Chimeric antigen receptors targeting human epcam, chimeric antigen receptor t cells, and uses thereof

Abstract

The application provides a chimeric antigen receptor targeting human EPCAM, a chimeric antigen receptor T cell and application thereof, the chimeric antigen receptor comprises an extracellular antigen recognition domain, a variable region sequence derived from an anti-EpCAM antibody, specifically a single-chain antibody fragment formed by a VH and a VL derived from a 3-17I anti-EpCAM antibody; a hinge region and a transmembrane region, comprising a hinge region and a transmembrane region derived from CD28; an intracellular signal transduction domain, comprising a CD28 costimulatory signal domain and a CD3 zeta signal domain. Meanwhile, the application also obtains an engineered T cell capable of recognizing and killing EpCAM positive target cells, and verifies that the CAR-T cell has an in-vivo tumor inhibition effect in an MC38-EpCAM transplanted tumor model, and is expected to be applied to a CAR-T therapy for clinical solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology, specifically relating to chimeric antigen receptors targeting human EPCAM, chimeric antigen receptor T cells, and their applications. Background Technology

[0002] Epithelial cell adhesion molecule (EpCAM), also known as CD326, is a 40kD transmembrane glycoprotein. EpCAM is highly expressed in various tumors derived from epithelial cells, including many adenocarcinomas such as those of the colon, stomach, pancreas, lung, ovary, and breast. As a signaling molecule, EpCAM activates the Wnt signaling pathway downstream, thereby regulating the cleavage of intracellular proteins and leading to tumorigenesis. In tumor tissues, EpCAM expression changes from the basal layer to uniform expression on the cell membrane surface, making it more suitable as a target in cell or antibody therapies.

[0003] Although various EpCAM CAR-T construction schemes have been disclosed in the existing technology, the following shortcomings still exist: (1) The antigen-binding domains used to construct EpCAM CARs in the prior art are diverse in origin, and there is a lack of construction schemes based on the variable region of specific anti-EpCAM antibodies and obtaining clear superior results in a specific CAR framework; (2) Although the prior art discloses that co-stimulatory domains such as CD28 or 4-1BB can be used for EpCAM CAR design, the functional differences of different co-stimulatory domain configurations under the same EpCAM antigen-binding domain background, especially the in vitro and in vivo comparative verification, are still not fully disclosed; (3) The prior art lacks a complete technical scheme for constructing an EpCAM CAR based on VH / VL derived from 3-17I and demonstrating its good in vitro killing activity and in vivo tumor suppression effect in mouse T cell system and MC38-EpCAM model; (4) There is still room for further optimization of the integrated protection layout of this type of CAR molecule, nucleic acid, vector, engineered T cells and pharmaceutical use in the prior art. Summary of the Invention

[0004] This invention provides a chimeric antigen receptor (CAR) constructed based on a variable region derived from a specific anti-EpCAM antibody and its engineered T cells, which can stably express and specifically recognize EpCAM-positive target cells in T cells, thereby effectively killing EpCAM-positive tumor cells and inhibiting the growth of EpCAM-positive tumors in vivo.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a chimeric antigen receptor targeting human EPCAM, the chimeric antigen receptor comprising a heavy chain variable region and a light chain variable region of an antibody targeting human EPCAM, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:13-15, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:16-18, respectively.

[0006] Furthermore, the chimeric antigen receptor comprises scFv, the amino acid sequence of which is shown in SEQ ID NO:10.

[0007] Furthermore, the heavy chain variable region also includes FR1, FR2, FR3, and FR4, wherein the amino acid sequences of FR1, FR2, FR3, and FR4 are as shown in SEQ ID NO:19-22 or are at least 80% identical to the amino acid sequences shown in SEQ ID NO:19-22.

[0008] Furthermore, the light chain variable region also includes FR1, FR2, and FR3, and the amino acid sequences of FR1, FR2, and FR3 are as shown in SEQ ID NO:23-25 ​​or are at least 80% identical to the amino acid sequences shown in SEQ ID NO:23-25.

[0009] Furthermore, the heavy chain variable region has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:12.

[0010] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0011] In this invention, the term "chimeric antigen receptor" refers to an artificially constructed fusion receptor molecule that endows immune cells with the ability to recognize and bind target antigens in a non-major histocompatibility complex (MHC)-dependent manner. The chimeric antigen receptor typically includes functional modules such as an extracellular antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular domain. The chimeric antigen receptor can be expressed on T cells, natural killer cells (NK cells), NKT cells, γδT cells, macrophages, or other immune effector cells to mediate an immune response against target cells.

[0012] The term "heavy chain variable region" refers to the variable amino acid sequence region in the antibody heavy chain that is responsible for antigen-specific recognition. These heavy chain variable regions typically include framework regions (FRs) and complementarity-determining regions (CDRs), with CDRs primarily involved in antigen binding and determining the antibody's specificity and affinity.

[0013] The term "light chain variable region" refers to the variable amino acid sequence region in the antibody light chain responsible for antigen recognition. This light chain variable region also includes a framework region and a complementarity-determining region, which, together with the heavy chain variable region, can form an antigen-binding site, thereby achieving specific recognition of the target antigen.

[0014] Furthermore, the chimeric antigen receptor also includes a hinge region.

[0015] Furthermore, the hinge region is selected from CD8α and CD28.

[0016] In a specific embodiment of the present invention, the hinge region is selected from CD28.

[0017] Furthermore, the chimeric antigen receptor also includes a transmembrane domain.

[0018] Furthermore, the transmembrane structural domain is selected from CD8α and CD28.

[0019] In a specific embodiment of the present invention, the transmembrane domain is selected from CD28.

[0020] Furthermore, the chimeric antigen receptor also includes an intracellular domain.

[0021] Furthermore, the intracellular domains include signal transduction domains and co-stimulatory molecular domains.

[0022] In a specific embodiment of the present invention, the signal transduction structure domain is selected from CD3ζ.

[0023] Furthermore, the co-stimulatory molecular domain is selected from one or more of 4-1BB, CD28, OX40, and ICOS.

[0024] In a specific embodiment of the present invention, the co-stimulatory molecular domain is selected from 4-1BB and CD28.

[0025] Furthermore, the chimeric antigen receptor also includes a detection tag.

[0026] The term "detection tag" refers to a labeled sequence, labeled protein, or labeled peptide that can be used to detect, identify, separate, purify, or quantitatively analyze the expression of a target molecule. The detection tag can be a fluorescent protein, enzyme label, antigen tag, or surface marker, such as, but not limited to, RQR8, GFP, EGFP, mCherry, FLAG tag, HA tag, Myc tag, V5 tag, His tag, Strep tag, EGFRt, LNGFR, CD34, or combinations thereof.

[0027] Furthermore, the detection tag is selected from RQR8.

[0028] Furthermore, the chimeric antigen receptor also includes a self-cleaving peptide.

[0029] The term "cleavage peptide" refers to a functional marker sequence or marker protein that is co-expressed with a chimeric antigen receptor, can be specifically recognized, and can be used for the detection, identification, sorting, enrichment, tracing, quantitative analysis, or clearance of target cells. The detection tag can be expressed on the cell surface, intracellularly, or secreted into the extracellular environment, and is preferably a marker molecule that can be expressed on the cell surface and recognized by specific antibodies.

[0030] Furthermore, the self-cleaving peptide is selected from T2A.

[0031] In this invention, the term "hinge region" refers to the connecting region located between the antigen recognition domain and the transmembrane domain. Its main function is to endow the chimeric antigen receptor with appropriate flexibility and spatial extensibility to promote the effective binding between the antigen recognition domain and the target antigen. The hinge region may be derived from, but is not limited to, CD8α, CD28, IgG1, IgG4, or functional variants thereof.

[0032] The term "transmembrane domain" refers to a hydrophobic amino acid sequence region that anchors a chimeric antigen receptor to the cell membrane. This transmembrane domain not only maintains stable receptor expression on the cell membrane but also influences receptor aggregation and signal transduction efficiency. The transmembrane domain may be derived from, but is not limited to, CD8α, CD28, CD4, CD3ζ, ICOS, or functional variants thereof.

[0033] The term "intrinsic domain" refers to a functional region located on the intracellular side of a chimeric antigen receptor that participates in cell activation and signal transduction. This intracellular domain typically includes one or more of a signal transduction domain and a co-stimulatory molecular domain. The signal transduction domain refers to an intracellular signaling element capable of mediating immune cell activation, proliferation, cytokine secretion, and cytotoxic effects. This signal transduction domain typically contains one or more immune receptor tyrosine activation motifs (ITAMs), which may be derived from, but are not limited to, CD3ζ, FcRγ, DAP10, DAP12, or their functional variants, with CD3ζ being one of the most widely used signal transduction domains. The co-stimulatory molecular domain refers to an intracellular functional domain capable of providing auxiliary activation signals to immune cells, thereby enhancing cell proliferation, survival, cytokine secretion, and sustained antitumor activity. This co-stimulatory molecular domain may be derived from, but is not limited to, CD28, 4-1BB (CD137), OX40 (CD134), ICOS, CD27, DAP10, DAP12, or their functional variants.

[0034] A second aspect of the invention provides an isolated polynucleotide encoding the chimeric antigen receptor described in the first aspect of the invention.

[0035] Furthermore, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO:9.

[0036] A third aspect of the present invention provides a recombinant vector comprising the polynucleotides described in the second aspect of the present invention.

[0037] Furthermore, the recombinant vector also includes one or more regulatory elements operatively linked to the polynucleotide.

[0038] Furthermore, the regulatory sequence includes a promoter sequence, a transcription terminator sequence, and a leader sequence.

[0039] Furthermore, the recombinant vector includes DNA vectors, RNA vectors, plasmids, and viral vectors.

[0040] Furthermore, the recombinant vector is a viral vector.

[0041] Furthermore, the viral vector includes retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, and herpesvirus vectors.

[0042] Furthermore, the vector is a retroviral vector; A fourth aspect of the present invention provides a recombinant host cell comprising the polynucleotide described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention.

[0043] Furthermore, the recombinant host cell is selected from eukaryotic cells.

[0044] Furthermore, the eukaryotic cells are selected from animal cells.

[0045] Furthermore, the animal cells are selected from mammalian cells.

[0046] Furthermore, the mammalian cells are selected from immune cells.

[0047] Furthermore, the immune cells include T cells, NK cells, or NKT cells.

[0048] Furthermore, the immune cells are T cells.

[0049] A fifth aspect of the present invention provides a pharmaceutical composition comprising any one of the chimeric antigen receptor described in the first aspect, the polynucleotide described in the second aspect, the recombinant vector described in the third aspect, and the host cell described in the fourth aspect.

[0050] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients. These excipients include, but are not limited to, buffers, stabilizers, cryoprotectants, osmotic pressure regulators, diluents, excipients, and combinations thereof.

[0051] Furthermore, the buffers include, but are not limited to, phosphate buffer, physiological saline, Plasma-Lyte A, lactated Ringer's solution, and combinations thereof; the stabilizers include, but are not limited to, human serum albumin, recombinant human albumin, human serum, and combinations thereof; the cryopreservation protectants include, but are not limited to, dimethyl sulfoxide (DMSO), trehalose, sucrose, dextran, hydroxyethyl starch, and combinations thereof; and the osmotic pressure regulators include, but are not limited to, sodium chloride, glucose, mannitol, and combinations thereof.

[0052] The sixth aspect of the invention provides any of the following methods: (1) A method for preparing recombinant host cells, characterized in that the preparation method includes the step of introducing the polynucleotide described in the second aspect of the present invention or the recombinant vector described in the third aspect of the present invention into a host cell; (2) A method for detecting the expression level of gene EpCAM in sample cells in vitro, characterized in that the method comprises contacting the chimeric antigen receptor described in the first aspect of the present invention or the recombinant host cell described in the fourth aspect with the cell sample to be tested, incubating under conditions suitable for forming antigen-receptor complexes, and detecting the binding rate of the chimeric antigen receptor to the cell sample to be tested to determine the expression level of gene EpCAM. The seventh aspect of the present invention provides any of the following applications: (1) Use of antibody 3-17I in the preparation of chimeric antigen receptor, wherein the amino acid sequence of antibody 3-17I is shown in SEQ ID NO:10; (2) The use of any one of the chimeric antigen receptor described in the first aspect of the present invention, the polynucleotide described in the second aspect, the recombinant vector described in the third aspect, the host cell described in the fourth aspect, the pharmaceutical composition described in the fifth aspect, and the method described in the sixth aspect in the preparation of a drug for treating human solid tumors. (3) The use of any one of the chimeric antigen receptor described in the first aspect of the present invention, the polynucleotide described in the second aspect, the recombinant vector described in the third aspect, the host cell described in the fourth aspect, the pharmaceutical composition described in the fifth aspect, and the method described in the sixth aspect in the preparation of a product for non-therapeutic in vitro detection of EpCAM gene expression level.

[0053] Furthermore, the solid tumors include breast cancer, pancreatic cancer, small bowel cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal carcinoma, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal cell carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP, thymic carcinoma, glioma, neuroglioma, astrocytoma, cervical cancer, or prostate cancer.

[0054] The advantages and beneficial effects of this invention are as follows: This invention does not simply provide known anti-EpCAM antibodies, but rather introduces the VH and VL sequences of existing patented anti-EpCAM antibodies into a CAR framework to construct an EpCAM-targeting CAR molecule capable of expression and effector function in T cells. The EpCAM CAR-T constructed in this invention has been validated in vitro to effectively kill EpCAM-positive target cells and in vivo to inhibit MC38-EpCAM tumor growth, demonstrating that this CAR design can be practically translated into an effective cell therapy product. To verify the effect of different intracellular co-stimulatory domains on CAR function, this invention also constructed a control CAR, which has the same antigen recognition domain and the same hinge / transmembrane region as the aforementioned CAR, but the intracellular co-stimulatory domain is replaced with a 4-1BB domain and linked to CD3ζ to form a control conformation. By comparing the two conformations under the same experimental system, the technical effectiveness of the preferred conformation of this invention is further demonstrated. Attached Figure Description

[0055] Figure 1 A schematic diagram of the CAR structure targeting EpCAM; Figure 2 Image of pMIG-EpCAM-28ZCAR plasmid; Figure 3The image shows the fold expansion results of EpCAM CAR-T cells; Figure 4 The graph shows the positive rate of EpCAM CAR-T cells; Figure 5 The image shows the results of EpCAM expression detection on the surface of MC38-EpCAM cells; Figure 6 The graph shows the statistical results of the killing effect of EpCAM 28Z CAR-T cells on MC38-EpCAM-GFP cells (killing statistics); Figure 7 A flowchart for animal experiments; Figure 8 The inhibitory effects of low-dose and high-dose mouse EpCAM CAR-T cells on MC38-EpCAM tumor growth are shown in the figure. Figure 9 A statistical graph showing the body weight of mice during the experimental procedure; Figure 10 Map of the pMIG plasmid, a retroviral expression vector; Figure 11 Map of the auxiliary plasmid pCL-Eco. Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0057] Example 1: Construction of pMIG-EpCAM-28Z CAR and pMIG-EpCAM-BBZ CAR retroviral expression vectors Experimental materials: a synthetic plasmid containing the EpCAM VH-linker-VL sequence synthesized by Genewiz, and the retroviral expression vector pMIG plasmid ( Figure 10 ), helper plasmid pCL-Eco( Figure 11 ).

[0058] Experimental Methods: The nucleotide sequence of EpCAM VH-linker-VL synthesized by Genewiz (EpCAM scFv, SEQ ID NO.1) was ligated with a pMIG plasmid containing the tag molecules RQR8, T2A, mouse CD28 hinge region, mouse CD28 transmembrane region, mouse CD28 intracellular region, and mouse CD3ζ intracellular region, which was stored in our group, to construct the pMIG-EpCAM-28Z CAR expression vector using a homologous recombination kit (Novizan, C112). Simultaneously, the nucleotide sequence encoding EpCAM VH-linker-VL was ligated with a pMIG plasmid containing the tag molecules RQR8, T2A, mouse CD28 hinge region, mouse CD28 transmembrane region, mouse 4-1BB intracellular region, and mouse CD3ζ intracellular region, which was stored in our group, to construct the pMIG-EpCAM-BBZ CAR control expression vector. The sequence information involved is shown in Table 1.

[0059] Table 1 Sequence Information

[0060] Prepare two large dishes of 293T cells. Preheat PEI at 65℃ for 15 min and DMEM at 65℃ for 30 min. Prepare solutions A and B, with specific components as shown in Table 2. Let each solution stand for 5 min, gently mix by pipetting, and incubate at 37℃ for 15 min. Transfect 293T cells with 500 μL / dish and incubate at 37℃. Prepare virus replacement medium (DMEM containing 10% FBS + 100× sodium pyruvate + 100× HEPES, 7 mL per dish). Change the medium every 6-8 h (preheat 20 min in a 37℃ water bath beforehand). After 48 h, centrifuge at 12000 rpm for 2 min, discard the supernatant, resuspend in 200 μL of virus dilution buffer, incubate overnight at 4℃, aliquot into 1.5 mL EP tubes, and store at -80℃.

[0061] Table 2 pMIG-EpCAM CAR retroviral packaging system

[0062] Experimental Results: After construction, sequencing confirmed the correct construction of the expression vector. This embodiment constructed two CAR configurations for subsequent functional comparisons; see reference [link to documentation] for details. Figure 1 pMIG-EpCAM-28Z CAR expression vector, such as Figure 2As shown, a complete CAR expression frame is thus formed, and its theoretically translated amino acid sequence and composition of each part are shown in Table 3, with reference to 3-17I scFv in US008637017B2.

[0063] Table 3. Amino acid sequence information

[0064] Example 2: Detection of the amplification capacity of mouse T cells after CAR transduction Experimental materials: C57BL / 6 mice, mouse EpCAM-28Z CAR-T cells (T cells infused with the pMIG-EpCAM-28Z CAR expression vector), mouse EpCAM-BBZ CAR-T cells (T cells infused with the pMIG-EpCAM-BBZ CAR expression vector), Mock T cells, trypan blue staining agent. Experimental methods: Inguinal and mesenteric lymph nodes of 6-8 week old C57BL / 6 mice were removed using sterile forceps, ground through a 0.45 μM filter membrane, and resuspended in mouse T cell culture medium; the mouse T cell suspension was centrifuged at 300 g for 5 min, and the supernatant was discarded; the mouse T cell density was adjusted to 1×10⁶ cells / mL. 6 Cells / mL were seeded into 48-well plates. After 24 h, the plates were sealed with sealing film. The above retroviruses and activated mouse T cells were centrifuged at 1500 g for 2 h at 26 °C. After centrifugation, the cells were cultured at 37 °C for another 48 h. After 48 h of infection, EpCAM-28Z CAR-T cells, EpCAM-BBZ CAR-T cells, and Mock T cells were obtained. The cells were cultured for 4 days, and stained with trypan blue daily. The number of CAR-T cells was counted using a cell counter.

[0065] Experimental results: Figure 3 The proliferation curves of EpCAM-28Z CAR-T cells, EpCAM-BBZ CAR-T cells, and Mock T cells during culture are shown. The horizontal axis represents the number of days of T cell proliferation, and the vertical axis represents the fold increase in cell size. EpCAM-28Z CAR-T cells, EpCAM-BBZ CAR-T cells, and Mock T cells all exhibited good proliferation capacity during culture, with similar overall expansion trends, indicating that the introduction of the constructed CAR did not significantly affect the in vitro expansion capacity of T cells.

[0066] Example 2: CAR Positive Rate Detection Experimental materials: mouse EpCAM-28Z CAR-T cells, EpCAM-41-BB CAR-T cells, PE-anti RQR8 (Cell Signaling Technology, 38907S), labeling reagents for detecting CAR surface expression, etc.

[0067] Experimental Methods: Cell Preparation: EpCAM-28Z CAR-T cells and EpCAM-BBZ CAR-T cells were prepared and labeled with RQR8 by flow cytometry. Flow Cytometry Analysis: The labeled cell samples were sent to a flow cytometer to detect the positivity rate of RQR8 on the cell surface. Data Collection and Analysis: The RQR8 expression level of each cell was recorded by flow cytometry, and the proportion of positive cells was calculated.

[0068] Experimental results: Figure 4 This is the flow cytometry analysis result of RQR8+ cells, where the horizontal axis represents the light scattering intensity of the cells and the vertical axis represents the fluorescence intensity of the cells. The results show that approximately 57.8% of EpCAM-28Z CAR-T cells and approximately 49% of EpCAM-BBZ CAR-T cells express CAR.

[0069] Example 4: Detection of EpCAM expression in MC38-EpCAM cells Experimental materials: MC38-EpCAM cells, anti-EpCAM-AF647 antibody (Biolegend, B250970) Experimental methods: Cell preparation: 4 × 10⁶ cells per well in a 6-well plate. 5 2 mL of MC38 parent cells to be overexpressed were inoculated into cells and cultured at 37°C for 16-24 h. EpCAM lentivirus concentrate was then added. After 24 h, the virus solution was removed, and the cells were expanded using complete culture medium. Flow cytometry was then performed using EpCAM-AF647 antibody.

[0070] MC38-EpCAM cells (overexpressing EpCAM) and MC38 cells (non-overexpressing maternal cells) were cultured and labeled with the EpCAM-AF647 antibody. Flow cytometry: After cell labeling, the expression of EpCAM on the cell surface of the two groups was detected by flow cytometry. Data analysis: The expression differences between MC38-EpCAM cells and Vehicle cells under EpCAM-AF647 fluorescent labeling were compared.

[0071] Experimental results: The results are as follows Figure 5As shown, the horizontal axis in flow cytometry represents the fluorescence intensity of EpCAM-AF647, and the vertical axis represents the number of cells. The experiment showed that MC38-EpCAM cells exhibited up to 95.5% EpCAM positive expression on their surface, providing clear target data for subsequent CAR-T cell killing experiments.

[0072] Example 5: Mouse EpCAM CAR-T cells effectively killed mouse MC38 cells overexpressing human EpCAM in vitro. Experimental materials: mouse EpCAM-28Z CAR-T cells, EpCAM-BBZ CAR-T cells, Mock T cells, MC38-EpCAM cells Experimental Methods: Cell Culture: MC38-EpCAM cells were seeded and cultured, and then co-cultured with CAR-T cells before use. EpCAM-28Z CAR-T cells, EpCAM-BBZ CAR-T cells, and MC38-EpCAM cells were co-cultured with MC38-EpCAM cells at different effector-to-target ratios (E:T = 1:1, 1:2, 1:4). Flow Cytometry: After culture, tumor cells were detected by flow cytometry. Analysis was performed at 24-hour time points. Data Analysis: The distribution of tumor cells was obtained by flow cytometry, and the number of tumor cells was obtained using absolute counting magnetic beads. The killing effects of CAR-T cells and Mock T cells on tumor cells at different E:T ratios were compared.

[0073] Experimental results: The results are as follows Figure 6 As shown, the killing effect of CAR-T cells increased with the increase of the E:T ratio, indicating that CAR-T cells have a dose-dependent anti-tumor effect. Furthermore, EpCAM28Z CAR-T cells using mouse CD28 as a co-stimulatory molecule showed significantly higher killing efficiency against MC38-EpCAM cells compared to EpCAM 4-1BBZ CAR-T cells using mouse 4-1BB as a co-stimulatory molecule. These results demonstrate that the preferred CAR configuration of this invention not only effectively recognizes EpCAM-positive target cells, but also exhibits superior in vitro anti-tumor activity compared to the control configuration in this experimental system.

[0074] Example 6: Low-dose EpCAM CAR-T cells significantly inhibited MC38-EpCAM tumor growth. Experimental materials: C57 mice, mouse EpCAM-28Z CAR-T cells, MC38-EpCAM cells, cyclophosphamide (Cy) (Tyce, C2236) Experimental methods: Tumor inoculation: 6-8 week old, female C57 mice were inoculated with MC38-EpCAM cells (2×10⁻⁶). 6 Cells / each or 5×10 6Subcutaneous tumor models were established using cells / animal. Treatment began when the tumor reached 100 mm³. Chemotherapy was administered: cyclophosphamide (150 mg / kg) was given, starting when the tumor size was 100-200 mm³, with injections every other day.

[0075] CAR-T cell reinfusion: 3 days after chemotherapy, mouse EpCAM CAR-T cells (2×10⁻⁶) were reinfused. 6 EpCAM CAR-T cells (2 × 10⁻⁶ cells / mouse) were injected into mice. Tumor volume changes were monitored every 3 days, and mouse weight was recorded. Experimental groups: Mice were divided into the following groups: NC group (mice not inoculated with CAR-T cells), EpCAM CAR-T (2 × 10⁻⁶ cells / mouse), and EpCAM CAR-T (2 × 10⁻⁶ cells / mouse). 6 Group ) (Experimental group inoculated with EpCAM CAR-T cells), EpCAM CAR-T (5×10) 6 The experimental groups (those inoculated with higher doses of EpCAM CAR-T cells) consisted of 3 mice per group, for a total of 16 mice, and each group included 2 NC control mice.

[0076] Experimental cycle: Process reference Figure 7 Tumor cells were inoculated 14 or 17 days before the start of the experiment, cyclophosphamide treatment was given on day 12 or 15, CAR-T cells were reinfused on day 0, and the experimental period was 28 days. Tumor volume and mouse weight changes were monitored every 3 days during the period.

[0077] Experimental results: Treatment outcomes are as follows Figure 8 As shown, at 2×10 6 Cells / each and 5×10 6 In tumor models constructed by inoculating cells with MC38-EpCAM at a dose of 2×10⁻⁶ cells / cells, reinfusion of low doses (2×10⁻⁶ cells / cells) was performed. 6 ) or high dose (5×10 6 EpCAM-28Z CAR-T cells from mice effectively cleared tumors, achieving a tumor clearance rate of 100%. Furthermore, ... Figure 9 The changes in mouse body weight shown indicate that no significant abnormalities were observed during the treatment period, suggesting that EpCAM-28Z CAR-T cells did not exhibit significant toxicity under this dosage model.

[0078] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A chimeric antigen receptor targeting human EpCAM, characterized in that, The chimeric antigen receptor comprises a heavy chain variable region and a light chain variable region of an antibody targeting human EpCAM, or an scFv constructed therefrom; the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:13-15, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:16-18, respectively. Preferably, the chimeric antigen receptor comprises scFv, the amino acid sequence of which is shown in SEQ ID NO:

10.

2. The chimeric antigen receptor according to claim 1, characterized in that, The heavy chain variable region further includes FR1, FR2, FR3, and FR4, wherein the amino acid sequences of FR1, FR2, FR3, and FR4 are as shown in SEQ ID NO:19-22 or are at least 80% identical to the amino acid sequences shown in SEQ ID NO:19-22. Preferably, the light chain variable region further includes FR1, FR2, and FR3, and the amino acid sequences of FR1, FR2, and FR3 are as shown in SEQ ID NO:23-25 ​​or are at least 80% identical to the amino acid sequences shown in SEQ ID NO:23-25.

3. The chimeric antigen receptor according to claim 1, characterized in that, The heavy chain variable region has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:12; Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

12.

4. The chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor further includes a hinge region, a transmembrane domain, a signal transduction domain, and a co-stimulatory molecule; the hinge region is selected from CD28, and the nucleotide sequence of the hinge region is shown in SEQ ID NO:2; the transmembrane domain is selected from CD28, and the nucleotide sequence of the transmembrane domain is shown in SEQ ID NO:5; the signal transduction domain is selected from CD3ζ, and the nucleotide sequence of the signal transduction domain is shown in SEQ ID NO:7; the co-stimulatory molecule domain is selected from 4-1BB and CD28, and the nucleotide sequence of the co-stimulatory molecule domain when it is 4-1BB is shown in SEQ ID NO:26, and the nucleotide sequence of the co-stimulatory molecule domain when it is CD28 is shown in SEQ ID NO:6; the chimeric antigen receptor further includes a detection tag selected from RQR8, and the nucleotide sequence of RQR8 is shown in SEQ ID NO:2; the chimeric antigen receptor further includes a self-cleaving peptide selected from T2A, and the nucleotide sequence of T2A is shown in SEQ ID NO:

3.

5. An isolated polynucleotide, characterized in that, The polynucleotide encodes the chimeric antigen receptor according to any one of claims 1 to 4; the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO:

9.

6. A recombinant vector, characterized in that, The carrier comprises the polynucleotide of claim 5; Preferably, the recombinant vector further includes one or more regulatory elements operatively linked to the polynucleotide; Preferably, the regulatory sequence includes a promoter sequence, a transcription terminator sequence, and a leader sequence; Preferably, the recombinant vector is a viral vector; Preferably, the viral vector is a retroviral vector.

7. A recombinant host cell, characterized in that, The host cell comprises the polynucleotide of claim 6 or the recombinant vector of claim 7; Preferably, the recombinant host cell is selected from eukaryotic cells; Preferably, the eukaryotic cells are selected from animal cells; Preferably, the animal cells are selected from mammalian cells; Preferably, the mammalian cells are selected from immune cells; Preferably, the immune cells include T cells, NK cells, or NKT cells; Preferably, the immune cells are T cells.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the chimeric antigen receptors of claims 1 to 4, the polynucleotide of claim 5, the recombinant vector of claim 6, and any one of the host cells of claim 7; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

9. Any of the following methods: (1) A method for preparing recombinant host cells, characterized in that, The preparation method includes the step of introducing the polynucleotide of claim 5 or the recombinant vector of claim 6 into a host cell; (2) A method for detecting EpCAM protein levels in vitro, characterized in that the method comprises contacting a chimeric antigen receptor as described in any one of claims 1 to 5 or a recombinant host cell as described in claim 7 with a sample to be tested, incubating under conditions suitable for forming an antigen-receptor complex, and detecting the binding rate of the chimeric antigen receptor to EpCAM protein in the sample to be tested, so as to determine the expression level of EpCAM protein.

10. Any of the following applications: (1) Use of antibody 3-17I in the preparation of chimeric antigen receptor, wherein the amino acid sequence of antibody 3-17I is shown in SEQ ID NO:10; (2) The use of any one of the following in the preparation of a human solid tumor treatment drug: antibody 3-17I, the chimeric antigen receptor of any one of claims 1 to 5, the polynucleotide of claim 6, the recombinant vector of claim 7, the host cell of claim 8, and the method of claim 9; the amino acid sequence of antibody 3-17I is shown in SEQ ID NO:

10. (3) The application of any one of the chimeric antigen receptors of any one of claims 1 to 5, the polynucleotide of claim 6, the recombinant vector of claim 7, the host cell of claim 8, and the preparation method of claim 9 in the preparation of a product for non-therapeutic in vitro detection of EpCAM gene expression level.

Images