An antibody or antigen-binding fragment thereof targeting dll3 and uses thereof

Abstract

This invention relates to the field of biomedical technology, specifically to an antibody or its antigen-binding fragment targeting DLL3 and its application. It also provides the application of the aforementioned antibody or fragment in the construction of chimeric antigen receptors (CARs). The antibody consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of VH is selected from any sequence shown in SEQ ID NO:1 to SEQ ID NO:7, and the amino acid sequence of VL is selected from any sequence shown in SEQ ID NO:8 to SEQ ID NO:14. After constructing a CAR from the scFv fragment of this antibody and introducing it into T cells, the resulting CAR-T cells exhibit highly efficient and specific killing activity against DLL3-positive target cells both in vitro and in vivo, showing promising clinical application prospects in the treatment of DLL3-positive tumors (such as small cell lung cancer).

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antibody or antigen-binding fragment thereof targeting DLL3 and its applications. Background Technology

[0002] Delta-like ligand 3 (DLL3) belongs to the Delta-like ligand (DLL) family and is a single-pass transmembrane protein anchored to the cell membrane surface. The human DLL3 protein contains 619 amino acid residues and its structural features include a conserved DSL (Delta, Serrate, Lag2) domain of 40 amino acids at the N-terminus, six consecutive EGF-like repeat sequences, and a transmembrane region. The DSL domain is highly conserved among ligand family members and is a key region for ligand binding to the Notch receptor.

[0003] From an evolutionary perspective, DLL3 exhibits extremely high conservation, with a homology of up to 82% between human and mouse DLL3 protein sequences. Under normal physiological conditions, DLL3 RNA transcripts are mainly distributed in brain tissue, endocrine organs, and the blood system, while their expression is almost undetectable in other normal tissues. However, multiple studies have confirmed that DLL3 shows significantly high levels of expression in approximately 80% of small cell lung cancer (SCLC) and other types of neuroendocrine tumors. This significant difference in DLL3 expression between normal cells and tumor cells endows DLL3 with unique value as a selective therapeutic target for tumors.

[0004] Small cell lung cancer (SCLC) is a poorly differentiated neuroendocrine malignancy and one of the most aggressive subtypes of lung cancer. The occurrence and development of SCLC are closely related to the Notch signaling pathway—which regulates many fundamental biological processes during normal development. Inactivation mutations in this pathway can induce non-neuroendocrine tumor cells or tumor progenitor cells to differentiate into neuroendocrine cells. Notably, DLL3 is the only known inhibitory ligand in the Notch signaling pathway, and it is highly expressed in approximately 85% of SCLC cases. The above research evidence strongly suggests that DLL3 could serve as an effective therapeutic target for small cell lung cancer and other neuroendocrine tumors. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an antibody or antigen-binding fragment targeting DLL3 and its application. The antibody or antigen-binding fragment can accurately identify the DLL3 target, thereby providing a new technical means for the treatment of DLL3-positive tumors.

[0006] To achieve the above objectives, in a first aspect of the present invention, an antibody or antigen-binding fragment thereof targeting DLL3 is provided. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0007] The amino acid sequence of the heavy chain variable region is selected from:

[0008] (1) Any one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:7;

[0009] (2) An amino acid sequence having at least 80% sequence identity with any of the sequences shown in SEQ ID NO:1 to SEQ ID NO:7;

[0010] The amino acid sequence of the light chain variable region is selected from:

[0011] (3) Any one of the sequences shown in SEQ ID NO:8 to SEQ ID NO:14;

[0012] (4) An amino acid sequence that has at least 80% sequence identity with any of the sequences shown in SEQ ID NO:8 to SEQ ID NO:14.

[0013] The amino acid sequence of any of the above heavy chain variable regions can be paired with the amino acid sequence of any of the light chain variable regions.

[0014] In one embodiment, the heavy chain variable region and the light chain variable region are paired one-to-one as shown in Table 1.

[0015] Table 1. Combinations of light and heavy chain amino acid sequences of antibodies

[0016] Combination number Heavy chain variable region (VH) SEQ ID NO Light chain variable region (VL) SEQ ID NO Corresponding antibody naming M1 SEQ ID NO:1 SEQ ID NO:8 SDL3-1 M2 SEQ ID NO:2 SEQ ID NO:9 SDL3-2 M3 SEQ ID NO:3 SEQ ID NO:10 SDL3-3 M4 SEQ ID NO:4 SEQ ID NO:11 SDL3-4 M5 SEQ ID NO:5 SEQ ID NO:12 SDL3-5 M6 SEQ ID NO:6 SEQ ID NO:13 SDL3-6 M7 SEQ ID NO:7 SEQ ID NO:14 SDL3-7

[0017] In one embodiment, the antibody or its antigen-binding fragment may be a fully human monoclonal antibody, a chimeric antibody, or a single-chain antibody (scFv).

[0018] When the heavy chain and light chain variable regions of the antibody or its antigen-binding fragment described in this invention are paired and used, a corresponding protein structure must be designed for them. In one embodiment, when it is a single-chain antibody, the heavy chain variable region and the light chain variable region are connected in series via a flexible linker.

[0019] In a second aspect of the invention, a chimeric antigen receptor fusion protein is provided. It comprises the antibody or its antigen-binding fragment described in the first aspect of the invention. Specifically, the extracellular antigen-binding domain is formed by the scFv form of the antibody, sequentially connected to a transmembrane domain and an intracellular signal transduction domain. The designed protein structure is arranged as follows:

[0020] SP (signal peptide)-VH-CH (human IgG1)-SP (signal peptide)-VL-CL (human Kappa);

[0021] The amino acid sequence of the signal peptide SP is shown in SEQ ID NO:15, the amino acid sequence of CH (heavy chain constant region) is shown in SEQ ID NO:16, and the amino acid sequence of CL (light chain constant region) is shown in SEQ ID NO:17.

[0022] In one embodiment, the transmembrane domain is selected from: the CD28 hinge region combined with the CD28 transmembrane domain, the CD8 hinge region combined with the 4-1BB transmembrane domain, or the CD8 hinge region combined with the ICOS transmembrane domain. The intracellular domain includes a co-stimulatory signal transduction region (selected from the intracellular domains of CD28, 4-1BB, or ICOS) and a CD3ζ chain activation domain.

[0023] In a third aspect of the invention, a plasmid expression cassette is provided, comprising a gene sequence encoding the antibody or its antigen-binding fragment described in the first aspect of the invention. After being introduced into cells via a delivery system (such as lentivirus, retrovirus, plasmid vector, nanodelivery system, electrotransduction, or transposon), the expression cassette can express the antibody or its antigen-binding fragment, or the chimeric antigen receptor fusion protein (CAR).

[0024] In a fourth aspect of the invention, a recombinant protein is provided, the recombinant protein comprising the antibody or antigen-binding fragment thereof described in the first aspect of the invention, the chimeric antigen receptor fusion protein described in the second aspect of the invention, or the plasmid expression cassette described in the third aspect of the invention.

[0025] In a fifth aspect of the invention, a polynucleotide is provided that encodes a polypeptide selected from the group consisting of: the antibody or antigen-binding fragment thereof described in the first aspect of the invention, the chimeric antigen receptor fusion protein described in the second aspect of the invention, or the recombinant protein described in the fourth aspect of the invention.

[0026] In a sixth aspect of the invention, a recombinant vector is provided, comprising the polynucleotide described in the fifth aspect of the invention. The recombinant vector is prepared from a plasmid, lentiviral vector, adenoviral vector, retroviral vector, or transposon vector.

[0027] In a seventh aspect of the invention, a host cell is provided, comprising the antibody or antigen-binding fragment thereof described in the first aspect of the invention, the chimeric antigen receptor fusion protein described in the second aspect of the invention, the plasmid expression cassette described in the third aspect of the invention, the recombinant protein described in the fourth aspect of the invention, or the recombinant vector described in the sixth aspect of the invention, or having the polynucleotide described in the fifth aspect of the invention integrated into its genome. The host cell is preferably an immune cell selected from T cells, NK cells, NKT cells, macrophages, γδT cells, TIL cells, or TCR-T cells.

[0028] In an eighth aspect of the invention, an antibody-drug conjugate is provided, comprising the antibody or its antigen-binding fragment described in the first aspect of the invention, the chimeric antigen receptor fusion protein described in the second aspect of the invention, the plasmid expression cassette described in the third aspect of the invention, the recombinant protein described in the fourth aspect of the invention, the polynucleotide described in the fifth aspect of the invention, the recombinant vector described in the sixth aspect of the invention, or the host cell described in the seventh aspect of the invention. The antibody-drug conjugate may further comprise a reagent for detecting DLL3, an antibody-drug conjugate toxin, a multispecific antibody, or a functional recombinant protein.

[0029] In a ninth aspect of the invention, a pharmaceutical composition is provided comprising the antibody or antigen-binding fragment thereof described in the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0030] The aforementioned antibodies, polynucleotides, plasmid expression cassettes, recombinant vectors, and host cells can all be used as raw material components for antibody-drug conjugates. Furthermore, these components can be widely used in the preparation of drugs for the prevention or treatment of DLL3-positive tumors or cancers; wherein the cancers or tumors can be selected from, but are not limited to, small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, glioblastoma multiforme, metastatic castration prostate cancer, small cell bladder cancer, neuroendocrine lung tumors, and other solid tumors.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] The DLL3-targeting antibody provided by this invention possesses a novel VH / VL sequence combination, which can flexibly pair to form various high-affinity antibodies. After constructing a chimeric antigen receptor from the scFv of this antibody and introducing it into T cells, the resulting CAR-T cells exhibit highly efficient and specific killing activity against DLL3-positive target cells, with no significant off-target toxicity to normal cells. It shows promising clinical application prospects in the treatment of DLL3-positive tumors such as advanced small cell lung cancer. Attached Figure Description

[0033] Figure 1 This is a bar chart showing the ELISA detection of the binding activity and specificity of the candidate antibody targeting DLL3 in Example 1;

[0034] Figure 2 This is a bar chart showing the MFI values ​​of the candidate antibody targeting DLL3 in Example 2 binding to T cells;

[0035] Figure 3 Flow cytometry plot of the CAR positivity rate of the first 6 types of T cells in Example 5;

[0036] Figure 4 Flow cytometry plot of the CAR positivity rate of the latter three types of T cells in Example 5;

[0037] Figure 5 The graph shows the killing rate curves of the seven types of CAR-T cells in Example 6. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The amino acid sequences of the heavy chain variable region and the light chain variable region designed in this invention are as follows.

[0040] (1) The amino acid sequence of the variable region of the heavy chain is as follows:

[0041] SEQ ID NO:1:QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDREDSGYDLPFWGQGTTVTVSS;

[0042] SEQ ID NO:2: EVQLVQSGGGLIQPGRSLRLSCKASGFIFRDHIMGWVRQAPGKGLEWVSAISANGGNTFYADSVKGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCAKDSGSFDYWGQGTTVTVSS;

[0043] SEQ ID NO:3: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQGTLVTVSS;

[0044] SEQ ID NO:4: EVHLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQGTLVTVSS;

[0045] SEQ ID NO:5: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARVPLYVGGAFDIWGQGTLVTVSS;

[0046] SEQ ID NO:6: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGPEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQGTLVTVSS;

[0047] SEQ ID NO:7: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQGTLVTVSS.

[0048] (2) The amino acid sequence of the above light chain variable region is as follows:

[0049] SEQ ID NO:8: NIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISNLQPDDFATYYCQQYNSYPYTFGQGTKVEIK;

[0050] SEQ ID NO:9: EIVLTQSPDFQSATPKEKVIITCRASQSIGTGLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGTDFTLTINSLEAEDAAAYYCHQSISLPTFGGGTKVDIK;

[0051] SEQ ID NO:10: DVVMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSRSGTDFTLTISSLQREDFATYYCQQSFSPLITFGQGTRLEIK;

[0052] SEQ ID NO:11: AIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLSVTFGQGTRLEIK;

[0053] SEQ ID NO:12: NIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQTPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFSISPLTFGGGTKVEIK;

[0054] SEQ ID NO:13: EIVMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGTAPKLLIYAASTLQSGVPSRFSGTGSGTEFTLTISSLQPEDSATYYCQQSYSTHSITFGQGTRLEIK;

[0055] SEQ ID NO:14: EIVLTQSPPSLSASVGDRVTITCRASQSISTYVNWYQQKPGKAPNLLIYAASSLQRGVPSRFSGSGSGTDFTLTLSSLQPEDSATYYCQQTFGTLYTFGQGTKLEIK.

[0056] The amino acid sequence of the signal peptide SP is shown in SEQ ID NO:15: MHSSALLCCLVLLTGVRA.

[0057] The amino acid sequence of CH (heavy chain constant region) is as SEQ ID NO:16 shows: ASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0058] The amino acid sequence of the CL (light chain constant region) is shown in SEQ ID NO:17: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0059] After constructing the gene of the antibody or its antigen-binding fragment described in this invention into a plasmid expression cassette, the expression cassette can be introduced into 293T cells for antibody expression via a delivery system. Selectable delivery systems include any one of lentivirus, retrovirus, conventional plasmid vectors, episome vectors, nanodelivery systems, electrotransduction, and transposons. The vector used for the expression cassette can be selected from one or more combinations of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, and other gene transfer systems; in a preferred embodiment, the vector is a viral vector.

[0060] Recombinant cells can be constructed using the aforementioned antibodies or their antigen-binding fragments through gene modification insertion technology. These recombinant cells are typically immune cells, which can be selected from any of the following: T cells, NK cells, NKT cells, macrophages, γδT cells, TIL cells, and TCR-T cells. When immune cells express chimeric antigen receptors (CARs), NK cells, NKT cells, TIL cells, and γδT cells are functionally equivalent to T cells (i.e., T cells can be replaced by NK cells, etc.).

[0061] The DLL3-targeting antibody or its antigen-binding fragment described in this invention can be used in combination with pharmaceutically acceptable carriers, diluents, or excipients to formulate a pharmaceutical preparation with good shape retention and efficacy retention, applicable to the fields of biomaterials and biopharmaceuticals. The biopharmaceutical may comprise an expression cassette (e.g., a gene expression frame), a recombinant vector (e.g., a plasmid), a recombinant protein (e.g., a fusion protein, an antibody protein, etc.), a recombinant microorganism (e.g., *Escherichia coli*, bacteriophages, etc.), or a recombinant cell line (e.g., immune cells, CHO cells, etc.) constructed from the aforementioned nucleic acid or amino acid sequences, and all of the aforementioned nucleic acid or amino acid sequences are derived from the monoclonal antibody described in this invention; wherein, the recombinant vector encompasses gene recombinant expression vectors and chimeric antigen receptors.

[0062] When the aforementioned antibodies or their antigen-binding fragments are used in biological agents, they function as an effective component of the agent. In addition, the biological agent may also contain the following functional components: reagents for detecting the concentration of human DLL3 protein, reagents for detecting the expression level of human DLL3 protein on the surface of tumor cells, antibody-drug conjugates (for killing human DLL3-positive cells), multispecific antibodies targeting human DLL3 and other antigens made by antibody-drug conjugates of other antibodies, and functional recombinant proteins targeting human DLL3 made by antibody-drug conjugates of other proteins.

[0063] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0064] Unless otherwise specified, all reagents and consumables used in the following experimental examples are commercially available.

[0065] Example 1

[0066] Screening of candidate antibodies targeting DLL3: Antibodies that specifically bind to DLL3 were screened from a fully human phage display library. Using a fully human library biopanning screening (order number: 22000134F; manufacturer: Pujian Biotechnology (Wuhan) Technology Co., Ltd.), 100 candidate antibodies that may bind to the DLL3 target antigen were selected from the human library.

[0067] (1) Using the fully human phage display library biopanning technology, 101 candidate antibodies capable of binding to the DLL3 target antigen were obtained in the first round of screening. After expressing and purifying the candidate antibodies in the CHO cell system, their binding activity and specificity to human DLL3 were evaluated by ELISA. At the same time, human DLL1 and DLL4 were selected as negative controls.

[0068] (2) ELISA verification:

[0069] (2-1) Coating: Dilute DLL3 protein to 5 μg / mL with carbonate coating buffer (0.05 mol / L, pH 9.6), add 100 μL to each well of a 96-well plate, and incubate at 4°C for 24 hours. Set up blank control wells with only coating buffer added;

[0070] (2-2) Blocking: Discard the coating solution, wash 3 times with PBST, add 250 μL of blocking solution (5% skim milk or 3% BSA PBST solution) to each well, and block at 37°C for 2 hours;

[0071] (2-3) Primary antibody incubation: 101 candidate antibodies were serially diluted 10-fold starting from 10 μg / mL, with 3 concentration points. 100 μL was added to each well and incubated at 37°C for 2 hours. DLL1 and DLL4 antibodies were set up as negative controls.

[0072] (2-4) Secondary antibody incubation: Add HRP-labeled secondary antibody (abcam, ab6789) and incubate at 37°C for 2 hours;

[0073] (2-5) Color development and detection: Wash 4 times with PBST, and wash the last time with PB to reduce air bubbles. Add 100 μL of freshly prepared TMB colorimetric solution to each well, and develop the color for 10 minutes at room temperature in the dark. Then add 50 μL of 2M H2SO4 to stop the reaction. Immediately read the absorbance at 450 nm using a microplate reader.

[0074] like Figure 1 As shown, the antibodies targeting DLL3 exhibited significantly better binding activity and specificity than those targeting DLL1 and DLL4. From the 101 candidate antibodies, nine more antibodies with even stronger binding activity and specificity were further screened: XCT04Ab19, XCT04Ab20, XCT04Ab25, XCT04Ab30, XCT04Ab42, XCT04Ab84, XCT04Ab89, XCT04Ab94, and XCT04Ab109. These antibodies were then used in subsequent validation experiments.

[0075] Example 2

[0076] The binding specificity of the candidate antibody to DLL3-positive cells:

[0077] This embodiment verifies the cellular-level binding specificity of the nine antibodies screened in Example 1.

[0078] 2.1 Antibody Coating

[0079] The primary antibody was diluted to 5 μg / mL using coating buffer (0.05 mol / L, pH 9.6). 100 μL of antibody solution was added to each well of a 96-well plate, grouped as follows:

[0080] Experimental group: 9 candidate antibodies (XCT04Ab19, XCT04Ab20, XCT04Ab25, XCT04Ab30, XCT04Ab42, XCT04Ab84, XCT04Ab89, XCT04Ab94, XCT04Ab109) and PC (AMG119);

[0081] Negative control group: DLL1 and DLL4 antibodies;

[0082] Blank control group (NC): Only coating buffer was added.

[0083] External environment: Incubate at 4℃ for 24 hours.

[0084] 2.2 Washing and Sealing

[0085] Discard the coating solution, add 300 μL of PBST to each well, let stand for 30 seconds, then aspirate and discard. Repeat 3 times. Then add 200 μL of blocking buffer (5% BSA / PBS solution) to each well, and block with gentle shaking on a shaker at room temperature for 2 hours. After blocking, wash 3 times with PBST again.

[0086] 2.3 Cell-antibody binding

[0087] After washing, bring the plate to room temperature and add 100 μL of cell suspension (293T-hDLL1 and 293T-hDLL4) to each well. Place the plate on a 4°C rotating shaker and incubate at 50 rpm for 60 minutes.

[0088] 2.4 Washing and Cell Recovery

[0089] After incubation, place the plate horizontally and slowly aspirate the supernatant. Tilt the plate at approximately 45° and very slowly add 200 μL of pre-cooled cell-binding wash buffer along the highest point of the well wall, avoiding rinsing the cell layer. Gently aspirate and repeat the wash 3-4 times. After the final wash, gently disperse and collect the cells.

[0090] 2.5 Flow Cytometry

[0091] The collected cells were centrifuged at 500g for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in PBS buffer containing 1% BSA and anti-CD16 / 32 antibody (FcBlock) and incubated on ice for 15 minutes.

[0092] After centrifugation, discard the supernatant, add staining buffer (PBS containing 1% BSA) and fluorescently labeled secondary antibody (Wuhan Sanying, SA00014-10), and incubate on ice in the dark for 30 minutes.

[0093] Wash once with 2 mL of staining buffer, then collect cells by centrifugation. Immediately after staining with 7-AAD live / dead dye, analyze by flow cytometry. Results are characterized by mean fluorescence intensity (MFI).

[0094] like Figure 2 As shown, among the nine candidate antibodies, seven antibodies—Ab20, Ab25, Ab30, Ab42, Ab84, Ab89, Ab94, and Ab109—almost did not bind to 293T-hDLL1 and 293T-hDLL4 cells, indicating that they have good specificity for DLL3. Therefore, the scFv sequences of these seven antibodies were selected for subsequent CAR molecule design and CAR-T cell preparation.

[0095] Example 3

[0096] Lentiviral packaging and CAR-T cell preparation:

[0097] 3.1 CAR Structural Design

[0098] Seven preferred candidates obtained from the screening in Example 2 (corresponding to the amino acid sequence combinations of the light and heavy chain variable regions in groups M1 to M7 in Table 1) were selected to design scFv sequences of the light and heavy chain variable regions of the antibody targeting DLL3. The corresponding antibody proteins were named SDL3-1, SDL3-2, SDL3-3, SDL3-4, SDL3-5, SDL3-6, and SDL3-7, respectively.

[0099] The seven scFv sequences were constructed into expression cassettes of second-generation CARs, named CAR-SDL3-1 to CAR-SDL3-7 respectively. Simultaneously, a CAR expression cassette targeting DLL3 was constructed using scFv as a control group, named CAR-SDL3-NT (i.e., NT cells). The specific structures of each CAR group are as follows:

[0100] 1#: CD8 SP / SDL3-1 scFv / CD8 Hinge / CD8 TM / 4-1BB / CD3ζ

[0101] 2#: CD8 SP / SDL3-2 scFv / CD8 Hinge / CD8 TM / 4-1BB / CD3ζ

[0102] 3#: CD8 SP / SDL3-3 scFv / CD8 Hinge / CD8 TM / 4-1BB / CD3ζ

[0103] 4#: CD8 SP / SDL3-4 scFv / CD8 Hinge / CD8 TM / 4-1BB / CD3ζ

[0104] 5#: CD8 SP / SDL3-5 scFv / CD8 Hinge / CD8 TM / 4-1BB / CD3ζ

[0105] 6#: CD8 SP / SDL3-6 scFv / CD8 Hinge / CD8 TM / 4-1BB / CD3ζ

[0106] 7#: CD8 SP / SDL3-7 scFv / CD8 Hinge / CD8 TM / 4-1BB / CD3ζ

[0107] 3.2 Lentiviral Packaging

[0108] The above 7 CAR expression frames were cloned into the PHBLV lentiviral vector backbone and placed under the EF1α promoter.

[0109] Each group of plasmids was mixed with a three-plasmid system: lentiviral envelope plasmid pMD2.G (Addgene#12259), lentiviral packaging plasmid psPAX2 (Addgene#12260), and a shuttle plasmid containing the DLL3 target gene, with a mass ratio of 1:1:1. The three-plasmid mixtures were introduced into 293T cells using Lipofectamine 3000 transfection reagent to prepare seven complete lentiviral expression vectors.

[0110] The lentiviral expression vector was co-cultured with 293T cells. At 48 and 72 hours of culture, samples were taken and centrifuged at 500g for 8 minutes to collect viral supernatants. Each supernatant was then concentrated by ultracentrifugation to obtain seven concentrated viral solutions for subsequent T cell infection.

[0111] Example 4

[0112] (1) Isolation of peripheral blood mononuclear cells (PBMCs) and culture of T cells:

[0113] Peripheral blood was collected from healthy donors, and mononuclear cells were isolated according to standard experimental procedures. Density gradient centrifugation was performed using Ficoll reagent, and T cells were enriched using CD3 MicroBeads (Miltenyi Biotec, 130-097-043). T cells were activated, cultured, and expanded from mononuclear cells using magnetic beads conjugated with anti-CD3 / anti-CD28. Culture conditions: TexMACS GMP Medium (Miltenyi Biotec, 170-076-309), containing 10% FBS, 2 mM L-glutamine, and 100 IU / mL rhIL2. All cells were cultured in a 37°C, 5% CO2 incubator.

[0114] (2) Transduction and expansion culture of T cells:

[0115] Eight isolates of purified primary T cells were taken and activated and cultured for one day. Seven lentiviruses prepared in step 3.2 of Example 3 were added to the seven activated T cells at an MOI ratio of 1 and cultured with lentiviral vectors for 13 days. Finally, seven CAR-T cells and one NT cell (NT cells were purified primary T cells without any treatment) were obtained.

[0116] Example 5

[0117] CAR positivity rate test:

[0118] Eight types of T cells (NT, CAR-SDL3-1 to CAR-SDL3-7) prepared in Example 4 were cultured for 13 days (the CAR positivity rate of each T cell type was detected on day 6). The flow cytometry results of the CAR positivity rates of the eight T cell types are as follows: Figure 3 and Figure 4 As shown in the figure (a to h correspond to the CAR positivity rates of NT cells and CAR-SDL3-1 to CAR-SDL3-7 on day 6, respectively, and the area to the right of the vertical line in each flow cytometry represents the CAR positivity rate).

[0119] Different scFv fragments did not have a significant effect on the CAR positivity rate (all greater than 85%), indicating that replacing the scFv fragment with the anti-DLL3 antibody had no significant effect on the proliferation and growth of CAR-T cells.

[0120] Therefore, it can be seen that the proliferation rates of the seven cell types CAR-SDL3-1 to CAR-SDL3-7 prepared in this invention are basically consistent with the proliferation rates of the NT control group cells. This indicates that the antibody or its fragment targeting human DLL3 protein, after being embedded in chimeric antigen receptor-immunized T cells (CAR-T), does not significantly interfere with the proliferation of CAR-T cells.

[0121] Example 6

[0122] In vitro lethality experiment (short-term lethality)

[0123] The eight types of T cells (seven types of CAR-T cells and one type of NT cell) obtained in Example 4 were used as effector cells, and the killing effect of each CAR-T cell was detected using an RTCA device. K562 target cells (DLL3 positive) were co-incubated with the above eight types of effector cells for 24 hours, and the effector-to-target ratio (E:T) was set at 1:1.

[0124] The results of the lethality test are as follows Figure 5As shown, the in vitro killing ability of the seven CAR-T cells was significantly superior to that of NT cells. Among them, the CAR-SDL3-5 CAR-T cells exhibited the best killing efficiency, reaching 79%. This result fully demonstrates that the design of the light and heavy chain amino acid sequences of the DLL3-targeting antibody in this invention was successful. After the selected DLL3 antibody was embedded into CAR-T cells, it could express good specific killing activity, and the selected DLL3 antibody had clear targeting specificity, capable of targetedly eliminating target cells expressing the DLL3 antigen.

[0125] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. An antibody or antigen-binding fragment thereof targeting DLL3, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is any one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:7; The amino acid sequence of the light chain variable region is any one of the sequences shown in SEQ ID NO:8 to SEQ ID NO:

14.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment simultaneously comprises: The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:8; or The amino acid sequence is the heavy chain variable region as shown in SEQ ID NO:2 and the amino acid sequence is the light chain variable region as shown in SEQ ID NO:9; or The heavy chain variable region having the amino acid sequence as shown in SEQ ID NO:3 and the light chain variable region having the amino acid sequence as shown in SEQ ID NO:10; or The heavy chain variable region having the amino acid sequence as shown in SEQ ID NO:4 and the light chain variable region having the amino acid sequence as shown in SEQ ID NO:11; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5 and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:12; or The heavy chain variable region having the amino acid sequence as shown in SEQ ID NO:6 and the light chain variable region having the amino acid sequence as shown in SEQ ID NO:13; or The amino acid sequence is the heavy chain variable region as shown in SEQ ID NO:7 and the amino acid sequence is the light chain variable region as shown in SEQ ID NO:

14.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment is a fully human monoclonal antibody, a chimeric antibody, or a single-chain antibody.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody or its antigen-binding fragment is a single-chain antibody, and the heavy chain variable region and the light chain variable region are connected in series via a linker.

5. A polynucleotide, characterized in that, The polynucleotide encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

6. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell expresses an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, or contains a polynucleotide as described in claim 5, or contains a recombinant vector as described in claim 6.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

9. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a medicament for the prevention or treatment of DLL3-positive tumors.

10. The application according to claim 9, characterized in that, The DLL3-positive tumors include small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, glioblastoma multiforme, metastatic castration prostate cancer, small cell bladder cancer, or neuroendocrine lung tumors.

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