Fully human specific chimeric antigen receptors targeting human dll3 antigen and uses thereof

Abstract

This invention relates to a specific chimeric antigen receptor (CAR) based on a fully human anti-DLL3 single-chain antibody and its applications. The CAR comprises a CD8 signal peptide, a fully human single-chain antibody specific to the human DLL3 antigen, a CD8 transmembrane region, and 41BB and CD3ζ intracellular signal transduction domains sequentially connected in series. This invention provides immune cells modified with a human DLL3 chimeric antigen receptor by constructing and screening different DLL3 antigen-specific humanized single-chain antibodies, which can specifically bind to the human DLL3 antigen. In in vitro and in vivo killing experiments on human DLL3 antigen-positive tumor models, the CAR-T cells targeting the human DLL3 antigen of this invention significantly and specifically killed human DLL3-positive small cell lung cancer (SCLC) cell lines. Animal model experiments show that after administration of the preferred DLL3 CAR-T cells of this invention, the various drug groups showed significant efficacy, with tumor inhibition rates ranging from 25% to 97%.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine or biopharmaceutical technology, and in particular to a chimeric antigen receptor targeting human DLL3, its preparation method, and its use in preparing drugs, treating and preventing diseases. Background Technology

[0002] Small cell lung cancer (SCLC) is a type of lung cancer that, according to the NCCN guidelines, accounts for approximately 15% of all lung cancers. It is characterized by rapid progression, high recurrence rate, early metastasis, and poor prognosis, with a 5-year survival rate of less than 5%, and limited treatment options. Traditional radiotherapy and chemotherapy only provide short-term benefits and currently lack effective means to significantly prolong patient survival.

[0003] Human DLL3 protein (delta-like ligand 3, DLL3) is a single-pass transmembrane protein composed of 619 amino acids and belongs to the Notch ligand family. It is a highly tumor-selective cell surface target, highly expressed in most small cell lung cancer (SCLC) and carcinoid subgroups (Lung Cancer; 135:73-79; 2019), but not expressed in normal lung cancer tissue or adjacent normal tissue. In one study, an independent tumor specimen analysis of 1073 SCLC patients showed that DLL3 positive expression (≥25%) reached 85%, and DLL3 high expression (≥75%) reached 68% (Lung Cancer; 147: 237-243; 2020). Chimeric antigen receptor T cells targeting human DLL3 can be stimulated to specifically kill tumor cells by recognizing the DLL3 antigen on the tumor surface.

[0004] Amgen's AMG 757 project is validating its DLL3 target in clinical trials. AMG757 is a bispecific antibody that binds to both DLL3 and CD3. In an interim Phase 1 dose-finding study for the treatment of SCLC, it demonstrated a disease control rate (stable disease / CR / PR) of 51%, a median PFS of 3.5 months, and a median OS of 12.3 months, showing preliminary evidence of efficacy and acceptable safety for AMG 757 (NCT03319940). Meanwhile, Amgen's DLL3 CAR-T project, AMG 119, is also in a Phase I clinical trial evaluating its safety, tolerability, and efficacy for the treatment of SCLC (NCT03392064). Harpoon Therapeutics' HPN-328 and Boehringer Ingelheim's OBT-620 are also in Phase I / II clinical trials. DLL3, as an important Notch ligand, is closely related to the development and progression of various tumors, and the development of multiple therapies targeting DLL3 holds promise for providing more treatment options for clinical oncology.

[0005] In light of the above, CAR-T cells targeting DLL3 are of great significance as an anti-cancer cell therapy, especially in cancers that highly express DLL3. This invention provides chimeric antigen receptor T cells targeting human DLL3 that exhibit good anti-human DLL3-positive tumor activity, bringing new hope to patients with advanced DLL3-positive tumors. Summary of the Invention

[0006] To address the technical problem of providing more anticancer drugs, particularly drugs targeting cancers that highly express DLL3, such as lung cancer, especially small cell lung cancer, the present invention provides a chimeric antigen receptor (CAR) targeting DLL3, and an immune cell, such as a T cell, containing the CAR. This invention also provides nucleic acids encoding the CAR; expression cassettes, vectors, and cells containing the nucleic acids; pharmaceutical compositions containing the CAR, the nucleic acids, the expression cassettes, the vectors, and the cells; kits containing the CAR, the nucleic acids, the expression cassettes, the vectors, the cells, and the pharmaceutical compositions; and the use of the CAR, immune cells containing the CAR, the nucleic acids, the expression cassettes, the vectors, the cells, and the pharmaceutical compositions in the prevention, treatment, detection, or diagnosis of DLL3-related diseases, or in the preparation of drugs or formulations for the prevention and / or treatment of cancer or tumors, wherein the DLL3-related diseases are DLL3-overexpressing diseases, further wherein the diseases are DLL3-overexpressing cancers or tumors, further wherein the cancer or tumor is selected from one or more of lung cancer, melanoma, medullary thyroid carcinoma, glioblastoma, prostate cancer, and neuroendocrine carcinoma; and further wherein the cancer or tumor is lung cancer, particularly small cell lung cancer. This invention also provides a method for preparing engineered immune cells.

[0007] The first aspect of the present invention provides a chimeric antigen receptor (CAR) targeting the DLL3 antigen, characterized in that the CAR comprises an anti-DLL3 antibody scFv, the scFv comprising three light chain complementarity-determining regions and / or three heavy chain complementarity-determining regions, wherein:

[0008] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20, and LCDR3 shown in SEQ ID NO:21, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, and HCDR3 shown in SEQ ID NO:25; preferably, the CAR number is 332;

[0009] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:27, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:29, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, and HCDR3 shown in SEQ ID NO:33; preferably, the CAR number is 244;

[0010] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:36, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:38, HCDR2 shown in SEQ ID NO:39, and HCDR3 shown in SEQ ID NO:40; preferably, the CAR number is 306;

[0011] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:42, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:29, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:44, HCDR2 shown in SEQ ID NO:45, and HCDR3 shown in SEQ ID NO:46; preferably, the CAR number is 304;

[0012] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:50, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:52, HCDR2 shown in SEQ ID NO:53, and HCDR3 shown in SEQ ID NO:54; preferably, the CAR number is 229;

[0013] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:56, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:58, HCDR2 shown in SEQ ID NO:59, and HCDR3 shown in SEQ ID NO:60; preferably, the CAR number is 564;

[0014] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:93, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:58, HCDR2 shown in SEQ ID NO:59, and HCDR3 shown in SEQ ID NO:60; preferably, the CAR number is 654;

[0015] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:62, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:38, HCDR2 shown in SEQ ID NO:64, and HCDR3 shown in SEQ ID NO:65; preferably, the CAR number is 108;

[0016] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:67, LCDR2 shown in SEQ ID NO:68, and LCDR3 shown in SEQ ID NO:69, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:44, HCDR2 shown in SEQ ID NO:71, and HCDR3 shown in SEQ ID NO:46; preferably, the CAR number is 83;

[0017] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:73, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:74, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:76, HCDR2 shown in SEQ ID NO:71, and HCDR3 shown in SEQ ID NO:77; preferably, the CAR number is 131;

[0018] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:79, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:81, HCDR2 shown in SEQ ID NO:71, and HCDR3 shown in SEQ ID NO:82; preferably, the CAR number is 136;

[0019] The three light chain complementary determinant regions include LCDR1 shown in SEQ ID NO:84, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:79, and / or the three heavy chain complementary determinant regions include HCDR1 shown in SEQ ID NO:86, HCDR2 shown in SEQ ID NO:71, and HCDR3 shown in SEQ ID NO:82; preferably, the CAR number is 412; or...

[0020] The three light chain complementary determination regions include LCDR1 shown in SEQ ID NO:67, LCDR2 shown in SEQ ID NO:68, and LCDR3 shown in SEQ ID NO:88, and / or the three heavy chain complementary determination regions include HCDR1 shown in SEQ ID NO:90, HCDR2 shown in SEQ ID NO:91, and HCDR3 shown in SEQ ID NO:92; preferably, the CAR number is 303.

[0021] The present invention further provides a chimeric antigen receptor targeting the DLL3 antigen, characterized in that,

[0022] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:18, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:22; preferably, the CAR number is 332;

[0023] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:30; preferably, the CAR number is 244;

[0024] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:34, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:37; preferably, the CAR number is 306;

[0025] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:41, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:43; preferably, the CAR number is 304;

[0026] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:47, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:51; preferably, the CAR number is 229;

[0027] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:55, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:57; preferably, the CAR number is 564;

[0028] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:61, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:94; preferably, the CAR number is 654;

[0029] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:95, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:63; preferably, the CAR number is 108;

[0030] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:66, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:70; preferably, the CAR number is 83;

[0031] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:72, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:75; preferably, the CAR number is 131;

[0032] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:78, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:80; preferably, the CAR number is 136;

[0033] The scFv of the anti-DLL3 antibody comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:83, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:85; preferably, the CAR number is 412; or,

[0034] The scFv of the anti-DLL3 antibody contains a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:87, and / or a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:89; preferably, the CAR number is 303.

[0035] Furthermore, the CAR also includes a transmembrane domain, preferably the CD8 transmembrane region. The transmembrane domain may also be selected from the transmembrane regions of the following proteins: the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, PD1, or combinations thereof.

[0036] Furthermore, the CAR also includes an intracellular signal transduction domain. Preferably, the intracellular signal transduction domain is the CD3ζ intracellular signal domain. The intracellular signal transduction domain may also be selected from the signal transduction domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, CD66d, or combinations thereof.

[0037] Furthermore, the CAR also includes a hinge region, preferably a CD8 hinge region, and the hinge region may also be selected from the hinge regions of the following proteins: CD28, GM-CSF, CD4, CD137, or combinations thereof.

[0038] Furthermore, the CAR also includes a co-stimulatory signal domain. Preferably, the co-stimulatory signal domain is a 4-1BB co-stimulatory signal. The co-stimulatory signal may also be selected from the co-stimulatory signals of the following proteins: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), HVEM, or a combination thereof.

[0039] Furthermore, the CAR also includes a signal peptide, preferably a CD8α signal peptide, and the signal peptide may also be selected from the signal peptides of the following proteins: CD8, CD28, GM-CSF, CD4, CD137, or combinations thereof.

[0040] Further, the CD8α signal peptide comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 1; the CD8 hinge region and transmembrane region comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 2; the 4-1BB co-stimulatory signal domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 3; or, the CD3ζ intracellular signal domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 4.

[0041] Furthermore, the CAR contains, from the N-terminus to the C-terminus, a CD8α signal peptide, a DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory signal, and a CD3ζ intracellular signal domain; optionally, the CAR does not contain the amino acid sequence of the CD8α signal peptide.

[0042] Further, the amino acid sequence of the CAR is as shown in any of SEQ ID NO: 5-17; more preferably, the CAR has the amino acid sequence of the CD8α signal peptide removed from the amino acid sequence shown in any of SEQ ID NO: 5-17.

[0043] A second aspect of the invention provides a nucleic acid that encodes the CAR.

[0044] A third aspect of the invention provides an expression cassette comprising the nucleic acid.

[0045] A fourth aspect of the present invention provides a vector comprising a nucleic acid encoding the CAR or the expression cassette. The vector can be used to express the CAR. Preferably, the vector is a viral vector; preferably, the viral vector includes, but is not limited to, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or retroviral vectors; preferably, the vector is a non-viral vector; preferably, the vector is a mammalian cell expression vector; preferably, the expression vector is a bacterial expression vector; preferably, the expression vector is a fungal expression vector.

[0046] A fifth aspect of the present invention provides a cell comprising the nucleic acid, or the expression cassette, or the vector, wherein the cell is capable of expressing the CAR. Preferably, the cell is a bacterial cell; preferably, the bacterial cell is Escherichia coli, etc.; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast cell is Pichia pastoris, etc.; preferably, the cell is a mammalian cell; preferably, the mammalian cell is Chinese hamster ovary cell (CHO), human embryonic kidney cell (293), B cell, T cell, DC cell, or NK cell, etc. Preferably, the cell is an engineered immune cell; more preferably, the engineered immune cell is a T cell; most preferably, the T cell is a primary T cell, γ-δ T cell, or NK T cell. A sixth aspect of the present invention provides a pharmaceutical composition comprising the CAR, nucleic acid, expression cassette, vector, or cell. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier comprises one or more of the following: pharmaceutically acceptable solvents, dispersants, excipients, plasticizers, and pharmaceutical excipients.

[0047] A seventh aspect of the present invention provides a kit comprising the CAR of the present invention, or comprising nucleic acid encoding the CAR, or the expression cassette.

[0048] The eighth aspect of the invention provides the use of the CAR, nucleic acid, expression cassette, vector, or cell in the preparation of pharmaceutical compositions for treating or preventing diseases.

[0049] The ninth aspect of the present invention provides the application of the CAR or nucleic acid or expression cassette in the preparation of diagnostic and detection kits.

[0050] The tenth aspect of the present invention provides a method for treating or preventing a disease, comprising administering the CAR, nucleic acid, expression cassette, vector, cell or pharmaceutical composition of the present invention to a subject in need.

[0051] The eleventh aspect of the present invention provides a method for diagnosis and detection, comprising administering the CAR, nucleic acid, expression cassette, reagent kit or pharmaceutical composition of the present invention to a subject or sample in need.

[0052] The twelfth aspect of the present invention provides the use of the CAR, nucleic acid, expression cassette, vector, cell or pharmaceutical composition for the treatment or prevention of diseases.

[0053] The thirteenth aspect of the present invention provides the use of the CAR, nucleic acid, expression cassette, kit, or pharmaceutical composition for detection and diagnosis.

[0054] The fourteenth aspect of the invention provides the use of the CAR, the nucleic acid, or the expression cassette, or the vector, or the pharmaceutical composition for the prevention, treatment, detection, or diagnosis of diseases related to DLL3.

[0055] In the present invention, the DLL3-related disease is a DLL3-overexpressing disease; preferably, the disease is a DLL3-overexpressing cancer or tumor; more preferably, the cancer or tumor is selected from one or more of lung cancer, melanoma, medullary thyroid carcinoma, glioblastoma, prostate cancer, and neuroendocrine carcinoma; most preferably, the cancer is lung cancer, particularly small cell lung cancer.

[0056] The fifteenth aspect of the present invention provides a method for preparing engineered immune cells, characterized by comprising the following steps:

[0057] (1) Provide an immune cell to be modified; and

[0058] (2) The nucleic acid, or the expression cassette, or the vector is introduced into the immune cells.

[0059] Preferably, the immune cells are T cells; more preferably, the T cells are primary T cells, γ-δ T cells, or NK T cells.

[0060] The fully human-specific chimeric antigen receptor targeting human DLL3 antigen provided by this invention has one or more of the following advantages:

[0061] 1. The human DLL3 chimeric antigen receptor-modified T cells (DLL3 CAR-T) of the present invention are capable of specifically binding to human DLL3 antigens.

[0062] 2. The human DLL3 chimeric antigen receptor modified T cell (DLL3 CAR-T) of the present invention uses a fully human anti-DLL3 antibody scfv structure, which theoretically has the advantage of low immunogenicity.

[0063] 3. The CAR expression of the 13 DLL3 CAR-T cells of the present invention remained basically stable under different culture days, and no abnormal phenomena such as decline were observed.

[0064] 4. In vitro toxicity tests of the 13 DLL3 CAR-T cells of this invention showed that, with a fixed number of tumor cells, except for 564 scfv CAR-T, the other 12 DLL3 CAR-T cells, when co-incubated with tumor cells at an effector-to-target ratio of 5:1, exhibited a killing efficiency of 18%-48% after 24 hours for SHP-77 cell lines expressing human DLL3 antigen; and a killing efficiency of 21%-56% after 24 hours for SHP-77-hDLL3 cell lines with high expression of human DLL3 antigen; while showing no significant killing effect on the human DLL3-negative cell line NCI-H460.

[0065] 5. The long-term anti-tumor effects of different scfv DLL3 CAR-T cells in vitro were evaluated. With an effector cell:target cell ratio of 1:10, the 108, 131 and 654 scfv DLL3 CAR-T cells of this invention were able to completely eliminate tumor cells.

[0066] 6. Animal model experiments show that after 17 days of administration of the six preferred DLL3 CAR-Ts (332, 304, 229, 654, 108, 131scfv DLL3 CAR-Ts) of this invention, the efficacy of each drug group is significant, with tumor inhibition rates ranging from 25% to 97%. Attached Figure Description

[0067] Figure 1 A schematic diagram of the structure of the recombinant plasmid pRRLSIN-DLL3 CAR-EGFP (PB DLL3 CAR) is shown.

[0068] Figures 2A-2N This diagram illustrates a flow cytometry study of CAR-positive T lymphocytes expressing different DLL3 chimeric antigen receptors, detected using anti-human IgG (Fab)2 antibody. Figure 2A Results for T cells without CAR transduction are shown. Figure 2B The results of 332 DLL3 CAR-T are shown. Figure 2C The results of 244 DLL3 CAR-T studies are shown. Figure 2D The results of 306 DLL3 CAR-T are shown. Figure 2E The results of 304 DLL3 CAR-T are shown. Figure 2F The results of 229 DLL3 CAR-T studies are shown. Figure 2G The results of 564DLL3 CAR-T are shown. Figure 2H The results of 654 DLL3 CAR-T studies are shown. Figure 2I The results of 108 DLL3 CAR-T were shown. Figure 2J The results of 83 DLL3 CAR-T were shown. Figure 2KThe results of 131 DLL3 CAR-T are shown. Figure 2L The results of 136 DLL3 CAR-T were shown. Figure 2M The results of 412 DLL3 CAR-T were shown. Figure 2N The results for 303 DLL3 CAR-T are shown.

[0069] Figures 3A-3N This diagram illustrates a flow cytometry assay for CAR-positive T cells expressing different DLL3 chimeric antigen receptors, using DLL antigen detection. Figure 3A Results for T cells without CAR transduction are shown. Figure 3B The results of 332 DLL3 CAR-T are shown. Figure 3C The results of 244 DLL3 CAR-T studies are shown. Figure 3D The results of 306 DLL3 CAR-T are shown. Figure 3E The results of 304 DLL3 CAR-T are shown. Figure 3F The results of 229 DLL3 CAR-T studies are shown. Figure 3G The results of 564 DLL3 CAR-T studies are shown. Figure 3H The results of 654 DLL3 CAR-T studies are shown. Figure 3I The results of 108 DLL3 CAR-T were shown. Figure 3J The results of 83 DLL3CAR-T are shown. Figure 3K The results of 131 DLL3 CAR-T are shown. Figure 3L The results of 136 DLL3 CAR-T were shown. Figure 3M The results of 412 DLL3 CAR-T were shown. Figure 3N The results for 303 DLL3 CAR-T are shown.

[0070] Figures 4A-4B ,in Figure 4A The results of CAR positivity rates of T cells targeting DLL3 chimeric antigen receptors, as detected by an anti-human IgG (Fab)2 antibody assay, are shown. Figure 4B The results of CAR positivity rates of 13 T cells targeting DLL3 chimeric antigen receptors were shown by a human DLL3 antigen protein detection method.

[0071] Figure 5 A- Figure 5B , Figure 5A The results of the CAR positivity rate of 13 T cells targeting DLL3 chimeric antigen receptors under different culture days are shown in the detection method of anti-human IgG (Fab)2 antibody. Figure 5BThe results show the changes in CAR positivity rates of 13 T cells targeting the DLL3 chimeric antigen receptor under different culture days using the human DLL3 antigen protein detection method.

[0072] Figure 6 The results of flow cytometry analysis of DLL3 antigen expression in human NCI-H460, SHP-77, and SHP-77-hDLL3 lung cancer cell lines are shown.

[0073] Figure 7 A- Figure 7C , Figure 7A The in vitro specific killing results of 13 DLL3 CAR-T cells against the NCI-H460 (non-small cell lung cancer) cell line are shown. Figure 7B The in vitro specific killing results of 13 DLL3 CAR-T cells against the SHP-77 (small cell lung cancer) cell line are shown. Figure 7C The in vitro specific killing results of 13 types of DLL3 CAR-T cells against human DLL3-positive SHP-77-hDLL3 (small cell lung cancer) are shown.

[0074] Figure 8 A- Figure 8C , Figure 8A The study shows the release of IFN-γ from 13 DLL3 CAR-T cells co-cultured with NCI-H460 cell lines. Figure 8B The study shows the release of IFN-γ from 13 DLL3 CAR-T cells co-cultured with SHP-77 cell lines. Figure 8C The release of IFN-γ from 13 DLL3 CAR-T cells co-cultured with SHP-77-hDLL3 cell lines is shown.

[0075] Figures 9A-9C , Figure 9A The in vitro killing results of 12 types of DLL3 CAR-T cells on the 293T cell line are shown. Figure 9B The in vitro killing results of 12 types of DLL3 CAR-T cells on the human DLL3 protein-overexpressing 293T cell line (293T-hDLL3) are shown. Figure 9C The in vitro killing results of 12 types of DLL3 CAR-T cells on the mouse DLL3 protein-overexpressing 293T cell line (293T-mDLL3) are shown.

[0076] Figures 10A-10C , Figure 10A The results of IFN-γ release after in vitro killing of 293T cell line by 12 types of DLL3 CAR-T cells are shown. Figure 10B The results of IFN-γ release after in vitro killing of human DLL3 protein-overexpressing 293T cell line (293T-hDLL3) by 12 types of DLL3 CAR-T cells are shown. Figure 10C The results of IFN-γ release after in vitro killing of mouse DLL3 protein-overexpressing 293T cell line (293T-mDLL3) by 12 types of DLL3 CAR-T cells are shown.

[0077] Figures 11A-11B The study presented the long-term antitumor effects of eight DLL3 CAR-T cell lines on DLL3-expressing positive cells, among which... Figure 11A The results show the changes in the number of tumor cells after long-term anti-tumor effects of eight types of DLL3 CAR-T cells on DLL3-expressing positive cells. Figure 11B The results show the changes in the number of CAR-positive T cells in response to the long-term antitumor effect of eight types of DLL3 CAR-T cells on DLL3-expressing positive cells.

[0078] Figures 12A-12G The results of different DLL3 CAR-T cell positivity rate assays used in in vivo efficacy studies are shown, among which... Figure 12A Results for T cells without CAR transduction are shown. Figure 12B The results of 332 DLL3 CAR-T are shown. Figure 12C The results of 304 DLL3 CAR-T are shown. Figure 12D The results of 229 DLL3 CAR-T studies are shown. Figure 12E The results of 654 DLL3 CAR-T studies are shown. Figure 12F The results of 108 DLL3 CAR-T were shown. Figure 12G The results of 131 DLL3 CAR-T are shown.

[0079] Figure 13 This study demonstrates the efficacy of six DLL3 CAR-T cells in reducing body weight in NCG mice bearing SHP-77 small cell lung cancer tumors.

[0080] Figure 14 This study demonstrates the efficacy of six DLL3 CAR-T cells in inducing tumor size changes in NCG mice bearing SHP-77 small cell lung cancer.

[0081] Figure 15 This study demonstrates the efficacy of six DLL3 CAR-T cells in improving the survival rate of NCG mice bearing SHP-77 tumors in small cell lung cancer. Detailed Implementation

[0082] The present invention will be further illustrated below with reference to specific embodiments. The described embodiments are only some, not all, of the embodiments of the present invention. It should be understood that the following embodiments are provided to give a complete disclosure and description to those skilled in the art on how to utilize the methods and compositions of the present invention, and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0083] Example 1. Preparation of gene fragment targeting DLL3 chimeric antigen receptor (CAR)

[0084] This invention designs a fusion gene fragment according to the following coding gene sequence: CD8α signal peptide, DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL, CD8 hinge region, CD8 transmembrane region, and 4-1BB co-stimulatory signal and CD3ζ intracellular signal domain. The fusion gene is directly synthesized using gene synthesis technology, resulting in an expressed chimeric antigen receptor with the amino acid structure scFvVH-linker-scFv VL-CD8 hinge-CD8TM-4-IBB-CD3ζ. The linker amino acid sequence is (G4S)3, i.e., GGGGSGGGGSGGGGS; the CD8α signal peptide amino acid sequence is SEQ ID NO: 1; the CD8 hinge region (CD8 hinge) and transmembrane region (CD8 TM) amino acid sequences are SEQ ID NO: 2; the 4-1BB amino acid sequence is SEQ ID NO: 3; and the CD3ζ amino acid sequence is SEQ ID NO: 4. Specific sequences of SEQ ID NO: 1-4 are shown in Table 1-1. Gene fragments targeting the DLL3 chimeric antigen receptor were prepared using gene synthesis technology, and their amino acid sequences are shown in SEQ ID NO: 5-17, with specific sequences listed in Tables 1-2. The amino acid sequences of the different active clones of anti-DLL3 antibodies scFv VH and VL used in CAR assays, as well as LCDR1-3 and HCDR1-3, are shown in Table 2. The analysis system used was the IMGT system.

[0085] Table 1-1. Amino acid sequences of some CAR elements

[0086] CD8α signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 1) CD8 hinge area and transmembrane area TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 2) 4-1BB costimulatory signal KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 3) CD3ζ Intracellular Signaling Domain RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 4)

[0087] Table 1-2. Amino acid sequence of DLL3 CAR

[0088] 332 MALPVTALLLPLALLLHAARPEVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARPNWGLAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPDSLAVSLGERATINCRSSQSVLYSSNNKNYLAWYQQRPGQPPKLLINWASTRESGVPDRFSGSGSGTDFSLTISSLQAEDVAVYYCQQYYGIPTFGQGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ IDNO: 5) 244 MALPVTALLLPLALLLHAARPEVQLQQWGAGLSKPSETLSLTCAVYGGSFSDYDWSWIRQPPGKGLEWIGEITHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGYSGYPYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLLSGGPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:6) 306 MALPVTALLLPLALLLHAARPEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVTSDDGSNKYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDETYYFGSGGYYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCHQYHTFPFTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 7) 304 MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGSSVKVSCKTSGGTFSNYAINWVRQAPGQGLEWMGRIIPILDITNYAQKFQGSVTITADKSTSTAYMELSSLRSEDTAVYYCATYSSSFDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:8) 229 MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGSSVKVSCKASGGTFGSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREDYFDSGNSYKNYFYYGLDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLTKGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQLYGSSITFGQGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 9) 564 MALPVTALLLPLALLLHAARPEVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYFWSWIRQPPGKGLEWIGEINPSGNTIYNPSLKSRVTISLDTSKNQFSLKLSSVTAADTAVYYCARNDYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASVGDRVTITCRASQGISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:10)

[0089] 108 MALPVTALLLPLALLLHAARPEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVILYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGDGSESPYYYDYGLDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASVGDRVTITCRASQGISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPFTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQID NO: 12) 83 MALPVTALLLPLAALLHAARPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGRIIPILDIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATYSSSFDAFDIWGQGTMVTVSSGGGGSGGGGSGIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYSSITFGQGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLRGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 13) 131 MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILDIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYCARGGDWRDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QANSFPFTGFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:14) 136 MALPVTALLLPLALLHAARPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFAINWVRQAPGQGLEWMGRIIPILDIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATYSAYDFADAFDIWGQGTTVTVSSGGGGSGGGGSGIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSQPTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNENLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 15) 412 MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPILDIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATYSAYDFDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSQPTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 16) 303 MALPVTALLLPLALLLHAARPEVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYQWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTTSVDTSKNQFSLKLSSVTAADTAVYYCARDWGGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 17)

[0090] Table 2. Amino acid sequences of the variable region and CDR of the active clone of anti-DLL3 antibody

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Note: The underlined part in VL and VH is CDR.

[0097] Example 2. Preparation of T cells modified with DLL3-specific chimeric antigen receptor

[0098] 2.1 Construction of non-viral piggybac (PB) transposon vector

[0099] Using the pBluescirpt vector (Universal Biosynthesis) as the starting backbone, the gene insulator sequence cHS4 was identified and placed at both ends of the multiple cloning site. The 5' ITR and 3' ITR sequences of the PB transposon were then identified and constructed inside the cHS4 sequence within the vector. Inside the ITR, an EF1a promoter was inserted at the 5' end, and a polyA signal was inserted at the 3' end. The multiple cloning sequence was retained in the middle. The gene sequence CD8α-DLL3 antibody ScFv HV-linker-ScFv VL-CD8 hinge-CD8TM-4-1BB-CD3ζ (DLL3 CAR) was inserted into the multiple cloning sequence. Simultaneously, an EGFP tag gene sequence was inserted after the DLL3 CAR sequence via P2A linkage. These sequences formed the pRRLSIN-DLL3 CAR-EGFP plasmid structure (abbreviated as PB DLL3 CAR), as shown in the schematic diagram. Figure 1 As shown.

[0100] The PB DLL3 CAR vector constructed from 13 different DLL3 antibodies (active clone numbers 332, 244, 306, 304, 229, 564, 654, 108, 83, 131, 136, 412, 303) was extracted (completed by Nanjing GenScript) to obtain PB DLL3CAR transfection-grade plasmids.

[0101] 2.2 Preparation of T lymphocytes

[0102] Peripheral blood mononuclear cells (PBMCs) from Shanghai Aoneng Biotechnology were labeled with magnetic beads using the CD3 MicroBeads human-lyophilized Kit (Miltenyi Biotech) to isolate highly pure CD3-positive T lymphocytes, with the proportion of CD3-positive T cells exceeding 95%. The purified T cells were then activated and proliferated using Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher, 11132D).

[0103] 2.3 CAR gene transduction of T cells

[0104] 72-96 hours after T cell stimulation and activation, electroporation was performed using PB DLL3 CAR plasmids constructed from the scfv of the 13 DLL3 antibodies (332, 244, 306, 304, 229, 564, 654, 108, 83, 131, 136, 412, 303) obtained in Example 2.1. First, the T cells used for electroporation were resuspended, and the T cell clumps were dispersed using pipette tips or pipettes. The T cell resuspension was counted, and 5 x 10⁻⁶ cells were used for electroporation. 6 T cells were used in a single electroporation experiment. 5 x 10⁵ cells were prepared using DPBS (GIBCO, 14190-144). 6 Dilute the cells to 5 mL, centrifuge at 300 xg for 10 minutes at room temperature, discarding as much supernatant as possible to avoid contact with the T cell pellet. Resuspend the T cells in 5 mL of DPBS and wash them. Centrifuge at 300 xg for 10 minutes at room temperature, discarding as much supernatant as possible to avoid contact with the T cell pellet. Resuspend the cells in 100 μL of electroporation buffer Entranster-E (Engreen, 98668-20). Transfer the cell suspension to a 1.5 mL centrifuge tube. Add the components from Table 3 to the centrifuge tube and mix well.

[0105] Table 3. Electro-electric system for each group

[0106] PB DLL3 CAR plasmid (1ug / uL) 5 Transposase mRNA (1ug / uL) 5 T cell suspension 100 Total volume 110

[0107] Electroporation was performed using a 4D-Nucleofector transducer (Lonza). The cell / plasmid suspension was rapidly transferred to an electroporation cuvette, and the cuvette was gently tapped to ensure a balanced surface. Electroporation was performed using the EO115 program. After electroporation, the cuvette was carefully removed. 500 μL of pre-warmed T-cell medium X-VIVO 15 (Lonza, 04-418Q) was added, and the cells were equilibrated at 37°C for 5 minutes. The cells were resuspended using a micropipette tip, gently pipetting 2-3 times. The cells were then transferred to 12-well plates containing 2 mL of pre-warmed medium and incubated at 37°C. Changing the medium 4-6 hours after electroporation helps increase cell viability. The supernatant was carefully discarded, and pre-warmed fresh medium was added. The cells were incubated at 37°C, 5% CO2 for 48-72 hours until detection.

[0108] 2.4 Detection of DLL3 CAR-T cell electroporation efficiency

[0109] After electroporation for 48-72 hours, 13 groups of T cells electroporated with PB DLL3 CAR plasmid and T cells without CAR plasmid electroporation were obtained for CAR positivity rate analysis by flow cytometry. Biotin-labeled anti-human IgG(Fab)2 antibody (Jackson Immunosorbent Assay, 109-065-006) or human DLL3 antigen protein (ACRO, DL3-H82E4) were used as CAR-binding proteins. Chimeric antigen receptor (CAR) expression was detected by flow cytometry using avidin-conjugated PE fluorescent dye. Untransduced T lymphocytes were used as negative controls. The CAR positivity rates of T lymphocytes expressing different chimeric antigen receptors are shown in Table 4, ranging from 10-50%. The experimental results indicate that CAR structures constructed with different SCFVs in this invention can all be used to prepare CAR-T cells, and under the same conditions, DLL3 CAR-T cells with higher transduction efficiency are more beneficial for subsequent applications. Figures 2A-2N This diagram illustrates a flow cytometry study of CAR-positive T lymphocytes expressing different DLL3 chimeric antigen receptors, detected using anti-human IgG (Fab)2 antibody. Figure 2A Results for T cells without CAR transduction are shown. Figure 2B The results of 332 DLL3 CAR-T are shown. Figure 2C The results of 244 DLL3 CAR-T studies are shown. Figure 2D The results of 306 DLL3 CAR-T are shown. Figure 2E The results for DLL3CAR-T 304 are shown. Figure 2F The results of 229 DLL3 CAR-T studies are shown. Figure 2G The results of 564 DLL3 CAR-T studies are shown. Figure 2HThe results of 654 DLL3 CAR-T studies are shown. Figure 2I The results of 108 DLL3 CAR-T were shown. Figure 2J The results of 83 DLL3 CAR-T were shown. Figure 2K The results of 131 DLL3 CAR-T are shown. Figure 2L The results of 136 DLL3 CAR-T were shown. Figure 2M The results of 412DLL3 CAR-T are shown. Figure 2N The results for 303 DLL3 CAR-T are shown. Figures 3A-3N This diagram illustrates a flow cytometry study of CAR-positive T cells expressing different DLL3 chimeric antigen receptors, using DLL antigen detection. Figure 3A Results for T cells without CAR transduction are shown. Figure 3B The results of 332 DLL3 CAR-T are shown. Figure 3C The results of 244 DLL3 CAR-T studies are shown. Figure 3D The results of 306 DLL3 CAR-T are shown. Figure 3E The results of 304 DLL3 CAR-T are shown. Figure 3F The results of 229 DLL3 CAR-T studies are shown. Figure 3G The results of 564 DLL3 CAR-T studies are shown. Figure 3H The results of 654 DLL3 CAR-T studies are shown. Figure 3I The results of 108DLL3 CAR-T are shown. Figure 3J The results of 83 DLL3 CAR-T were shown. Figure 3K The results of 131 DLL3 CAR-T are shown. Figure 3L The results of 136 DLL3 CAR-T were shown. Figure 3M The results of 412 DLL3 CAR-T were shown. Figure 3N The results for 303 DLL3 CAR-T are shown. Figure 4A The results of CAR positivity rates of 13 T cells targeting DLL3 chimeric antigen receptors were shown by an anti-human IgG (Fab)2 antibody detection method. Figure 4B The results of CAR positivity rates of 13 T cells targeting DLL3 chimeric antigen receptors were shown by a human DLL3 antigen protein detection method.

[0110] Table 4. Positive rates of T lymphocytes expressing different chimeric antigen receptors

[0111]

[0112]

[0113] 2.5 Detection of CAR expression stability in DLL3 CAR-T cells

[0114] T cells from 13 groups of PB DLL3 CAR plasmids electroporated and T cells without CAR plasmid electroporation were continuously cultured. CAR positivity was detected on days 4, 6, 8, and 11 after electroporation. Biotin-labeled anti-human IgG (Fab)2 antibody and human DLL3 antigen protein were used as CAR binding proteins, respectively. Chimeric antigen receptor (CAR) expression was detected by flow cytometry using avidin-conjugated PE fluorescent dye. Untransduced T lymphocytes were used as negative controls. The changes in CAR positivity of 13 DLL3CAR-T cells at different culture days were recorded. Figure 5A The results of the CAR positivity rate of 13 T cells targeting DLL3 chimeric antigen receptors under different culture days are shown in the detection method of anti-human IgG (Fab)2 antibody. Figure 5B The results show the changes in CAR positivity rates of 13 T cells targeting the DLL3 chimeric antigen receptor using a human DLL3 antigen protein detection method at different culture days. The results indicate that within a certain time range, the CAR expression of different DLL3 CAR-T cells remained relatively stable, without any abnormalities such as a decrease.

[0115] Example 3. In vitro toxicity test of DLL3-specific chimeric antigen receptor-modified T cells.

[0116] In vitro efficacy studies were conducted using a mechanism of action (MOA) model of the product. Lung cancer cell lines NCI-H460 (non-small cell lung cancer), SHP-77 (small cell lung cancer), and SHP-77-hDLL3 (small cell lung cancer) were used as target cells for validating DLL3 CAR-T cell function. The SHP-77-hDLL3 cell line was obtained by infection and selection with a lentivirus overexpressing human DLL3 protein, resulting in high expression of human DLL3 protein. Flow cytometry was used to detect human DLL3 antigen in these three lung cancer cell lines. The NCI-H460 cell line showed low expression of human DLL3 antigen, the SHP-77 cell line showed moderate expression, and the SHP-77-hDLL3 cell line showed high expression. Figure 6 The flow cytometry results of DLL3 antigen expression in human NCI-H460, SHP-77, and SHP-77-hDLL3 lung cancer cell lines are shown.

[0117] By setting different E:T (effective cells: target cells) ratios, a co-culture system of CAR-T cells and targeted tumor cells was established. The biological efficacy of CAR-T was evaluated by detecting the tumor cell killing rate. At the same time, a control system of co-culturing untransduced T cells and tumor cells was established. Figure 7A -C shows the in vitro specific killing results of different DLL3 CAR-T cells on lung cancer cell lines. Figure 7A The in vitro specific killing results of 13 DLL3 CAR-T cells against the NCI-H460 (non-small cell lung cancer) cell line are shown. Figure 7B The in vitro specific killing results of 13 DLL3 CAR-T cells against the SHP-77 (small cell lung cancer) cell line are shown. Figure 7C The in vitro specific killing results of 13 types of DLL3 CAR-T cells against human DLL3-positive SHP-77-hDLL3 (small cell lung cancer) are shown. Figure 7A -C indicates that T represents the experimental results of T cells without CAR transduction. In vitro experimental results show that, with a fixed number of tumor cells, except for 564 scfv CAR-T, the other 12 DLL3 CAR-T cells, when co-incubated with tumor cells at an effector-to-target ratio of 5:1, exhibited a killing efficiency of 18%-48% after 24 hours for SHP-77 cells expressing human DLL3 antigen; for SHP-77-hDLL3 cells with high human DLL3 antigen expression, the killing efficiency was 21%-56% after 24 hours; while there was no significant killing effect on the NCI-H460 cell line with low human DLL3 antigen expression. These results indicate that the 12 DLL3 CAR-T cells can specifically kill tumor cells expressing human DLL3 antigen, and the killing efficiency is positively correlated with the expression level of human DLL3 antigen. Table 5 shows the killing effects of 13 DLL3 CAR-T cells on the above three lung cancer cell lines.

[0118] Table 5. Killing effects of 13 DLL3 CAR-T cells on the above three lung cancer cell lines.

[0119]

[0120] Simultaneously, the biological efficacy of CAR-T cells was assessed by detecting the levels of cytokines (IFN-γ) secreted into the culture supernatant. After co-culturing DLL3 CAR-T cells with human DLL3-overexpressing tumor cells (SHP-77-hDLL3), except for 564 scfv CAR-T cells, the IFN-γ release levels of the other 12 DLL3 CAR-T cell groups after killing tumor cells were at least twice that of the negative control (untransduced CAR T cells) (see [link to relevant documentation]). Figure 8CTable 6 shows the IFN-γ release from untransduced CAR T cells after co-culturing the 13 DLL3 CAR-T cells of this invention with three cell lines: NCI-H460, SHP-77, and SHP-77-hDLL3. Figure 8A -C presents the IFN-γ release results after different DLL3 CAR-T cells killed three types of lung cancer cells, among which... Figure 8A The study shows the release of IFN-γ from 13 DLL3 CAR-T cells co-cultured with NCI-H460 cell lines. Figure 8B The study shows the release of IFN-γ from 13 DLL3 CAR-T cells co-cultured with SHP-77 cell lines. Figure 8C The study showed the release of IFN-γ from 13 DLL3 CAR-T cells co-cultured with SHP-77-hDLL3 cell lines. Figure 8A In -C, T represents the release of IFN-γ from T cells that have not been transduced with CAR.

[0121] Table 6. IFN-γ release from co-culture supernatant

[0122]

[0123]

[0124] Based on the above in vitro cytotoxicity test, it was shown that, except for 564 scfv CAR-T, the 12 types of T lymphocytes of the present invention expressing different DLL3 chimeric antigen receptors all had good killing effects on DLL3-positive lung cancer cells, which provides a basis for in vivo efficacy studies in animals.

[0125] The specific killing assay was performed using the LDH Release Assay Kit (Dojin Chemical, CK12), a diaphorase-catalyzed INT colorimetric reaction. This method detects the activity of lactate dehydrogenase released during cytotoxicity through colorimetric analysis. The principle is based on the disruption of cell membrane structure caused by apoptosis or necrosis, which leads to the release of enzymes from the cytoplasm into the culture medium, including the relatively stable enzyme lactate dehydrogenase (LDH). By detecting the activity of LDH released from ruptured cells into the culture medium, quantitative analysis of cytotoxicity can be achieved. LDH release is considered an important indicator of cell membrane integrity and is widely used in cytotoxicity detection.

[0126] The cytokine detection method utilizes a human IFN-γ enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, SIF50), based on the immobilization of antigens or antibodies and enzyme labeling of these antibodies. The antigens or antibodies bound to the solid-phase carrier retain their immunological activity, while the enzyme-labeled antigens or antibodies retain both their immunological and enzyme activity. During detection, the analyte (antigen or antibody) in the sample binds to the immobilized antibody or antigen. Unbound material is removed by washing the plate, and then enzyme-labeled antigen or antibody is added. The amount of enzyme that is immobilized is related to the amount of the analyte in the sample. After adding a substrate that reacts with the enzyme, color development occurs, and the intensity of the color indicates the concentration of the substance in the sample, allowing for qualitative or quantitative analysis.

[0127] Example 4: Cross-reactivity test of DLL3-specific chimeric antigen receptor-modified T cells to DLL3-positive cells of different species

[0128] The most crucial aspect of evaluating the safety of CAR-T products using immunodeficient mice is the cross-reactivity of their antibody scfv with the mice's own target proteins. Therefore, this invention conducted a cross-reactivity test of DLL3-specific chimeric antigen receptor-modified T cells with DLL3-positive cells from different species.

[0129] High-expressing human or mouse DLL3 proteins were obtained by infecting and screening 293T cells with human or mouse DLL3 protein lentiviruses (purchased from Jimon Biotechnology), and were named 293T-hDLL3 and 293T-mDLL3, respectively. Using 12 different SCFVs (excluding 564) prepared above as effector cells, 293T cells, 293T cells overexpressing human DLL3 protein, and 293T cells overexpressing mouse DLL3 protein were used as target cells. Co-culture systems of CAR-T cells and target cells were established at different E:T ratios (effector cells: target cells = 5:1, 2.5:1, 1.25:1). The specific response of CAR-T cells to the two proteins was evaluated by detecting tumor cell killing rate. In vitro experimental results showed that... Figures 9A-9C As shown, Figure 9A The in vitro killing results of 12 types of DLL3 CAR-T cells on the 293T cell line are shown. Figure 9B The in vitro killing results of 12 types of DLL3 CAR-T cells on the human DLL3 protein-overexpressing 293T cell line (293T-hDLL3) are shown. Figure 9CThe in vitro killing results of 12 types of DLL3 CAR-T cells against the mouse DLL3 protein-overexpressing 293T cell line (293T-mDLL3) are shown. Tables 7-1 to 7-3 present the experimental results of killing assays of the 12 types of DLL3 CAR-T cells and untransduced CAR T cells against the 293T, 293T-hDLL3, and 293T-mDLL3 cell lines. In vitro experimental results showed that, with a fixed number of tumor cells, when DLL3 CAR-T cells prepared with different scfvs were co-incubated with 293T-hDLL3 tumor cells at an effector-to-target ratio of 5:1, the tumor cell killing efficiency reached 15%-20% after 24 hours. When co-incubated with 293T-mDLL3 tumor cells at effector-to-target ratios of 1.25:1, 2.5:1, and 5:1, the DLL3 CAR-T cells prepared with different scfvs all showed significant differences in specific killing effect, indicating that DLL3 CAR-T cells may attack tissues with DLL3 target distribution in mice in vivo. Therefore, it can be used to evaluate the in vivo safety of DLL3 CAR-T cells.

[0130] Table 7-1. Results of DLL3 CAR-T cell line killing assay

[0131]

[0132]

[0133] Meanwhile, the CAR-T specific response was further evaluated by detecting the content of cytokines (IFN-γ) secreted into the culture supernatant. The results showed that when T cells modified with DLL3-specific chimeric antigen receptors of different scfvs were co-cultured with DLL3-positive cells of different species, they not only killed human or mouse DLL3-positive cells (see Figures 9A-C, Tables 7-1 to 7-3), but also released a large amount of IFN-γ cytokines (see Figures 10A-C, Table 8). Figure 10A The results of IFN-γ release after in vitro killing of 293T cell line by 12 types of DLL3 CAR-T cells are shown. Figure 10B The results of IFN-γ release after in vitro killing of human DLL3 protein-overexpressing 293T cell line (293T-hDLL3) by 12 types of DLL3 CAR-T cells are shown. Figure 10C The results of IFN-γ release after in vitro killing of mouse DLL3 protein-overexpressing 293T cell lines (293T-mDLL3) by 12 types of DLL3 CAR-T cells are shown. Table 8 presents the specific experimental results of IFN-γ release after killing 293T, 293T-hDLL3, and 293T-mDLL3 cell lines by the 12 types of DLL3 CAR-T cells.

[0134] The results showed that the different scfv DLL3 CAR-T cells involved in the present invention all killed human DLL3 protein-overexpressing 293T cells and released cytokines. At the same time, they could also kill mouse DLL3 protein-overexpressing 293T cells and release cytokine IFN-γ, proving that they have cross-reactivity with mouse target proteins, providing a theoretical basis for subsequent safety assessment using mouse animal models.

[0135] Table 8. Effects of DLL3 CAR-T on 293T, 293T-hDLL3, and 293T-mDLL3 cell lines

[0136] Results of IFN-γ release detection test after killing

[0137]

[0138] Example 5: Test on the sustained inhibitory effect of DLL3-specific chimeric antigen receptor-modified T cells on human small cell lung cancer SHP-77 cell line.

[0139] To evaluate the long-term antitumor effects of different scfv DLL3 CAR-T cells in vitro, this invention presents Example 5. Using the aforementioned eight different scfv DLL3 CAR-T cells as effector cells and human small cell lung cancer SHP-77 cell line as target cells, a cytokine-free co-culture system was employed at an E:T (effective cell: target cell) ratio of 1:10. The sustained tumor-killing ability of DLL3 CAR-T cells was assessed by detecting changes in the number of CAR-T cells and tumor cells in the co-culture system.

[0140] Figure 11A -B shows the long-term anti-tumor effects of eight DLL3 CAR-T cells on DLL3-expressing SHP-77 cells, among which... Figure 11A The results show the changes in the number of tumor cells after long-term anti-tumor effects of eight types of DLL3 CAR-T cells on DLL3-expressing positive SHP-77 cells. Figure 11B The study presents the changes in the number of CAR-positive T cells in the long-term antitumor effect of eight DLL3 CAR-T cell lines on DLL3-expressing SHP-77 cells. The results show that, at an effector cell:target cell ratio of 1:10, different scfv DLL3 CAR-T cells exhibited varying degrees of sustained inhibitory activity against the human small cell lung cancer SHP-77 cell line. Specifically, 108, 131, and 654 scfv DLL3 CAR-T cells were able to completely eliminate tumor cells.

[0141] Example 6: In vivo efficacy test of DLL3 CAR-T in animals

[0142] In Example 6, an immunodeficient mouse model of human small cell lung cancer tumor cell burden was established to evaluate the efficacy and safety of different scfv DLL3 CAR-T cells in mice.

[0143] 6.1 Preparation of DLL3 CAR-T cells

[0144] 6.1.1 Sorting and Activation of T Cells

[0145] Peripheral blood mononuclear cells (PBMCs) were provided (separation services were provided by Shanghai Aoneng Company). Cells were magnetically labeled using the CD3 MicroBeads human-lyophilized Kit (purchased from Miltenyi Biotech), resulting in high-purity CD3+ T lymphocytes that were positively sorted, with the proportion of CD3-positive T cells exceeding 95%. The purified T cells were then activated and proliferated using Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher, 11132D).

[0146] 6.1.2 CAR gene transduction of T cells

[0147] PB DLL3 CAR plasmids constructed from the scfv of six selected DLL3 antibodies (332, 304, 229, 654, 108, and 131) were electroporated. First, the cells used for electroporation were resuspended, and cell clumps were dispersed using pipette tips or pipettes. The cell resuspension was counted, and 5 x 10⁻⁶ cells were used. 6 Cells were used for a single electroporation experiment. 5 x 10⁵ cells were incubated in DPBS (GIBCO, 14190-144). 6 Dilute the cells to 5 mL, centrifuge at 300 xg for 10 minutes at room temperature, discarding as much supernatant as possible to avoid contact with the cell pellet. Resuspend the cells in 5 mL of DPBS and wash them. Centrifuge at 300 xg for 10 minutes at room temperature, discarding as much supernatant as possible to avoid contact with the cell pellet. Resuspend the cells in 100 μL of electroporation buffer Entranster-E (Engreen, 98668-20). Transfer the cell suspension to a 1.5 mL centrifuge tube. Add the components from Table 9 (for each electroporation system) to the centrifuge tube and mix well.

[0148] Table 9. Electro-electric system for each group

[0149] PB DLL3 CAR plasmid (1ug / uL) 5 Transposase mRNA (1ug / uL) 5 cell suspension 100 Total volume 110

[0150] 6.1.3 Detection of DLL3 CAR-T cell electroporation efficiency

[0151] After 48-72 hours of electroporation, T cells transfected with the PB DLL3 CAR plasmid in six groups, along with T cells as a control without CAR plasmid transfection, were obtained for CAR positivity rate analysis by flow cytometry. Biotin-labeled human DLL3 antigen protein (ACRO, DL3-H82E4) was used as the CAR-binding protein, and chimeric antigen receptor (CAR) expression was detected by flow cytometry using avidin-conjugated PE fluorescent dye. Untransduced T lymphocytes served as a negative control. The positivity rates of T lymphocytes expressing six different chimeric antigen receptors are shown in Table 10. Figures 12A-12G The results of positivity rate assays for six different DLL3 CAR-T cells used in in vivo efficacy studies are shown, with positivity rates ranging from 10% to 40%. Figure 12A Results for T cells without CAR transduction are shown. Figure 12B The results of 332 DLL3 CAR-T are shown. Figure 12C The results of 304 DLL3 CAR-T are shown. Figure 12D The results of 229DLL3 CAR-T are shown. Figure 12E The results of 654 DLL3 CAR-T studies are shown. Figure 12F The results of 108 DLL3 CAR-T were shown. Figure 12G The results of 131 DLL3 CAR-T are shown.

[0152] Table 10. Positive rates of T lymphocytes expressing different chimeric antigen receptors

[0153] Untransduced CAR T cells (Mock T) 0.12% 332 DLL3 CAR-T 24.18% 304 DLL3 CAR-T 26.50% 229 DLL3 CAR-T 26.92% 654 DLL3 CAR-T 12.77% 108 DLL3 CAR-T 33.72% 131 DLL3 CAR-T 24.63%

[0154] 6.1.4 DLL3 CAR-T Animal Model Experiment

[0155] Based on in vitro studies, female NCG mice (purchased from Jicui Yaokang) were injected subcutaneously with 5×10 6 Personal small cell lung cancer cells SHP-77, day 11 after inoculation (tumor volume 60mm) 3 The drug was administered at approximately the same size (likely referring to a specific drug or treatment unit), with the Mock-T (untransduced CAR T cell group) receiving 5 × 10⁻⁶ cells. 6 The number of T cells was determined by administering 5 × 10⁻⁶ T cells to different scfv DLL3 CAR-T cell groups (332, 304, 229, 654, 108, and 131 scfv DLL3 CAR-T cells). 6CAR-positive T cells were administered in 200 μL volumes to all groups. Five animals were included in each conditional group. Tumors were measured twice weekly after administration; tumor growth curves were plotted, and TGI and T / C were calculated. All tumors were photographed at the experimental endpoint. Blood samples were collected on days 2, 14, and 28 after CAR-T administration, as well as at the endpoint. The copy number of CAR in peripheral blood (VCN) was detected by qPCR to confirm CAR-T cell expansion. Results showed that after 17 days of CAR-T administration, all drug groups exhibited significant efficacy, with tumor inhibition rates ranging from 25% to 97%, specifically including the following indicators:

[0156] (1) Body weight: Compared with the Mock T group (T cells without CAR transduction), there was no significant difference in body weight among the different scfv DLL3 CAR-T administration groups. Figure 13 This study demonstrates the efficacy of six DLL3 CAR-T cells in reducing body weight in NCG mice bearing SHP-77 small cell lung cancer tumors.

[0157] (2) Tumor inhibition rate (TGI) statistics: By day 17 after medication, all drug groups showed significant efficacy, with tumor inhibition rates ranging from 25% to 97%. Among them, drug 108 showed the best efficacy, with a tumor inhibition rate of 96.53%. Figure 14 This study demonstrates the efficacy of six DLL3 CAR-T cells in inducing tumor size changes in NCG mice bearing SHP-77 small cell lung cancer.

[0158] (3) Mortality rate: Up to 17 days after drug administration, mice in the Mock-T cell group and different scfv DLL3 CAR-T drug administration groups were in good condition and no mice died. Figure 15 This study demonstrates the efficacy of six DLL3 CAR-T cells in improving the survival rate of NCG mice bearing SHP-77 tumors in small cell lung cancer.

Claims

1. A chimeric antigen receptor (CAR) targeting DLL3 antigen, characterized in that, The CAR comprises an anti-DLL3 antibody scFv, wherein the scFv contains three light chain complementarity-determining regions and three heavy chain complementarity-determining regions, wherein The three light chain complementary determination regions are LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:62, and the three heavy chain complementary determination regions are HCDR1 shown in SEQ ID NO:38, HCDR2 shown in SEQ ID NO:64, and HCDR3 shown in SEQ ID NO:

65.

2. The chimeric antigen receptor targeting DLL3 antigen according to claim 1, characterized in that, The scFv of the anti-DLL3 antibody contains a light chain variable region of the amino acid sequence shown in SEQ ID NO:95 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:

63.

3. The chimeric antigen receptor targeting DLL3 antigen according to claim 2, characterized in that, The CAR, from N-terminus to C-terminus, sequentially comprises a CD8α signal peptide, a DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular signaling domain, and the amino acid sequence of the CAR is shown in SEQ ID NO:

12. The amino acid sequence of the CD8α signal peptide is SEQ ID NO: 1; The amino acid sequences of the CD8 hinge region and CD8 transmembrane region are SEQ ID NO: 2; The amino acid sequence of the 4-1BB co-stimulatory signal domain is SEQ ID NO: 3; and The amino acid sequence of the CD3ζ intracellular signaling domain is SEQ ID NO:

4.

4. A nucleic acid encoding a chimeric antigen receptor targeting the DLL3 antigen as described in any one of claims 1-3.

5. An expression cassette comprising the nucleic acid of claim 4.

6. A vector containing the nucleic acid of claim 4 or the expression cassette of claim 5.

7. A cell comprising the nucleic acid of claim 4, the expression cassette of claim 5, or the vector of claim 6, wherein the cell is a CAR-T cell.

8. A pharmaceutical composition comprising the cells of claim 7.

9. A kit comprising a chimeric antigen receptor targeting DLL3 antigen as described in any one of claims 1-3, or a nucleic acid as described in claim 4, or an expression cassette as described in claim 5, or a vector as described in claim 6.

10. A kit comprising the cells of claim 7.

11. Use of the cells of claim 7, the pharmaceutical composition of claim 8, or the kit of claim 10 in the preparation of a medicament for treating a DLL3-related disease, wherein the DLL3-related disease is small cell lung cancer or non-small cell lung cancer.

12. A method for preparing engineered immune cells, characterized in that, Includes the following steps: (1) Provide an immune cell to be modified; and (2) The nucleic acid of claim 4, or the expression cassette of claim 5, or the vector of claim 6 is introduced into the immune cells; The immune cells mentioned are T cells.

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