20, EGFR, Her2, PSCA, CD123, CEA (carcinoembryonic antigen), FAP, CD133, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, LICAM, VEGFR, CS1, ROR1, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, and CD171. Preferably, the antigen is MAdCAM-1.

[0009] Part B may originate from the hinge region of CD8α, CD28, or IgG. Preferably, the hinge region is the hinge region of CD8α.

[0010] Part C may originate from the transmembrane region of CD8α or CD28. Preferably, the transmembrane region is the transmembrane region of CD8α.

[0011] Part D may originate from any one or more intracellular domains of IL1RAP, IL1RL1, IL17RA, IL17RB, ICOS, and CD3ζ. Preferably, the intracellular signaling domain is the intracellular domain of IL1RL1, IL1RAP, and IL1RL1 combined.

[0012] A second aspect of the invention provides an isolated polynucleotide encoding a chimeric antigen receptor in the aforementioned CAR-ILC2 cells.

[0013] A third aspect of the present invention provides a recombinant vector comprising the above-described polynucleotide.

[0014] A fourth aspect of the present invention provides a pharmaceutical composition comprising any one or more of the following: the above-described polynucleotide, recombinant vector, or the above-described CAR-ILC2 cells.

[0015] A fifth aspect of the present invention provides the use of the above-described CAR-ILC2 cells, polynucleotides, recombinant vectors, or pharmaceutical compositions in the preparation of a medicament.

[0016] The drugs include those for treating inflammatory bowel disease and cancer, with inflammatory bowel disease being preferred.

[0017] A sixth aspect of the present invention provides an immunotherapy for a disease, the immunotherapy comprising administering to a subject the above-described CAR-ILC2 cells, polynucleotides, recombinant vectors, or pharmaceutical compositions.

[0018] The diseases mentioned include cancer and inflammatory bowel disease, without being specifically limited to these.

[0019] A seventh aspect of the present invention provides the use of the above-described CAR-ILC2 cells, polynucleotides, recombinant vectors, or pharmaceutical compositions in the preparation of products for detecting target antigen expression.

[0020] The beneficial technical effects of one or more of the above technical solutions are as follows: The CAR molecules constructed using the above-mentioned technical approach can be efficiently expressed on ILC2 cells, overcoming the limitation of natural ILC2 cells failing to accumulate at the lesion site after reinfusion. In a DSS-induced mouse model of acute colitis, mice in the CAR-ILC2 treatment group experienced less weight loss than the control group, and their colon length recovered significantly. HE staining showed that the intestinal epithelial structure in the treatment group was more intact, with less inflammatory cell infiltration and submucosal edema compared to the control group. Pathological scores were significantly reduced, and biocompatibility was good. This technical approach also provides CAR sequences targeting mouse and human MAdCAM-1, providing a molecular basis for subsequent clinical translation and demonstrating promising application prospects. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is a flow cytometry graph showing the expression efficiency of CAR molecules in ILC2 cells according to the present invention. In the graph, A represents the flow cytometry detection of GFP expression in ILC2 cells; B represents the flow cytometry detection of the linker peptide G4S expression in ILC2 cells.

[0023] Figure 2 This diagram shows the flow cytometry results of the present invention for detecting the specificity of CAR-ILC2 cells binding to MAdCAM-1 protein; where A is the experimental flowchart and B is the flow cytometry result analysis diagram and quantitative results.

[0024] Figure 3 This image shows the therapeutic effect of CAR-ILC2 cells on a DSS-induced mouse model of acute colitis. In the image, A is a schematic diagram of the animal experimental protocol; B is a curve showing the weight change of mice in each group; C is a representative image of the colon of each group and a statistical chart of colon length; D is a representative image of HE-stained colon tissue of each group; and E is a statistical chart of the pathological scores of colon tissue of each group.

[0025] Figure 4 This is a graph showing the results of flow cytometry detection of the specificity of human CAR-ILC2 cells binding to MAdCAM-1 protein according to the present invention.

[0026] Figure 5 This invention uses flow cytometry to detect the expression of cytokines in human CAR-ILC2 cells and control cells. Wherein, A represents IL-5 expression; B represents IL-13 expression; and C represents Areg expression. Detailed Implementation

[0027] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0029] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0030] In a typical embodiment of the present invention, a CAR-ILC2 cell is provided, wherein the CAR molecule is composed of four parts: A, B, C, and D, wherein A is the antigen recognition domain, B is the hinge region, C is the transmembrane region, and D is the intracellular signaling domain.

[0031] Part A can be configured to use different antigen recognition domains for different targets. Specifically, for inflammatory bowel disease, Part A can be an antigen recognition domain targeting targets such as MAdCAM-1, CD13, CD146, ICAM-1, VCAM-1, E-selectin, P-selectin, CMTM3, αvβ3, and αvβ5. For cancer treatment, Part A can be an antigen recognition domain targeting targets such as CD19, CD20, EGFR, Her2, PSCA, CD123, CEA (carcinoembryonic antigen), FAP, CD133, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, LICAM, VEGFR, CS1, ROR1, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, and CD171. Preferably, the antigen is MAdCAM-1.

[0032] Part B may originate from the hinge region of CD8α, CD28, or IgG. Preferably, the hinge region is the hinge region of CD8α.

[0033] Part C may originate from the transmembrane region of CD8α or CD28. Preferably, the transmembrane region is the transmembrane region of CD8α.

[0034] Part D may originate from any one or more intracellular domains of IL1RAP, IL1RL1, IL17RA, IL17RB, ICOS, and CD3ζ. Preferably, the intracellular signaling domain is the intracellular domain of IL1RL1, IL1RAP, and IL1RL1 combined.

[0035] In another specific embodiment of the present invention, the antigen recognition domain includes a single-chain antibody (ScFv) targeting MAdCAM-1, the single-chain antibody including a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1 or SEQ ID NO.2; DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRVSITRDTSKNHFFLKLSSVTTEDTATYYCARASDSDGFAYWGQGTLVTVSA (SEQ ID NO. 1); QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGINWVRQAPGQGLEWMGWISVYSGNTNYAQKVQGRVTMTADTSTSTAYMDLRSLRSDDTAVYYCAREGSSSSSGDYYYGMDVWGQGTTVTVSS (SEQ ID NO. 2).

[0036] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0037] DILMTQSPSSMSVSLGDTVSFTCHASQGIGRNIGWLQQKPGKSFKGLIYHGTNLKDGVPSRFSGSGSGADYSLTISRIESEDFADYYCIQYVQFPYTFGGGTKLEIK (SEQ ID NO. 3); DIVMTQTPLSLSVTPGQPASISCKSSQSLLHTDGTTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQNIQLPWTFGQGTKVEIK (SEQ ID NO. 4).

[0038] In another specific embodiment of the present invention, in the single-chain antibody, the light chain variable region and the heavy chain variable region are connected by a linker peptide; the linker peptide may be (G4S)n, where n is a positive integer, and further, n is less than 10, preferably 3.

[0039] Therefore, the single-chain antibody can be composed of the aforementioned light chain variable region, linker peptide, and heavy chain variable region in series.

[0040] In another specific embodiment of the present invention, the single-chain antibody may be a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of these antibodies, without specific limitation herein.

[0041] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned functional fragments.

[0042] In another specific embodiment of the present invention, The hinge region is the CD8α hinge region; The transmembrane region is the CD8α transmembrane region; The intracellular signaling domain is the IL1RL1 intracellular domain and / or the IL1RAP intracellular domain. When the intracellular signaling domain is the IL1RL1 intracellular domain and the IL1RAP intracellular domain, i.e., a dual intracellular domain, the two can be connected by a linker peptide; the linker peptide can be (G4S)n, where n is a positive integer, and further, n is less than 10, preferably 3.

[0043] The amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.5 or SEQ ID NO.6.

[0044] SSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACD (SEQ ID NO.5); KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO. 6).

[0045] The amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0046] IYIWAPLAGICVALLLSLIITLIKVFWIE (SEQ ID NO.7); IYIWAPLAGTCGVLLLSLVITLKMFWIE (SEQ ID NO. 8).

[0047] The amino acid sequence of the intracellular domain of said IL1RL1 is as shown in SEQ ID NO. 9 or SEQ ID NO. 10.

[0048] VALFWRDIVTPYKTRNDGKLYDAYIIYPRVFRGSAAGTHSVEYFVHHTLPDVLENKCGYKLCIYGRDLLPGQDAATVVESSIQNSRRQVFVLAPHMMHSKEFAYEQEIALHSALIQNNSKVILIEMEPLGEASRLQVGDLQDSLQHLVKIQGTIKWREDHVADKQSLSSKFWKHVRYQMPVPERASKTASVAAPLSGKACLDLKHF (SEQ ID NO. 9); ATLLWRDIAKPYKTRNDGKLYDAYVVYPRNYKSSTDGASRVEHFVHQILPDVLENKCGYTLCIYGRDMLPGEDVVTAVETNIRKSRRHIFILTPQITHNKEFAYEQEVALHCALIQNDAKVILIEMEALSELDMLQAEALQDSLQHLMKVQGTIKWREDHIANKRSLNSKFWKHVRYQMPVPSKIPRKASSLTPLAAQKQ (SEQ ID NO. 10).

[0049] The amino acid sequence of the intracellular domain of said IL1RAP is as shown in SEQ ID NO. 11.

[0050] YRAHFGTDETILDGKEYDIYVSYARNVEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLVVLSPNYVLQGTQALLELKAGLENMASRGNINVILVQYKAVKDMKVKELKRAKTVLTVIKWKGEKSKYPQGRFWKQLQVAMPVKKSPRWSSNDKQGLSYSSLKNV (SEQ ID NO. 11).

[0051] In order to guide the nascent peptide chain into the endoplasmic reticulum for processing and membrane localization, the present invention provides a signal peptide at its N-terminus when preparing the synthetic chimeric antigen receptor. Specifically, the amino acid sequence of the signal peptide is shown in SEQ ID NO.12 (mouse) or SEQ ID NO.13 (human).

[0052] MASPLTRFLSLNLLLLGESIILGSGEA (SEQ ID NO. 12); MALPVTALLLPLALLLHAARP (SEQ ID NO. 13).

[0053] In another specific embodiment of the present invention, an isolated polynucleotide is provided, the polynucleotide encoding the chimeric antigen receptor in the above-mentioned CAR-ILC2 cells.

[0054] Considering the degeneracy of codons, the gene sequence encoding the above antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0055] In another specific embodiment of the present invention, a recombinant vector is provided, the recombinant vector comprising the above-mentioned polynucleotide.

[0056] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector. It can refer to any recombinant polynucleotide construct that can directly or indirectly (e.g., packaged as a virus) introduce the target DNA fragment into host cells for target gene expression via transformation, transfection, or transduction. One type of vector is a plasmid, a circular double-stranded DNA molecule, which can ligate the target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package the target DNA fragment into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus, etc.). After entering the host cell, these vectors can express the target gene.

[0057] According to the present invention, the recombinant expression vector can be a viral vector, including retroviral vectors and lentiviral vectors; more preferably, it is a lentiviral vector, wherein the recombinant expression vector is obtained by inserting a polynucleotide encoding the above-mentioned chimeric antigen receptor into a virus to obtain a recombinant viral expression vector expressing the chimeric antigen receptor.

[0058] In another specific embodiment of the present invention, a pharmaceutical composition is provided, comprising any one or more of the following: the above-mentioned polynucleotide, recombinant vector or the above-mentioned CAR-ILC2 cells.

[0059] The pharmaceutical composition refers to a combination of at least one drug, optionally a pharmaceutically acceptable carrier or excipient, combined together to achieve a specific purpose. In some embodiments, the pharmaceutical composition includes combinations that are separate in time and / or space, provided they can work together to achieve the objectives of the invention. Some pharmaceutical compositions enhance the biological efficacy of the invention or reduce drug side effects by combining several pharmaceutically acceptable ingredients or compounds. Other pharmaceutical compositions aim to promote administration to the organism, facilitate the absorption of the active ingredient, enhance stability or targeting, prolong half-life, and thus better exert the biological efficacy of the invention.

[0060] The pharmaceutical composition further includes at least one of a pharmaceutical excipient, a carrier, and a diluent.

[0061] In another specific embodiment of the invention, the drug can be administered into the body by known means, such as intravenous systemic delivery or local injection into the tissue of interest. Such administration can be performed via single or multiple doses. Those skilled in the art will understand that the actual dose to be administered in this invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0062] In another specific embodiment of the present invention, the drug may be administered to humans or non-human mammals, including mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc., without specific limitations.

[0063] In another specific embodiment of the present invention, the use of the above-mentioned CAR-ILC2 cells, polynucleotides, recombinant vectors or pharmaceutical compositions in the preparation of drugs is provided.

[0064] The drugs include those for treating inflammatory bowel disease and cancer, with inflammatory bowel disease being preferred.

[0065] As mentioned above, the enteritis includes inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. No specific limitations are made here.

[0066] This invention demonstrates through research that the MAdCAM-1-targeting CAR-ILC2 cells provided by this invention can utilize the biological functions of naturally secreted cytokines such as Areg in ILC2 cells to promote intestinal epithelial repair and alleviate colitis. Combined with the specific targeting ability of the CAR structure to MAdCAM-1, CAR-ILC2 cells can precisely target the lesions of enteritis, exerting intestinal damage repair and anti-inflammatory effects. Therefore, the pharmaceutical composition described herein can be used to treat enteritis, especially inflammatory bowel diseases, including ulcerative colitis and Crohn's disease.

[0067] In another specific embodiment of the present invention, an immunotherapy method for a disease is provided, the immunotherapy method comprising: administering the above-mentioned CAR-ILC2 cells, polynucleotides, recombinant vectors or pharmaceutical compositions to a subject.

[0068] The diseases mentioned include cancer and inflammatory bowel disease, without being specifically limited to these.

[0069] In another specific embodiment of the present invention, the use of the above-mentioned CAR-ILC2 cells, polynucleotides, recombinant vectors or pharmaceutical compositions in the preparation of products for detecting target antigen expression is provided.

[0070] The product may be a test kit, a test device, or an equipment, and there is no limitation thereof. The test product may be used to qualitatively or quantitatively detect the expression of target antigens, and may be used for basic research related to target antigens or for clinical applications, such as screening, (auxiliary) diagnosis, monitoring or predicting the progression of diseases mediated by high / low expression of target antigens, etc., and there is no specific limitation thereof.

[0071] The target antigen has been described as above and will not be repeated here. Preferably, the target antigen is MAdCAM-1.

[0072] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The following examples illustrate test methods with specific conditions, which are generally performed under conventional conditions.

[0073] Example Materials Methods 1. Isolation and culture of mouse ILC2 cells (1) Remove the intestinal segment, remove the surface connective tissue and place it in pre-cooled PBS. Cut the intestine longitudinally, clean the internal fecal residue and cut it into small segments of 0.5-1.0cm. Place them in pre-cooled PBS and shake to clean 2-3 times.

[0074] (2) After cleaning, transfer to digestion solution containing 0.5 mM EDTA and 1 M 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and shake for 30 min.

[0075] (3) After the shaking is finished, shake vigorously up and down for 1 minute, filter, transfer the remaining tissue to 5 mL of RPMI 1640 complete culture medium, and shake gently up and down manually for 2 minutes or on a flat plate shaker at low speed for 5 minutes.

[0076] (4) After filtering through a tissue filter, transfer 5 mL of DNase and collagenase digestion solution into a six-well plate and place it in a 37°C carbon dioxide incubator for 1.5 h of digestion.

[0077] (5) After digestion, the digestion solution and tissue were transferred to a 15 mL centrifuge tube and shaken vigorously to obtain single cells. After no colon tissue was visible, the cells were filtered through a 100 μm cell filter, washed with 5 mL PBS, and centrifuged. After centrifugation, the supernatant was removed, and the precipitate was resuspended in 4 mL of 40% Percoll (Stopfan, 17089109). 3 mL of 80% Percoll was added to the bottom of the tube with a dropper and the cells were centrifuged at 2500 rpm for 15 min.

[0078] (6) Obtain the upper, middle and lower three layers of liquid. Use a pipette to draw up the middle cell layer, add 10 mL of PBS for rinsing, and centrifuge. The precipitate is the single lymphocyte of the intestinal lamina propria.

[0079] (7) The lymphocytes obtained above were first transferred to ep tubes for live and dead staining, and stained at room temperature in the dark for 25 minutes.

[0080] (8) After the end, add 400 μL of MACS buffer to each tube, mix thoroughly to wash the cells, centrifuge (1400g, 5min) and remove the supernatant.

[0081] (9) Surface antibody staining: First, use Fc Block (final concentration 1:200, to block non-specific antibodies) to stain at room temperature in the dark for 5 min. At the same time, use Macs buffer to prepare surface antibodies (FITC-Lineage final concentration 1:100, Percp5.5-KLRG1 final concentration 1:400, APC-CD127 final concentration 1:200) and stain at 4℃ in the dark for 25 min.

[0082] (10) After the end, add 400 μL of MACS buffer to each tube, mix thoroughly to wash the cells, centrifuge (Eppendorf, 1400 g, 5 min) and remove the supernatant.

[0083] (11) Add 400-1000 μL of MACS Buffer according to the amount of cell pellet. Before running the flow cytometer, filter the buffer through a 40 μm white nylon mesh and transfer it to the flow cytometer to prevent blockage.

[0084] (12) Use blank sample tubes without any labeled antibodies to determine the cell voltage on the sorting instrument, and adjust the main cell population to be analyzed to the center of the logic gate. Use individually labeled antibody tubes to determine the positive signal position, and at the same time, adjust the compensation to avoid mutual interference between different laser channels to ensure the specificity and accuracy of the results. After adjustment, the sample tubes can be sorted on the machine according to the experimental purpose and situation.

[0085] (13) Cell sorting was performed according to the Lineage-CD127+KLRG1+ cell population strategy. The sorted cells were received in autoclaved ep tubes using RPMI complete medium containing 15% FBS and immediately inverted to mix before being placed on ice.

[0086] (14) After centrifugation at 4℃ (Eppendorf, 1400g, 5min), discard the supernatant and the precipitate is ILC2.

[0087] 2. Plasmid construction and lentivirus production (1) Plasmid construction: Construction of the target mouse MAdCAM-1 plasmid: The antigen recognition domain, hinge region, CD8 transmembrane region, and IL1RL1 intracellular region or immediately following the IL1RAP intracellular region were sequentially linked to synthesize the corresponding target sequence fragments, as shown in SEQ ID NO.14 and SEQ ID NO.15. The target sequence was then ligated into the lentiviral expression vector pLVX-IRES-ZsGreen1 to obtain the CAR target plasmid. The plasmid was extracted by column centrifugation. The two sets of plasmids were named αMAd.IL1RL1 (containing SEQ ID NO.14) and αMAd.IL1RAP (containing SEQ ID NO.15), respectively.

[0088] Construction of the human MAdCAM-1 target plasmid: The antigen recognition domain, hinge region, CD8 transmembrane region, and IL1RL1 intracellular region were sequentially ligated to synthesize the corresponding target sequence fragment (SEQ ID NO.16). The target sequence was then ligated into the lentiviral expression vector pLVX-IRES-ZsGreen1 to obtain the CAR target plasmid. The plasmid was then extracted using column centrifugation.

[0089] SEQ ID NO.14: MASPLTRFLSLNLLLLGESIILGSGEADILMTQSPSSMSVSLGDTVSFTCHASQGIGRNIGWLQQKPGKSFKGLIYHGTNLKDGVPSRFSGSGSGADYSLTISRIESEDFADYYCIQYVQFPYTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRVSITRDTSKNHFFLKLSSVTTEDTATYYCARASDSDGFAYWGQGTLVTVSASSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLIKVFWIEVALFWRDIVTPYKTRNDGKLYDAYIIYPRVFRGSAAGTHSVEYFVHHTLPDVLENKCGYKLCIYGRDLLPGQDAATVVESSIQNSRRQVFVLAPHMMHSKEFAYEQEIALHSALIQNNSKVILIEMEPLGEASRLQVGDLQDSLQHLVKIQGTIKWREDHVADKQSLSSKFWKHVRYQMPVPERASKTASVAAPLSGKACLDLKHF SEQ ID NO.15: MASPLTRFLSNLLLLLGESIILGSGEADILMTQSPSSMSVSLGDTVSFTCHASQGIGRNIGWLQQKPGKSFKGLIYHGTNLKDGVPSRFSGSGSGADYSLTISRIESEDFADYYCIQYVQFPYTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIR QFPGNKLEWMGYISYDGSNNYNPSLKNRVSITRDTSKNHFFLKLSSVTTEDTATYYCARASDSDGFAYWGQGTLVTVSASSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLIKVFWIEVALFWRDIVTPPYKTRNDGKLYDAYII YPRVFRGSAAGTHSVEYFVHHTLPDVLENKCGYKLCIYGRDLLPGQDAATVVESSIQNSRRQVFVLAPHMMHSKEFAYEQEIALHSALIQNNSKVILIEMEPLGEASRLQVGDLQDSLQHLVKIQGTIKWREDHVADKQSLSSKFWKHVRYQMPVPERASKTASVAAPLSGKACLDLKHFGGGGGSGG GSGGGGSYRAHFGTDETILDGKEYDIYVSYARNVEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRRLVVLSPNYVLQGTQALLELKAGLENMASRGNINVILVQYKAVKDMKVKELKRAKTVLTVIKWKGEKSKYPQGRFWKQLQVAMPVKKSPRWSSNDKQGLSYSSLKNV SEQ ID NO.16: MALPVTALLLPLALLLHAARPDIVMTQTPLSLSVTPGQPASISCKSSQSLLHTDGTTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQNIQLPWTFGQGTKVEIKGGG GSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGINWVRQAPGQGLEWMGWISVYSGNTNYAQKVQGRVTMTADTSTSTAYMDLRSLRSDDTAVYYCAREGSSSSGDYYYGMDVWGQGTTVTVSSK PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLKMFWIEATLLWRDIAKPYKTRNDGKLYDAYVVYPRNYKSSTTDGASRVEHFVHQILPDVLENKCGYTLCI YGRDMLPGEDVVTAVETNIRKSSRRHIFILTPQITHNKEFAYEQEVALHCALIQNDAKVILIEMEALSELDMLQAEALQDSLQHLMKVQGTIKWREDHIANKRSLNSKFWKHVRYQMPVPSKIPRKASSLTPLAAQKQ (2) Virus emergence: After mixing the control plasmid, αMAd.IL1RL1, αMAd.IL1RAP and packaging plasmid, they were added to Lipo2000 and incubated together. Then, they were co-transfected into 293T cells. The cell supernatant was collected and filtered after 48 h and 72 h.

[0090] 3. Preparation of CAR-ILC2 cells The expanded ILC2 cells were seeded into 24-well plates at a density of 200,000 per well, along with 1 mL of virus solution. The plates were then centrifuged (Eppendorf, 1400g, 3 h, 30℃). After 24 h, the medium was replaced with fresh medium, and the plates were incubated. The positivity rate was measured after 48 h.

[0091] Experimental results 1. CAR infection efficiency detection Cell washing: Collect cells in a flow cytometry tube, add 2 mL of PBS washing buffer containing 1% BSA, vortex to mix, centrifuge at 300g for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of washing buffer.

[0092] Antibody incubation: Add an appropriate volume of antibody according to the antibody instructions. Some CAR molecules in this invention contain a (G4S)3 linker in their extracellular recognition region, and the lentiviral vector carries a GFP fluorescent tag. Therefore, PE-Labeled Monoclonal Anti-G4S linker (Acro) or GFP is used to detect CAR expression. Washing procedure: Add 2 mL of washing buffer, vortex to mix, centrifuge at 300g for 5 minutes, discard the supernatant, and repeat this step once to thoroughly remove unbound antibodies. Sample preparation and detection: Resuspend cells in 300 μL of washing buffer, filter through a 300-mesh nylon filter, and transfer to a detection tube for immediate analysis (if delayed detection is required, it is recommended to store at 4°C protected from light for no more than 6 hours).

[0093] The results showed that various CAR molecules were highly expressed in ILC2 cells. Figure 1 ).

[0094] 2. CAR-ILC2 cells specifically bind to the MAdCAM-1 target. CAR-ILC2 cells were co-incubated with biotin-labeled mouse MAdCAM-1 protein for 1 h, followed by washing with PBS. Then, streptavidin-labeled fluorescent secondary antibody (Streptavidin PE-Cyanine, eBioscience) was added, and staining was performed for 1 h. Figure 2 A). Wash twice with PBS before instrumental analysis. Results showed that, compared with the unstained control group and the group stained with streptavidin-labeled fluorescent secondary antibody (Isotype), CAR-ILC2 cells (αMAd.IL1RL1) specifically bound to the MAdCAM-1 target. Figure 2 B).

[0095] 3. Establishment and treatment of experimental colitis model in mice: Establishing a DSS-induced mouse model of acute colitis Twelve 6-week-old male C57BL / 6J mice were housed in an SPF-grade environment for one week, during which time they were given 3% dextran sulfate sodium (DSS) in their drinking water to establish an ulcerative colitis model.

[0096] To investigate the therapeutic effect of engineered ILC2 in colitis, three groups were set up: a PBS group, a Control group, and a CAR-ILC2 cell therapy group.

[0097] In the αMAd.IL1RL1 treatment group, CAR-ILC2 cells (200,000 cells per mouse) were infused via tail vein on days 1, 3, and 5. Mouse weight was measured daily in each group, and mice were sacrificed on day 7. Colons were collected for evaluation, and pathological scoring was performed after HE staining. All measured mouse weights were statistically analyzed using GraphPad Prism 9 software to plot weight change curves.

[0098] The control group received 200,000 control ILC2 cells via tail vein infusion on days 1, 3, and 5. Mice in each group were weighed daily, and sacrificed on day 7. Colons were collected for evaluation, and pathological scoring was performed after HE staining. All mouse weights were statistically analyzed using GraphPad Prism 9 software to plot weight change curves. Lentiviral cells were constructed using the empty vector pLVX-IRES-ZsGreen 1 and transfected into ILC2 cells to obtain control ILC2 cells.

[0099] In the PBS group, 100 µL of control PBS was reinfused via the tail vein on days 1, 3, and 5. Mice in each group were weighed daily, and sacrificed on day 7. Colons were collected for evaluation, and pathological scoring was performed after HE staining. All measured mouse weights were statistically analyzed using GraphPad Prism 9 software to plot weight change curves.

[0100] In this invention, the experimental scheme is illustrated as follows: Figure 3 As shown in Figure A.

[0101] Mouse weight as Figure 3 As shown in Figure B, the treatment group experienced less weight loss compared to the control group. The colon images also show that the colon in the gene-modified ILC2 treatment group was significantly longer than the control group. Figure 3 C). HE staining results showed that, compared with the control group, the intestinal epithelial structure of the gene-modified ILC2 treatment group was more intact, with less inflammatory cell infiltration and less submucosal edema. Figure 3 D). Pathological scoring was performed according to the literature "Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines". Higher scores indicate more severe intestinal tissue damage. Statistical analysis of pathological scores showed that, compared with the control group, infusion of genetically modified ILC2 significantly alleviated colitis in mice. Figure 3 E).

[0102] 4. Human CAR-ILC2 cells specifically bind to the human MAdCAM-1 target. Human CAR-ILC2 cells were co-incubated with biotin-labeled human MAdCAM-1 protein for 1 h, followed by washing once with PBS. Then, streptavidin-labeled fluorescent secondary antibody (Streptavidin PE-Cyanine, eBioscience) was added, and staining was performed for 1 h. After washing twice with PBS, the cells were ready for analysis. Results showed that, compared to the unstained control group and the group stained only with streptavidin-labeled fluorescent secondary antibody (Isotype), human CAR-ILC2 cells (human αMAd.IL1RL1) specifically bound to the MAdCAM-1 target. Figure 4 ).

[0103] 5. Detection of human CAR-ILC2 cell function Human CAR-ILC2 cells (human αMAd.IL1RL1) and control ILC2 cells were stimulated with medium containing phorbol ester and iomycin for 4 h, respectively, followed by the addition of brevidin A and monensin for the next 2 h. Cells were collected after 4 h for flow cytometry antibody staining to assess CAR-ILC2 cell function. The results show that... Figure 5 Compared with Control, human αMAd.IL1RL1 expressed higher levels of IL-5, IL-13, and Areg, indicating that the modified human CAR-ILC2 cells have stronger clinical therapeutic potential.

[0104] Matters not covered in this invention are common knowledge.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A CAR-ILC2 cell, characterized in that, The CAR molecule is composed of four parts: A, B, C, and D. Among them, A is the antigen recognition domain, B is the hinge region, C is the transmembrane region, and D is the intracellular signaling domain. For inflammatory bowel disease, A is an antigen recognition domain targeting MAdCAM-1, CD13, CD146, ICAM-1, VCAM-1, E-selectin, P-selectin, CMTM3, αvβ3, and αvβ5. For tumors, A is an antigen recognition domain targeting CD19, CD20, EGFR, Her2, PSCA, CD123, CEA, FAP, CD133, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, LICAM, VEGFR, CS1, ROR1, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, and CD171. Preferably, the antigen is MAdCAM-1.

2. The CAR-ILC2 cells as described in claim 1, characterized in that, Part B is the hinge region of CD8α, CD28, and IgG; preferably, the hinge region is the hinge region of CD8α. Part C is the transmembrane region of CD8α and CD28; preferably, the transmembrane region is the transmembrane region of CD8α; Part D is any one or more intracellular domains of IL1RAP, IL1RL1, IL17RA, IL17RB, ICOS, and CD3ζ; preferably, the intracellular signaling domain is the intracellular domain of IL1RL1, IL1RAP, and IL1RL1 combined.

3. The CAR-ILC2 cells as described in claim 1, characterized in that, The antigen recognition domain includes a single-chain antibody targeting MAdCAM-1, and the single-chain antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1 or 2; The amino acid sequence of the light chain variable region is shown in SEQ ID NO.3 or 4; Furthermore, in the single-chain antibody, the light chain variable region and the heavy chain variable region are connected by a linker peptide; the linker peptide is (G4S)n, where n is a positive integer, and more specifically, n is less than 10, preferably 3.

4. An isolated polynucleotide, characterized in that, The polynucleotide encodes the chimeric antigen receptor in CAR-ILC2 cells according to any one of claims 1-3.

5. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 4.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one or more of the following: CAR-ILC2 cells as described in any one of claims 1-3, the polynucleotide as described in claim 4, and the recombinant vector as described in claim 5.

7. The use of the CAR-ILC2 cells according to any one of claims 1-3, the polynucleotide according to claim 4, the recombinant vector according to claim 5, or the pharmaceutical composition according to claim 6 in the preparation of a medicament.

8. The application as described in claim 7, characterized in that, The drug is used to treat inflammatory bowel disease and cancer; preferably inflammatory bowel disease.

9. The use of the CAR-ILC2 cells according to any one of claims 1-3, the polynucleotide according to claim 4, the recombinant vector according to claim 5, or the pharmaceutical composition according to claim 6 in the preparation of a product for detecting target antigen expression.

10. The application as described in claim 9, characterized in that, The product is a test kit, test device, or equipment; the target antigen is preferably MAdCAM-1.

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